Evaluating the Growth Potential of Pathogenic Bacteria in Water

The degree to which a water sample can potentially support the growth of human pathogens was evaluated. For this purpose, a pathogen growth potential (PGP) bioassay was developed based on the principles of conventional assimilable organic carbon (AOC) determination, but using pure cultures of selected pathogenic bacteria (Escherichia coli O157, Vibrio cholerae, or Pseudomonas aeruginosa) as the inoculum. We evaluated 19 water samples collected after different treatment steps from two drinking water production plants and a wastewater treatment plant and from ozone-treated river water. Each pathogen was batch grown to stationary phase in sterile water samples, and the concentration of cells produced was measured using flow cytometry. In addition, the fraction of AOC consumed by each pathogen was estimated. Pathogen growth did not correlate with dissolved organic carbon (DOC) concentration and correlated only weakly with the concentration of AOC. Furthermore, the three pathogens never grew to the same final concentration in any water sample, and the relative ratio of the cultures to each other was unique in each sample. These results suggest that the extent of pathogen growth is affected not only by the concentration but also by the composition of AOC. Through this bioassay, PGP can be included as a parameter in water treatment system design, control, and operation. Additionally, a multilevel concept that integrates the results from the bioassay into the bigger framework of pathogen growth in water is discussed. The proposed approach provides a first step for including pathogen growth into microbial risk assessment.Pathogenic bacteria can survive and also grow in low-nutrient aquatic environments, such as surface waters or man-made water treatment systems (2, 17, 30). Studies on pathogen survival and/or die-off (including disinfection) in water are common, but little is known about the fundamental factors governing their growth in the environment (34, 35). Understanding the growth of pathogenic bacteria in aquatic ecosystems is essential for a holistic approach to microbial risk assessment as well as for improving drinking water treatment design and operation.A key factor governing growth of all organisms is nutrient availability. All human pathogens are heterotrophs, utilizing organic compounds as their carbon and energy source. Natural organic matter in water comprises a broad spectrum of many different compounds; it is usually determined as a bulk parameter, such as dissolved organic carbon (DOC). Only a fraction (0.1 to 44%) of this DOC pool is readily available for bacterial growth (18, 33). This bioavailable fraction is quantified using bioassays, such as the biodegradable dissolved organic carbon (BDOC) assay (27) or the assimilable organic carbon (AOC) assay (31). Typically, AOC represents small molecules readily available for growth, whereas BDOC can also include larger molecular compounds, which require predegradation before they can be taken up by microbial cells. Results from both of these assays are commonly used as indicators for bacterial growth potential and have previously been associated with regrowth and biofilm formation in drinking water distribution systems (7, 20, 32).Previous studies have pointed toward an apparent correlation between the concentration of AOC and the presence of enteric bacteria. For example, during two large surveys of drinking water treatment systems across North America, the occurrence (presence/absence) of coliform bacteria was found to be elevated above an AOC concentration of 100 μg liter⁻¹ (4, 21). Other studies also found that AOC concentrations were directly correlated to growth of pathogenic bacteria (30, 34, 35). However, AOC is a bulk parameter, which includes many different substrates (e.g., amino acids, sugars, and fatty acids) readily available for heterotrophic growth. Hence, its composition can differ distinctly, and it is assumed that every aquatic environment carries a complex and unique “fingerprint” of utilizable organic carbon compounds (22). Moreover, the spectrum of growth-supporting substrates (carbon compounds) of individual bacterial strains is specific—a fact also used for the classification of bacteria for taxonomic purposes. This principle has been integrated into conventional AOC assays, where the specific substrate spectrum of different pure cultures can be used to quantify different types of compounds present in water (26, 33). The term “pathogenic bacteria” is a collective term for many different bacterial species that can all cause disease in humans but their individual substrate spectra are unique for each species. Thus, we have hypothesized that the total concentration of AOC alone is not a sufficient parameter for describing the growth potential of pathogenic bacteria; the quality of the available carbon compounds has to be considered as well.There is no existing method that is capable of fractionating organic carbon in a way that allows for the quantification of individual compounds that support growth of specific pathogens. In this study, we have developed a pathogen growth potential (PGP) assay by combining the conventional AOC assay (31) with flow cytometric quantification of bacterial growth (11) and using pathogens as inocula. The PGP assay yields two main results, namely, (i) the extent of pathogen growth, and (ii) the relative fraction of AOC consumed by a pathogen. With this approach, we investigated the growth potential of three model pathogens from three different genera, namely, Escherichia coli O157, Vibrio cholerae O1, and Pseudomonas aeruginosa, in a broad range of water samples, differing considerably in their origin and quality.     

Bioluminescence Imaging of Clavibacter michiganensis subsp. michiganensis Infection of Tomato Seeds and Plants

Clavibacter michiganensis subsp. michiganensis is a Gram-positive bacterium that causes wilting and cankers, leading to severe economic losses in commercial tomato production worldwide. The disease is transmitted from infected seeds to seedlings and mechanically from plant to plant during seedling production, grafting, pruning, and harvesting. Because of the lack of tools for genetic manipulation, very little is known regarding the mechanisms of seed and seedling infection and movement of C. michiganensis subsp. michiganensis in grafted plants, two focal points for application of bacterial canker control measures in tomato. To facilitate studies on the C. michiganensis subsp. michiganensis movement in tomato seed and grafted plants, we isolated a bioluminescent C. michiganensis subsp. michiganensis strain using the modified Tn1409 containing a promoterless lux reporter. A total of 19 bioluminescent C. michiganensis subsp. michiganensis mutants were obtained. All mutants tested induced a hypersensitive response in Mirabilis jalapa and caused wilting of tomato plants. Real-time colonization studies of germinating seeds using a virulent, stable, constitutively bioluminescent strain, BL-Cmm17, showed that C. michiganensis subsp. michiganensis aggregated on hypocotyls and cotyledons at an early stage of germination. In grafted seedlings in which either the rootstock or scion was exposed to BL-Cmm17 via a contaminated grafting knife, bacteria were translocated in both directions from the graft union at higher inoculum doses. These results emphasize the use of bioluminescent C. michiganensis subsp. michiganensis to help better elucidate the C. michiganensis subsp. michiganensis-tomato plant interactions. Further, we demonstrated the broader applicability of this tool by successful transformation of C. michiganensis subsp. nebraskensis with Tn1409::lux. Thus, our approach would be highly useful to understand the pathogenesis of diseases caused by other subspecies of the agriculturally important C. michiganensis.Clavibacter michiganensis subsp. michiganensis is a Gram-positive, aerobic bacterium that belongs to a group of plant-pathogenic actinomycetes (37). Infections by C. michiganensis subsp. michiganensis cause bacterial canker and wilt in tomato, which is considered one of the most destructive and economically significant diseases of this crop. Severe epidemics can cause up to 80% yield loss, mainly due to wilting and death of plants and lesions on fruit. Bacterial canker was first discovered in Michigan greenhouses in 1909 and has now been reported to occur in most tomato production areas around the world (11, 40).Plant wounds facilitate but are not required for infection by C. michiganensis subsp. michiganensis, which invades the xylem vessels and causes vascular disease with high titers (10⁹ bacteria/g of plant tissue) (2, 29), impairing water transport and leading to plant wilting, canker stem lesions, and death (17, 23). Alternatively, asymptomatic infections can be induced by C. michiganensis subsp. michiganensis during late stages of plant development, resulting in the production of contaminated seeds, a major source of outbreaks of C. michiganensis subsp. michiganensis infections in tomato production (13, 34). Traditional bacterial-disease management measures, such as applications of antibiotics and copper bactericides, have not been successful against this disease, and canker-resistant tomato cultivars are not available. As a result, C. michiganensis subsp. michiganensis has been included under international quarantine regulation (10, 11). Consequently, seed testing and maintaining pathogen-free seeds and transplants is currently the most appropriate approach to minimize the spread of disease (23). However, even a low C. michiganensis subsp. michiganensis transmission rate (0.01%) from seed to seedling can cause a disease epidemic under favorable conditions (5). Due to overcrowding of seedlings during transplant production, the pathogen can easily spread through splashing of irrigation water and leaf contact. Despite its apparent significance in C. michiganensis subsp. michiganensis epidemiology, the mechanism of seed-to-seedling transmission of C. michiganensis subsp. michiganensis is not well understood.Another critical point for disease spread is the grafting process, which is now a common practice for the majority of plants used in production greenhouses. Desirable tomato cultivars (scions) are grafted onto rootstocks that provide greater vigor, longevity, or, in some cases, disease resistance (26). Grafting requires cutting both rootstock and scion, providing a quick way for C. michiganensis subsp. michiganensis to spread from plant to plant. However, grafting is a relatively recent innovation in tomato production, and little is known about how grafting affects the dynamics of C. michiganensis subsp. michiganensis infection. Developing adequate control measures for C. michiganensis subsp. michiganensis is complicated by the complexity of genetic manipulation of Gram-positive bacteria, which impairs analysis and characterization of pathogenesis mechanisms (23). Consequently, there is a need to develop molecular techniques that would allow a better understanding of C. michiganensis subsp. michiganensis infections.One method of interest is using engineered bioluminescent bacteria to monitor plant-pathogen interactions in real time. By exploiting natural light-emitting reactions that are encoded by the luxCDABE genes, bioluminescent bacteria have been used to assess gene expression and to monitor the internalization and distribution of bacteria in hosts (3, 6, 7, 8, 9, 12, 15, 24, 31, 35, 36). In particular, bioluminescent phytopathogenic Xanthomonas campestris pathovars and Pseudomonas spp. have been used to track bacterial movement and distribution in host plants (7, 8, 15, 31, 36), as well as to assess host susceptibility quantitatively (15). Likewise, the lux genes have also been transferred to beneficial bacteria, such as Rhizobium leguminosarum and Pseudomonas spp. to visualize colonization patterns in rhizospheres (3, 9).The genes that carry the function of light emission are luxAB, which express luciferase enzymes that catalyze the bioluminescent reaction, while luxCDE encode the enzymes required for biosynthesis of a fatty aldehyde substrate necessary for the reaction (28, 39). Bioluminescence involves an intracellular oxidation of the reduced form of flavin mononucleotide and the fatty aldehyde by luciferase in the presence of molecular oxygen; therefore, bacterial bioluminescence also requires oxygen, a source of energy (38). Cells that express the lux operon spontaneously emit photons that can be captured by a sensitive charge-coupled-device (CCD) camera, enabling imaging and visualization of bacterial cells (22). Luciferase activity depends on the metabolic integrity of the cell, while the number of photons emitted correlates with the biomass of living bacteria (12, 31). Furthermore, since the half-life of luciferase binding to its substrate is several seconds (28), captured light events reflect processes in real time and are not artifacts of accumulated signals. Consequently, live imaging of bioluminescence provides a sensitive means of visualizing bacterial colonization and invasion of hosts and allows real-time representation and examination of pathogen-plant interactions (24, 36).Very little information is available about the mechanisms of C. michiganensis subsp. michiganensis pathogenesis and its colonization of seeds and subsequent transmission to seedlings. This is largely attributable to a lack of tools and difficulties in genetically manipulating this Gram-positive bacterium (30). However, recent development of an insertion sequence element IS1409 (Tn1409)-based efficient transposon mutagenesis system for C. michiganensis subsp. michiganensis has increased our knowledge of the pathogenesis of tomato canker (16, 25). To better understand the dynamics of seed-to-seedling transmission of C. michiganensis subsp. michiganensis, as well as movement of C. michiganensis subsp. michiganensis in grafted plants, we constructed a bioluminescent C. michiganensis subsp. michiganensis strain using the Tn1409 transposon mutagenesis system. Our results demonstrated the utility of using a bioluminescent C. michiganensis subsp. michiganensis strain as a novel approach to elucidate the interaction of plants with this economically important pathogen.     

Effects of Ammonium and Nitrite on Growth and Competitive Fitness of Cultivated Methanotrophic Bacteria

The effects of nitrite and ammonium on cultivated methanotrophic bacteria were investigated. Methylomicrobium album ATCC 33003 outcompeted Methylocystis sp. strain ATCC 49242 in cultures with high nitrite levels, whereas cultures with high ammonium levels allowed Methylocystis sp. to compete more easily. M. album pure cultures and cocultures consumed nitrite and produced nitrous oxide, suggesting a connection between denitrification and nitrite tolerance.The application of ammonium-based fertilizers has been shown to immediately reduce the uptake of methane in a number of diverse ecological systems (3, 5, 7, 8, 11-13, 16, 27, 28), due likely to competitive inhibition of methane monooxygenase enzymes by ammonia and production of nitrite (1). Longer-term inhibition of methane uptake by ammonium has been attributed to changes in methanotrophic community composition, often favoring activity and/or growth of type I Gammaproteobacteria methanotrophs (i.e., Gammaproteobacteria methane-oxidizing bacteria [gamma-MOB]) over type II Alphaproteobacteria methanotrophs (alpha-MOB) (19-23, 25, 26, 30). It has been argued previously that gamma-MOB likely thrive in the presence of high N loads because they rapidly assimilate N and synthesize ribosomes whereas alpha-MOB thrive best under conditions of N limitation and low oxygen levels (10, 21, 23).Findings from studies with rice paddies indicate that N fertilization stimulates methane oxidation through ammonium acting as a nutrient, not as an inhibitor (2). Therefore, the actual effect of ammonium on growth and activity of methanotrophs depends largely on how much ammonia-N is used for assimilation versus cometabolism. Many methanotrophs can also oxidize ammonia into nitrite via hydroxylamine (24, 29). Nitrite was shown previously to inhibit methane consumption by cultivated methanotrophs and by organisms in soils through an uncharacterized mechanism (9, 17, 24), although nitrite inhibits purified formate dehydrogenase from Methylosinus trichosporium OB3b (15). Together, the data from these studies show that ammonium and nitrite have significant effects on methanotroph activity and community composition and reveal the complexity of ammonia as both a nutrient and a competitive inhibitor. The present study demonstrates the differential influences of high ammonium or nitrite loads on the competitive fitness of a gamma-MOB versus an alpha-MOB strain.     

Zoosporic Tolerance to pH Stress and Its Implications for Phytophthora Species in Aquatic Ecosystems

Phytophthora species, a group of destructive plant pathogens, are commonly referred to as water molds, but little is known about their aquatic ecology. Here we show the effect of pH on zoospore survival of seven Phytophthora species commonly isolated from irrigation reservoirs and natural waterways and dissect zoospore survival strategy. Zoospores were incubated in a basal salt liquid medium at pH 3 to 11 for up to 7 days and then plated on a selective medium to determine their survival. The optimal pHs differed among Phytophthora species, with the optimal pH for P. citricola at pH 9, the optimal pH for P. tropicalis at pH 5, and the optimal pH for the five other species, P. citrophthora, P. insolita, P. irrigata, P. megasperma, and P. nicotianae, at pH 7. The greatest number of colonies was recovered from zoospores of all species plated immediately after being exposed to different levels of pH. At pH 5 to 11, the recovery rate decreased sharply (P ≤ 0.0472) after 1-day exposure for five of the seven species. In contrast, no change occurred (P ≥ 0.1125) in the recovery of any species even after a 7-day exposure at pH 3. Overall, P. megasperma and P. citricola survived longer at higher rates in a wider range of pHs than other species did. These results are generally applicable to field conditions as indicated by additional examination of P. citrophthora and P. megasperma in irrigation water at different levels of pH. These results challenge the notion that all Phytophthora species inhabit aquatic environments as water molds and have significant implications in the management of plant diseases resulting from waterborne microbial contamination.Phytophthora species, a group of oomycetes in the kingdom of Stramenopila and well-known plant pathogens, were first listed as “water molds” by Blackwell in 1944 (5), and this notion has since been generally accepted. These species are phylogenetically close to golden-brown algae, although morphologically and physiologically, they resemble fungi. Most algae are aquatic in nature. Phytophthora species produce flagellate zoospores as their primary dispersal structure (35-37, 39). Zoospores can travel in aquatic environments actively on their own locomotion and passively through water movement (12, 13, 41).More than 20 species of Phytophthora, including P. ramorum, the sudden oak death pathogen, have been isolated from irrigation reservoirs and natural waterways (20-22, 30, 40, 43), and a number of previously unknown taxa also have been documented in aquatic environments (8, 24). These pathogens pose a threat to agricultural sustainability and natural ecosystems, as agriculture increasingly depends on recycled water for irrigation in light of rapidly spreading global water scarcity (19, 22). Recycling irrigation systems provide an efficient means of pathogen dissemination from a single point of infection to an entire farm and from a single farm to other farms sharing the same water resources (22, 24).A search of science-based solutions to this crop health issue reveals a surprising lack of information on the aquatic ecology of Phytophthora species. For instance, hydrogen ion concentration (pH) is among the most important water quality parameters which influence sporangium production and germination (1-3, 6, 32, 34, 38), survival of thick-walled chlamydospores and oospores in the soil environment, and disease development (2, 4, 33, 44). However, the effect of pH on the survival of zoospores and growth of germlings in aquatic environments is not known. As motile zoospores lack cell walls and encysted spores or cysts have thin walls, they are presumably more vulnerable to pH stress than chlamydospores and oospores are. On the other hand, the pH level is likely to fluctuate more regularly and at a greater range in aquatic systems, such as irrigation reservoirs, than in soil systems. pH can change diurnally due to respiration of aquatic plants and seasonally due to rain, oxidation of sulfide-containing sediments through the production of sulfuric acid, algal blooms, and released bases or acids from residues of fertilizer and pesticides. Thus, zoospores and aquatic systems are more prone to the influence of wide pH changes than chlamydospores/oospores in soil systems are. The aim of this study was to determine the impact of pH on zoospore survival and understand the aquatic ecology of different Phytophthora species.     

Role of Plant Residues in Determining Temporal Patterns of the Activity,Size, and Structure of Nitrate Reducer Communities in Soil

The incorporation of plant residues into soil not only represents an opportunity to limit soil organic matter depletion resulting from cultivation but also provides a valuable source of nutrients such as nitrogen. However, the consequences of plant residue addition on soil microbial communities involved in biochemical cycles other than the carbon cycle are poorly understood. In this study, we investigated the responses of one N-cycling microbial community, the nitrate reducers, to wheat, rape, and alfalfa residues for 11 months after incorporation into soil in a field experiment. A 20- to 27-fold increase in potential nitrate reduction activity was observed for residue-amended plots compared to the nonamended plots during the first week. This stimulating effect of residues on the activity of the nitrate-reducing community rapidly decreased but remained significant over 11 months. During this period, our results suggest that the potential nitrate reduction activity was regulated by both carbon availability and temperature. The presence of residues also had a significant effect on the abundance of nitrate reducers estimated by quantitative PCR of the narG and napA genes, encoding the membrane-bound and periplasmic nitrate reductases, respectively. In contrast, the incorporation of the plant residues into soil had little impact on the structure of the narG and napA nitrate-reducing community determined by PCR-restriction fragment length polymorphism (RFLP) fingerprinting. Overall, our results revealed that the addition of plant residues can lead to important long-term changes in the activity and size of a microbial community involved in N cycling but with limited effects of the type of plant residue itself.Modern agricultural practices include a return of plant residues to soil, as this is considered sustainable to the environment. It is now recognized that the conversion of native land into cultivated systems leads to carbon losses, which can be up to 20 to 40% (17). Postharvest plant residues therefore represent an important source of carbon, helping to replenish soil organic matter that decomposes as a result of cultivation. Decomposing plant residues are also a source of nutrients, such as nitrogen, with reduced nitrate leaching compared to mineral fertilizers, which is beneficial for water quality (3). In addition, leaving the plant residue on the soil surface limits water losses by evaporation and prevents soil erosion by wind or water (15).The biochemical composition of plant residues is one of the most important factors influencing their decomposition in soil (14, 28, 29, 51). Indeed, Manzoni et al. (28), using a data set of 2,800 observations, showed previously that the patterns of decomposition were regulated by the initial residue stoichiometry. Several other factors such as climatic conditions, soil type, or localization of the residue in the soil (incorporated or on the soil surface) were also reported previously to influence decomposition (2, 24, 29, 44). Microorganisms are the major decomposers of organic matter in soil, and therefore, the diversity and activity of the microbial community during plant residue decomposition has received much attention (6, 23, 26, 27, 35). It was shown previously that the biochemical composition of plant residues influences microbial respiration (8) and microbial community structure (7, 37). The recent development of carbon-labeling approaches has furthered our knowledge of the microorganisms that actively assimilate the carbon derived from various plant residues (10, 31). However, most of those studies focused on microorganisms involved in C mineralization, and in contrast, very little is known about the effect of plant residue decomposition on the microbial communities involved in biochemical cycles other than the carbon cycle. Thus, despite the influence of plant residues on nitrogen cycling (1, 4, 5, 16, 20), studies assessing the effect of the presence and composition of plant residues on the ecology of microbial communities involved in nitrogen cycling are rare (21, 32, 36).The dissimilatory reduction of nitrate into nitrite is the first step in the processes of denitrification and the dissimilatory reduction of nitrate to ammonium (33, 41). The reduction of nitrate by denitrification leads to losses of nitrogen, which is often a limiting nutrient for plant growth in agriculture. Two types of dissimilatory nitrate reductases, differing in location, have been characterized: a membrane-bound nitrate reductase (Nar) and a periplasmic nitrate reductase (Nap) (9, 53). Nitrate reducers can harbor either Nar, Nap, or both (40, 47). Nitrate reducers are probably the most taxonomically diverse functional community within the nitrogen cycle, with members in most bacterial phyla and also archaea (42). Because of this high level of diversity of heterotrophs sharing the ability to produce energy from nitrate reduction, nitrate reducers are an excellent model system to investigate the response of the N-cycling community to plant residue addition.The aim of this work was to determine how the incorporation of plant residues with contrasting biochemical compositions into soil affects the nitrate-reducing community. For this purpose, we monitored the dynamics of the potential activity, size, and structure of the nitrate-reducing community after the addition of wheat, rape, or alfalfa residues to soil in a field experiment. As the nature and availability of the substrate change during residue decomposition (38, 39, 48), the influence of the incorporation of different plant residues on the nitrate-reducing community was investigated at several sampling times for 11 months.     

Aerobic Biodegradation of N-Nitrosodimethylamine by the Propanotroph Rhodococcus ruber ENV425

The propanotroph Rhodococcus ruber ENV425 was observed to rapidly biodegrade N-nitrosodimethylamine (NDMA) after growth on propane, tryptic soy broth, or glucose. The key degradation intermediates were methylamine, nitric oxide, nitrite, nitrate, and formate. Small quantities of formaldehyde and dimethylamine were also detected. A denitrosation reaction, initiated by hydrogen atom abstraction from one of the two methyl groups, is hypothesized to result in the formation of n-methylformaldimine and nitric oxide, the former of which decomposes in water to methylamine and formaldehyde and the latter of which is then oxidized further to nitrite and then nitrate. Although the strain mineralized more than 60% of the carbon in [¹⁴C]NDMA to ¹⁴CO₂, growth of strain ENV425 on NDMA as a sole carbon and energy source could not be confirmed. The bacterium was capable of utilizing NDMA, as well as the degradation intermediates methylamine and nitrate, as sources of nitrogen during growth on propane. In addition, ENV425 reduced environmentally relevant microgram/liter concentrations of NDMA to <2 ng/liter in batch cultures, suggesting that the bacterium may have applications for groundwater remediation.N-Nitrosodimethylamine (NDMA) is a potent carcinogen that has recently been detected in groundwater, wastewater, and drinking water (1, 2, 17, 18). It forms as a disinfection byproduct in wastewater and drinking water treated with chloramine and other disinfectants (17, 18, 43). NDMA has also been found to be present in aquifers at several military sites that have used 1,1-dimethylhydrazine, a component of liquid rocket propellant that contained NDMA as an impurity (6, 9). Although there is presently no federal maximum contaminant level for NDMA in drinking water, a risk assessment conducted by the U.S. Environmental Protection Agency suggested that concentrations as low as 0.7 ng/liter can increase lifetime cancer risk by 1 × 10⁻⁶ (34). In addition, California currently has a 10 ng/liter notification level for NDMA concentrations in drinking water and has recently recommended an even lower public health goal of 3 ng/liter (3, 20). Thus, the presence of even trace concentrations of this chemical in drinking water represents a potential public health concern.The rates and extents of NDMA biodegradation in natural environments, including surface water, sludges, and soils, are highly variable. In some studies, the compound has been reported to be recalcitrant or only partially biodegraded (16, 30, 31); in others, fairly rapid and extensive biodegradation has been previously observed (2, 13, 22, 40). Few studies have been conducted to examine NDMA biodegradation in groundwater. However, the persistence of NDMA derived originally from 1,1-dimethylhydrazine-based rocket fuel over decades in some groundwater aquifers (e.g., Rocky Mountain Arsenal, CO; former Air Force Plant PJKS, CO; and Aerojet Superfund Site, CA) suggests that this molecule can be very recalcitrant (8, 9, 35). At sites where biodegradation has been observed, the organisms responsible and the microbial degradation pathways are largely unknown.The metabolism of NDMA and other nitrosamines by mammals has received extensive study. NDMA requires metabolic activation to the methyldiazonium ion (a strong alkylating agent) to exert its genotoxic effects (1, 19, 34). This activation reaction is catalyzed by specific isozymes of the cytochrome P-450-dependent mixed-function oxidase system and proceeds through an initial α-hydroxylation reaction. Alternately, NDMA can be oxidized by the P-450 system via a denitrosation route, which does not result in the formation of a highly carcinogenic intermediate (11, 28, 37).The bacterial transformation of NDMA has not been studied in significant detail. Several bacteria expressing broad-specificity monooxygenase enzymes have been reported to degrade NDMA via cometabolism. These include the propanotrophs Rhodococcus sp. strain RHA1 (25, 26) and Rhodococcus ruber ENV425 (29) as well as Mycobacterium vaccae JOB5 (25), the methanotroph Methylosinus trichosporium OB3b (42), and the toluene oxidizer Pseudomonas mendocina KR1 (7). We recently characterized the pathway of NDMA transformation used by P. mendocina KR1, a bacterium that utilizes the enzyme toluene-4-monooxygenase (T4MO) to cometabolically degrade NDMA and other anthropogenic pollutants (7, 38). The pathway of NDMA transformation by KR1 differs from the two pathways described for mammals. A majority of the NDMA metabolized by T4MO in this strain is oxidized to N-nitrodimethylamine (NTDMA) and then further to N-nitromethylamine (NTMA), which accumulates as a terminal product (7).In this report, we describe the pathway used by the propanotroph R. ruber ENV425 to catabolize NDMA. This strain was originally isolated from turf soil, where propane was used as the sole carbon source, and was previously reported to oxidize methyl tertiary-butyl ether and other gasoline oxygenates (27). Our data show that the pathway of NDMA degradation mediated by strain ENV425 differs from that mediated by P. mendocina KR1. Rather, the pathway used for transformation of NDMA by ENV425 appears to be similar to the denitrosation pathway catalyzed by various P-450 isozymes in mammals, resulting in the production of nitric oxide (NO), nitrite, nitrate, formaldehyde, formate, and methylamine (MA) (11, 12, 28, 39). A significant fraction of the carbon in the NDMA molecule was released as CO₂ by strain ENV425, although growth on NDMA could not be confirmed. However, the bacterium was observed to utilize NDMA as well as the NDMA-degradation intermediates MA and nitrate as sources of nitrogen during growth on propane as a sole carbon and energy source.     

A New Borrelia Species Defined by Multilocus Sequence Analysis of Housekeeping Genes

Analysis of Lyme borreliosis (LB) spirochetes, using a novel multilocus sequence analysis scheme, revealed that OspA serotype 4 strains (a rodent-associated ecotype) of Borrelia garinii were sufficiently genetically distinct from bird-associated B. garinii strains to deserve species status. We suggest that OspA serotype 4 strains be raised to species status and named Borrelia bavariensis sp. nov. The rooted phylogenetic trees provide novel insights into the evolutionary history of LB spirochetes.Multilocus sequence typing (MLST) and multilocus sequence analysis (MLSA) have been shown to be powerful and pragmatic molecular methods for typing large numbers of microbial strains for population genetics studies, delineation of species, and assignment of strains to defined bacterial species (4, 13, 27, 40, 44). To date, MLST/MLSA schemes have been applied only to a few vector-borne microbial populations (1, 6, 30, 37, 40, 41, 47).Lyme borreliosis (LB) spirochetes comprise a diverse group of zoonotic bacteria which are transmitted among vertebrate hosts by ixodid (hard) ticks. The most common agents of human LB are Borrelia burgdorferi (sensu stricto), Borrelia afzelii, Borrelia garinii, Borrelia lusitaniae, and Borrelia spielmanii (7, 8, 12, 35). To date, 15 species have been named within the group of LB spirochetes (6, 31, 32, 37, 38, 41). While several of these LB species have been delineated using whole DNA-DNA hybridization (3, 20, 33), most ecological or epidemiological studies have been using single loci (5, 9-11, 29, 34, 36, 38, 42, 51, 53). Although some of these loci have been convenient for species assignment of strains or to address particular epidemiological questions, they may be unsuitable to resolve evolutionary relationships among LB species, because it is not possible to define any outgroup. For example, both the 5S-23S intergenic spacer (5S-23S IGS) and the gene encoding the outer surface protein A (ospA) are present only in LB spirochete genomes (36, 43). The advantage of using appropriate housekeeping genes of LB group spirochetes is that phylogenetic trees can be rooted with sequences of relapsing fever spirochetes. This renders the data amenable to detailed evolutionary studies of LB spirochetes.LB group spirochetes differ remarkably in their patterns and levels of host association, which are likely to affect their population structures (22, 24, 46, 48). Of the three main Eurasian Borrelia species, B. afzelii is adapted to rodents, whereas B. valaisiana and most strains of B. garinii are maintained by birds (12, 15, 16, 23, 26, 45). However, B. garinii OspA serotype 4 strains in Europe have been shown to be transmitted by rodents (17, 18) and, therefore, constitute a distinct ecotype within B. garinii. These strains have also been associated with high pathogenicity in humans, and their finer-scale geographical distribution seems highly focal (10, 34, 52, 53).In this study, we analyzed the intra- and interspecific phylogenetic relationships of B. burgdorferi, B. afzelii, B. garinii, B. valaisiana, B. lusitaniae, B. bissettii, and B. spielmanii by means of a novel MLSA scheme based on chromosomal housekeeping genes (30, 48).     

Free-Living Protozoa in Two Unchlorinated Drinking Water Supplies,Identified by Phylogenic Analysis of 18S rRNA Gene Sequences

Free-living protozoan communities in water supplies may include hosts for Legionella pneumophila and other undesired bacteria, as well as pathogens. This study aimed at identifying free-living protozoa in two unchlorinated groundwater supplies, using cultivation-independent molecular approaches. For this purpose, samples (<20°C) of treated water, distributed water, and distribution system biofilms were collected from supply A, with a low concentration of natural organic matter (NOM) (<0.5 ppm of C), and from supply B, with a high NOM concentration (7.9 ppm of C). Eukaryotic communities were studied using terminal restriction fragment length polymorphism and clone library analyses of partial 18S rRNA gene fragments and a Hartmannella vermiformis-specific quantitative PCR (qPCR). In both supplies, highly diverse eukaryotic communities were observed, including free-living protozoa, fungi, and metazoa. Sequences of protozoa clustered with Amoebozoa (10 operational taxonomic units [OTUs]), Cercozoa (39 OTUs), Choanozoa (26 OTUs), Ciliophora (29 OTUs), Euglenozoa (13 OTUs), Myzozoa (5 OTUs), and Stramenopiles (5 OTUs). A large variety of protozoa were present in both supplies, but the estimated values for protozoan richness did not differ significantly. H. vermiformis was observed in both supplies but was not a predominant protozoan. One OTU with the highest similarity to Acanthamoeba polyphaga, an opportunistic human pathogen and a host for undesired bacteria, was observed in supply A. The high level of NOM in supply B corresponded with an elevated level of active biomass and with elevated concentrations of H. vermiformis in distributed water. Hence, the application of qPCR may be promising in elucidating the relationship between drinking water quality and the presence of specific protozoa.Free-living protozoa are ubiquitous in natural freshwater environments (7, 38, 51, 71) but also proliferate in engineered water systems, including water treatment systems (3, 47, 70), distribution systems (6, 75), and tap water installations inside buildings (54, 69). Concentrations of protozoa, determined using cultivation methods and microscopy, range from <1 to 10⁴ cells liter⁻¹ in treated water (3, 47, 70, 75) and from <1 to 7 × 10⁵ cells liter⁻¹ in distribution systems (6, 61, 64, 75). Genera of free-living protozoa commonly observed in these systems and in tap water installations include Acanthamoeba, Echinamoeba, Hartmannella, Platyamoeba, Vahlkampfia, and Vannella (47, 58, 69, 70). In warm water systems, certain free-living protozoa, e.g., Acanthamoeba spp. (57), Balamuthia mandrillaris (62), Echinamoeba exandans (16), Hartmannella spp. (39, 56), Naegleria spp. (49, 57), Tetrahymena spp. (18, 33), and Vahlkampfia jugosa (56), serve as hosts for Legionella pneumophila, the etiologic agent of Legionnaires'' disease. High concentrations of L. pneumophila are generally associated with the proliferation of host protozoa in biofilms (38, 53). In addition, other amoeba-resistant, potentially pathogenic bacteria, e.g., Burkholderia spp. (28) and Mycobacterium spp. (37), have been observed in man-made aquatic environments (24). Free-living protozoa may enhance the multiplication of bacteria, serve as a transmission vector, or serve as a shelter against unfavorable environmental conditions, such as the presence of disinfectants. Furthermore, certain free-living protozoa are human pathogens, e.g., Naegleria fowleri (81), Balamuthia mandrillaris (77), and Acanthamoeba spp. (12) can cause encephalitis. Acanthamoeba spp. have also been associated with keratitis in persons wearing contact lenses (31).Free-living protozoa feed on bacteria, algae, fungi, other protozoa, and organic detritus in biofilms or in the planktonic phase, thereby affecting the structure of microbial communities. In turn, the community of free-living protozoa depends on the diversity and abundance of bacteria in the biofilm and in the planktonic phase (26, 50, 51, 55, 63, 65). Water quality is a critical factor for biofilm formation in distribution systems and tap water installations and therefore will affect the abundance and diversity of free-living protozoa in these systems (72, 78). However, information about the presence and identity of free-living protozoa in water supplies in relation to the quality of treated water is scarce, which may be attributed to the limitations of microscopic techniques and cultivation methods for detection and identification of these organisms, e.g., low detection limits and selectivity for specific groups (19).In this study, we applied a variety of cultivation-independent techniques, viz., quantitative PCR, terminal restriction fragment length polymorphism (T-RFLP) analysis, and cloning and sequencing of eukaryotic 18S rRNA gene fragments, for the detection and identification of free-living protozoa predominating in two unchlorinated groundwater supplies. The concentrations of dissolved natural organic matter (NOM) in treated water at the plant were <0.5 mg C liter⁻¹ and 7.9 mg C liter⁻¹, covering the entire range of NOM concentrations in drinking water in The Netherlands. The objectives of the study were (i) to elucidate the identities of and diversity in the free-living protozoa predominating in these two different water supplies and (ii) to trace the presence of host protozoa for L. pneumophila and pathogenic free-living protozoa. The study revealed that treated water and biofilms in the distribution systems of both water supplies contained a large variety of free-living protozoa, including protozoan hosts for Legionella bacteria.     

Roles of Small,Acid-Soluble Spore Proteins and Core Water Content in Survival of Bacillus subtilis Spores Exposed to Environmental Solar UV Radiation

Spores of Bacillus subtilis contain a number of small, acid-soluble spore proteins (SASP) which comprise up to 20% of total spore core protein. The multiple α/β-type SASP have been shown to confer resistance to UV radiation, heat, peroxides, and other sporicidal treatments. In this study, SASP-defective mutants of B. subtilis and spores deficient in dacB, a mutation leading to an increased core water content, were used to study the relative contributions of SASP and increased core water content to spore resistance to germicidal 254-nm and simulated environmental UV exposure (280 to 400 nm, 290 to 400 nm, and 320 to 400 nm). Spores of strains carrying mutations in sspA, sspB, and both sspA and sspB (lacking the major SASP-α and/or SASP-β) were significantly more sensitive to 254-nm and all polychromatic UV exposures, whereas the UV resistance of spores of the sspE strain (lacking SASP-γ) was essentially identical to that of the wild type. Spores of the dacB-defective strain were as resistant to 254-nm UV-C radiation as wild-type spores. However, spores of the dacB strain were significantly more sensitive than wild-type spores to environmental UV treatments of >280 nm. Air-dried spores of the dacB mutant strain had a significantly higher water content than air-dried wild-type spores. Our results indicate that α/β-type SASP and decreased spore core water content play an essential role in spore resistance to environmentally relevant UV wavelengths whereas SASP-γ does not.Spores of Bacillus spp. are highly resistant to inactivation by different physical stresses, such as toxic chemicals and biocidal agents, desiccation, pressure and temperature extremes, and high fluences of UV or ionizing radiation (reviewed in references 33, 34, and 48). Under stressful environmental conditions, cells of Bacillus spp. produce endospores that can stay dormant for extended periods. The reason for the high resistance of bacterial spores to environmental extremes lies in the structure of the spore. Spores possess thick layers of highly cross-linked coat proteins, a modified peptidoglycan spore cortex, a low core water content, and abundant intracellular constituents, such as the calcium chelate of dipicolinic acid and α/β-type small, acid-soluble spore proteins (α/β-type SASP), the last two of which protect spore DNA (6, 42, 46, 48, 52). DNA damage accumulated during spore dormancy is also efficiently repaired during spore germination (33, 47, 48). UV-induced DNA photoproducts are repaired by spore photoproduct lyase and nucleotide excision repair, DNA double-strand breaks (DSB) by nonhomologous end joining, and oxidative stress-induced apurinic/apyrimidinic (AP) sites by AP endonucleases and base excision repair (15, 26-29, 34, 43, 53, 57).Monochromatic 254-nm UV radiation has been used as an efficient and cost-effective means of disinfecting surfaces, building air, and drinking water supplies (31). Commonly used test organisms for inactivation studies are bacterial spores, usually spores of Bacillus subtilis, due to their high degree of resistance to various sporicidal treatments, reproducible inactivation response, and safety (1, 8, 19, 31, 48). Depending on the Bacillus species analyzed, spores are 10 to 50 times more resistant than growing cells to 254-nm UV radiation. In addition, most of the laboratory studies of spore inactivation and radiation biology have been performed using monochromatic 254-nm UV radiation (33, 34). Although 254-nm UV-C radiation is a convenient germicidal treatment and relevant to disinfection procedures, results obtained by using 254-nm UV-C are not truly representative of results obtained using UV wavelengths that endospores encounter in their natural environments (34, 42, 50, 51, 59). However, sunlight reaching the Earth''s surface is not monochromatic 254-nm radiation but a mixture of UV, visible, and infrared radiation, with the UV portion spanning approximately 290 to 400 nm (33, 34, 36). Thus, our knowledge of spore UV resistance has been constructed largely using a wavelength of UV radiation not normally reaching the Earth''s surface, even though ample evidence exists that both DNA photochemistry and microbial responses to UV are strongly wavelength dependent (2, 30, 33, 36).Of recent interest in our laboratories has been the exploration of factors that confer on B. subtilis spores resistance to environmentally relevant extreme conditions, particularly solar UV radiation and extreme desiccation (23, 28, 30, 34 36, 48, 52). It has been reported that α/β-type SASP but not SASP-γ play a major role in spore resistance to 254-nm UV-C radiation (20, 21) and to wet heat, dry heat, and oxidizing agents (48). In contrast, increased spore water content was reported to affect B. subtilis spore resistance to moist heat and hydrogen peroxide but not to 254-nm UV-C (12, 40, 48). However, the possible roles of SASP-α, -β, and -γ and core water content in spore resistance to environmentally relevant solar UV wavelengths have not been explored. Therefore, in this study, we have used B. subtilis strains carrying mutations in the sspA, sspB, sspE, sspA and sspB, or dacB gene to investigate the contributions of SASP and increased core water content to the resistance of B. subtilis spores to 254-nm UV-C and environmentally relevant polychromatic UV radiation encountered on Earth''s surface.     

Coral-Associated Bacteria and Their Role in the Biogeochemical Cycling of Sulfur

Marine bacteria play a central role in the degradation of dimethylsulfoniopropionate (DMSP) to dimethyl sulfide (DMS) and acrylic acid, DMS being critical to cloud formation and thereby cooling effects on the climate. High concentrations of DMSP and DMS have been reported in scleractinian coral tissues although, to date, there have been no investigations into the influence of these organic sulfur compounds on coral-associated bacteria. Two coral species, Montipora aequituberculata and Acropora millepora, were sampled and their bacterial communities were characterized by both culture-dependent and molecular techniques. Four genera, Roseobacter, Spongiobacter, Vibrio, and Alteromonas, which were isolated on media with either DMSP or DMS as the sole carbon source, comprised the majority of clones retrieved from coral mucus and tissue 16S rRNA gene clone libraries. Clones affiliated with Roseobacter sp. constituted 28% of the M. aequituberculata tissue libraries, while 59% of the clones from the A. millepora libraries were affiliated with sequences related to the Spongiobacter genus. Vibrio spp. were commonly isolated from DMS and acrylic acid enrichments and were also present in 16S rRNA gene libraries from coral mucus, suggesting that under “normal” environmental conditions, they are a natural component of coral-associated communities. Genes homologous to dddD, and dddL, previously implicated in DMSP degradation, were also characterized from isolated strains, confirming that bacteria associated with corals have the potential to metabolize this sulfur compound when present in coral tissues. Our results demonstrate that DMSP, DMS, and acrylic acid potentially act as nutrient sources for coral-associated bacteria and that these sulfur compounds are likely to play a role in structuring bacterial communities in corals, with important consequences for the health of both corals and coral reef ecosystems.Dimethylsulfoniopropionate (DMSP) is an organic sulfur compound implicated in the formation of clouds via its cleavage product dimethyl sulfide (DMS) and therefore has the potential to exert major cooling effects on climate (9, 38). The production of DMSP is mainly restricted to a few classes of marine macro- and microalgae (27, 68), with the main producers being phytoplankton species belonging to prymnesiophyte and dinoflagellate taxa (28, 62, 67). Recently, significant concentrations of DMSP and DMS have been recorded in association with animals that harbor symbiotic algae such as scleractinian corals and giant clams (7, 8, 68), raising questions about the role of coral reefs in sulfur cycling. The densities of symbiotic dinoflagellates (genus Symbiodinium, commonly known as zooxanthellae) in coral tissues are similar to those recorded for dinoflagellates in phytoplankton blooms (11, 68). Since dinoflagellates are among the most significant producers of DMSP and high intracellular concentrations of DMSP have been found in both cultured zooxanthellae (26) and scleractinian corals (6-8, 25), these observations suggest that endosymbiotic zooxanthellae have an integral role in sulfur cycling in oligotrophic reef waters.Most of the DMSP produced by planktonic dinoflagellates is exuded into the surrounding water, where it is degraded by bacteria via two possible pathways: the first one converts a large fraction (ca. 75%) of dissolved DMSP to methylmercaptopropionate, which is subsequently incorporated into the biomass of microbial cells (22, 27, 66). The second pathway transforms the remaining part of the dissolved DMSP to equimolar concentrations of DMS and acrylic acid (43, 66, 72). This metabolic pathway for DMSP degradation has been identified in the alphaproteobacterial species Sulfitobacter sp. and the enzyme involved (DMSP-dependent DMS lyase [DddL]) characterized (10). Another pathway for DMS formation (without production of acrylate) has been described for Marinomonas sp. and the gene responsible, dddD, identified. In addition, the protein DddR has been directly implicated in the regulation of the gene encoding DddD (66). The DMS produced by these enzymes are then released into the surrounding water (27). Prior to the 1980s, diffusion of supersaturated DMS from the oceans to the atmosphere was thought to be the major removal pathway of this compound from the oceans (35, 72). More recently, however, it has been estimated that between 50 and 80% of the DMS produced by DMSP-degrading bacteria is degraded directly by other types of bacteria (58, 59), although the populations and metabolic pathways involved in the degradation of DMS are still poorly understood.Coral-associated bacterial communities are known to be diverse and highly abundant (12, 30, 48, 49, 52). These dynamic communities exploit a number of habitats associated with corals, including mucus on coral surfaces (48), intracellular niches within coral tissues (3, 16, 45, 47, 52), spaces within coral skeletons (15, 51), and seawater surrounding corals (16, 61). Each of these habitats is believed to harbor different bacterial populations (4, 52). Despite high bacterial diversity, corals have been reported to harbor species-specific microbial communities for beneficial effects; however, their role in coral health is poorly understood (47-50). In coral reef environments, bacteria are dependent upon organic compounds produced by photoautotrophic organisms such as endosymbiotic zooxanthellae (48); therefore, photosynthates translocated to coral tissues and mucus may determine microbial communities closely associated with corals (48, 52). The high levels of DMSP and DMS produced by corals, coupled with the dependence of DMSP and DMS conversion on processes typically involving bacteria, suggest that corals are likely to harbor bacterial species involved in the cycling of these compounds. To investigate the potential of the organosulfur compound DMSP and its breakdown products, DMS and acrylic acid, to drive coral-associated microbial communities, we used these compounds as sole carbon sources to isolate bacteria from two coral species (Montipora aequituberculata and Acropora millepora) and then directly compared these microbial communities with coral-associated microbiota identified using culture-independent analyses. Genes implicated in the metabolism of DMSP were also characterized from isolated strains, confirming that bacteria associated with corals have the potential to metabolize organic sulfur compounds present in coral tissues.     

Detection of Toxoplasma gondii Oocysts in Water Sample Concentrates by Real-Time PCR

PCR techniques in combination with conventional parasite concentration procedures have potential for the sensitive and specific detection of Toxoplasma gondii oocysts in water. Three real-time PCR assays based on the B1 gene and a 529-bp repetitive element were analyzed for the detection of T. gondii tachyzoites and oocysts. Lower sensitivity and specificity were obtained with the B1 gene-based PCR than with the 529-bp repeat-based PCR. New procedures for the real-time PCR detection of T. gondii oocysts in concentrates of surface water were developed and tested in conjunction with a method for the direct extraction of inhibitor-free DNA from water. This technique detected as few as one oocyst seeded to 0.5 ml of packed pellets from water samples concentrated by Envirocheck filters. Thus, this real-time PCR may provide a detection method alternative to the traditional mouse assay and microscopy.Toxoplasma gondii is a ubiquitous parasite found in all classes of warm-blooded vertebrates. Nearly one-third of humans have been exposed to this parasite (15). In immunocompetent adults, acute infection normally results in transient influenza-like symptoms, but in immunocompromised persons retinochoroiditis and encephalitis are more common. Infected individuals can retain the parasite as quiescent tissue cysts for long periods, but invasive infection can occur if the immune status of the infected person deteriorates (42). If women become infected during pregnancy, the parasite can cause abortion or seriously damage the fetus. The potential morbidity from the ingestion of oocysts of T. gondii and the organism''s low infectious dose are a great concern for public health. There are at least four reported waterborne outbreaks of toxoplasmosis (2, 3, 14, 44), and endemic toxoplasmosis in Brazil is associated with the consumption of water or ice contaminated with T. gondii oocysts (1, 23), demonstrating the potential for the waterborne transmission of this disease (15).There is no rapid detection method for T. gondii oocysts recovered from water or other environmental samples. Traditionally, the detection of protozoa in water required their concentration from large volumes of water by filtration or centrifugation, isolation from concentrated particulates by immunomagnetic separation (IMS) or other methods, and detection by immunofluorescence microscopy, the infection of cultured cells, biochemistry, animal infection tests, molecular techniques, or combinations of these (17, 58). For T. gondii oocysts there are no commercially available IMS techniques, no widely available immunofluorescent staining reagents, and no standardized cultivation protocols. The identification of oocysts from environmental samples has included differential floatation and mouse inoculation (27). Recently, IMS techniques have been developed for the isolation of T. gondii oocysts and sporocysts in water (16, 18). Both the oocyst and sporocyst IMS assays, however, had poor specificity, because antibodies cross-reacted with water debris and the sporocyst wall of Hammondia hammondi, Hammondia heydorni, and Neospora caninum (16).PCR is becoming a favored technique for the detection of T. gondii oocysts in water (32, 35, 36, 46, 49, 55) over the conventional mouse bioassay (27, 55), as it reduces the detection time from weeks to 1 to 2 days. Although they have been developed for the detection of T. gondii in clinical specimens (50), no real-time PCR assays have been adapted for the detection of oocysts in water samples, possibly because of expected high concentrations of PCR inhibitors and low numbers of T. gondii oocysts in environmental samples (55).There are several unresolved issues regarding the effectiveness of the PCR detection of T. gondii oocysts in water. The most readily available method for the isolation of T. gondii oocysts from water samples is flocculation or sucrose floatation prior to DNA extraction (35, 36, 49, 55). Because sucrose flotation and flocculation result in oocyst losses, the recovery rate of using these methods is poor. For DNA extraction, the phenol-chloroform method or QIAamp mini kit frequently is used (16, 35, 36, 46, 55). When oocysts are recovered from water either by the Environmental Protection Agency (EPA) information collection rule method (53) or EPA Method 1623 (54) without purification by IMS, neither the conventional phenol-chloroform DNA extraction nor the QIAamp mini kit is effective at removing PCR inhibitors (30, 55, 57).Recently, a method was used effectively in the analysis of Cryptosporidium oocysts in surface water, storm water, and wastewater samples (30). This method extracted DNA directly from water concentrates without pathogen IMS, differential flotation, or enrichment cultures, and it utilized a commercial DNA extraction kit, the FastDNA spin kit for soil, and a high concentration of nonacetylated bovine serum albumin in PCR. The FastDNA soil kit has a higher capacity for PCR inhibitor removal than several other commercial extraction kits designed for environmental samples. The use of nonacetylated bovine serum in the PCR neutralizes residual PCR inhibitors that are coextracted with the DNA (30).In the present study, the performance of two published LightCycler real-time PCR assays based on the multicopy B1 gene and 529-bp repetitive element (13, 45) and a newly developed LightCycler real-time PCR assay using a common primer set were analyzed for the detection of T. gondii, using pure DNA and DNA extracted by the aforementioned extraction method (30) from water sample concentrates seeded with known number of oocysts.     

Long-Term Persistence and Leaching of Escherichia coli in Temperate Maritime Soils

Enteropathogen contamination of groundwater, including potable water sources, is a global concern. The spreading on land of animal slurries and manures, which can contain a broad range of pathogenic microorganisms, is considered a major contributor to this contamination. Some of the pathogenic microorganisms applied to soil have been observed to leach through the soil into groundwater, which poses a risk to public health. There is a critical need, therefore, for characterization of pathogen movement through the vadose zone for assessment of the risk to groundwater quality due to agricultural activities. A lysimeter experiment was performed to investigate the effect of soil type and condition on the fate and transport of potential bacterial pathogens, using Escherichia coli as a marker, in four Irish soils (n = 9). Cattle slurry (34 tonnes per ha) was spread on intact soil monoliths (depth, 1 m; diameter, 0.6 m) in the spring and summer. No effect of treatment or the initial soil moisture on the E. coli that leached from the soil was observed. Leaching of E. coli was observed predominantly from one soil type (average, 1.11 ± 0.77 CFU ml⁻¹), a poorly drained Luvic Stagnosol, under natural rainfall conditions, and preferential flow was an important transport mechanism. E. coli was found to have persisted in control soils for more than 9 years, indicating that autochthonous E. coli populations are capable of becoming naturalized in the low-temperature environments of temperate maritime soils and that they can move through soil. This may compromise the use of E. coli as an indicator of fecal pollution of waters in these regions.The contamination of groundwater, including potable water supplies, with microbial pathogens continues to be a global concern (52, 59). Of particular importance in developed countries are the high levels of contamination associated with small-scale and very-small-scale drinking water supplies (5, 19, 57), often groundwater, which serve an estimated 10% of the total population in the European Union (13). The high numbers of these water supplies found to be contaminated with fecal bacteria and thus considered to be unfit for human consumption are worrying because the water from them is often untreated or inadequately treated prior to consumption. Microbial pathogens are known to survive for considerable periods of time in groundwater (29), which increases the health risk due to utilization of contaminated supplies. There are various sources of contamination, but evidence suggests that contamination from the spreading of animal slurries and manures on land can be a significant contributor (3, 33, 53). Spreading of agricultural slurries and manures on land is used by the agricultural sector as a means of nutrient recycling. The health risks associated with the spreading of animal and human wastes containing enteric pathogens have been recognized for a long time (10, 18). Animal manure and wastewaters may contain a broad range of pathogenic microorganisms, including Escherichia coli O157:H7, Campylobacter, Cryptosporidium, Salmonella spp., and pathogenic viruses, which are released into the environment during spreading (15, 22, 55). The levels and incidence of pathogens present in animal manures and slurries are influenced by a number of factors, including herd health, age demographics, stress factors, diet, season, and manure management and storage (37, 39).Soils (and subsoils) often act as a zone for mitigating microbial contamination of groundwater associated with the spreading of animal slurries and manures on land. Some of the pathogenic microorganisms applied to agricultural soils have, however, been observed to leach through the soil into groundwater, which can affect drinking water quality and pose a risk to public health (16, 26, 28, 42, 50), confirming that soil is not always a sufficient obstruction for protection of groundwater (16, 53). Consequently, characterization of the movement of pathogens through the unsaturated soil and subsoil zone (vadose zone) has become critical for assessment of the risk to groundwater posed by agricultural activities (8, 14, 42). The soil and subsoil type is believed to be a major factor influencing the potential transfer of pathogens through soil to groundwater (3, 34, 41, 50). The preapplication moisture status of a soil, which may be influenced by the season, also impacts pathogen survival, fate, and transport (2, 11, 43, 54).E. coli is widely used as an indicator of fecal contamination of water, and certain strains are known to be pathogenic (12). Thus, characterizing this organism''s transport through soil is important because of the health risk posed by the organism itself and with regard to its validity as an indicator of the fate of enteropathogens in the environment. E. coli strains have diverse properties and capabilities that affect their survival and transport in soils (9, 36, 56, 60). Consequently, data obtained by using total E. coli rather than individual surrogate strains can be more representative of the fate and transport of E. coli present in animal slurries. E. coli O157 die-off in soils has been reported to be the same as or quicker than total E. coli die-off, suggesting that data for total E. coli provide a conservative estimate of the survival potential (38, 56). Although many field and laboratory studies have investigated E. coli transport through soil columns (4, 6, 16, 43, 46, 47, 50, 51), most studies have investigated transport through soil to a depth of less than 30 cm. For assessment of the risk of transport to groundwater, such studies may not take into account the variation in soil physical and chemical characteristics with depth (e.g., the frequency and continuity of macropores, organic matter, and moisture contents) that affect bacterial transport. Furthermore, rainfall was often simulated in previous studies, which allows experimental conditions to be controlled but may not be representative of the risk due to variable natural rainfall events over time. In this study, we used intact soil monoliths that were 1 m deep to assess the risk of leaching of total E. coli in four representative Irish soil types under natural rainfall and environmental conditions.The objective of this study was to quantitatively investigate the impact of soil type and season (soil moisture content) on the fate and transport of E. coli spread on four different temperate maritime soil types under natural rainfall conditions. We hypothesized that there would be a greater microbial risk to underlying groundwater with better-drained soil types than with relatively poorly drained soil types following the application of animal slurry. In addition, we hypothesized that E. coli cells spread on wetter spring soils would be transported in greater numbers than E. coli cells spread on drier soils in the summer.     

Effects of Aeration on the Synthesis of Poly(3-Hydroxybutyrate) from Glycerol and Glucose in Recombinant Escherichia coli

Bioreactor cultures of Escherichia coli recombinants carrying phaBAC and phaP of Azotobacter sp. FA8 grown on glycerol under low-agitation conditions accumulated more poly(3-hydroxybutyrate) (PHB) and ethanol than at high agitation, while in glucose cultures, low agitation led to a decrease in PHB formation. Cells produced smaller amounts of acids from glycerol than from glucose. Glycerol batch cultures stirred at 125 rpm accumulated, in 24 h, 30.1% (wt/wt) PHB with a relative molecular mass of 1.9 MDa, close to that of PHB obtained using glucose.Polyhydroxyalkanoates (PHAs), accumulated as intracellular granules by many bacteria under unfavorable conditions (5, 8), are carbon and energy reserves and also act as electron sinks, enhancing the fitness of bacteria and contributing to redox balance (9, 11, 19). PHAs have thermoplastic properties, are totally biodegradable by microorganisms present in most environments, and can be produced from different renewable carbon sources (8).Poly(3-hydroxybutyrate) (PHB) is the best known PHA, and its accumulation in recombinant Escherichia coli from several carbon sources has been studied (1, 13). In the last few years, increasing production of biodiesel has caused a sharp fall in the cost of its main by-product, glycerol (22). Its use for microbial PHA synthesis has been analyzed for natural PHA producers, such as Methylobacterium rhodesianum, Cupriavidus necator (formerly called Ralstonia eutropha) (3), several Pseudomonas strains (22), the recently described bacterium Zobellella denitrificans (7), and a Bacillus sp. (18), among others. Glycerol has also been used for PHB synthesis in recombinant E. coli (12, 15). PHAs obtained from glycerol were reported to have a significantly lower molecular weight than polymer synthesized from other substrates, such as glucose or lactose (10, 23).Apart from the genes that catalyze polymer biosynthesis, natural PHA producers have several genes that are involved in granule formation and/or have regulatory functions, such as phasins, granule-associated proteins that have been shown to enhance polymer synthesis and the number and size of PHA granules (17, 24). The phasin PhaP has been shown to exert a beneficial effect on bacterial growth and PHB accumulation from glycerol in bioreactor cultures of strain K24KP, a recombinant E. coli that carries phaBAC and phaP of Azotobacter sp. FA8 (6).Because the redox state of the cells is known to affect the synthesis of PHB (1, 4, 14), the present study investigates the behavior of this recombinant strain under different aeration conditions, by using two substrates, glucose and glycerol, with different oxidation states.     

mcrA-Targeted Real-Time Quantitative PCR Method To Examine Methanogen Communities

Methanogens are of great importance in carbon cycling and alternative energy production, but quantitation with culture-based methods is time-consuming and biased against methanogen groups that are difficult to cultivate in a laboratory. For these reasons, methanogens are typically studied through culture-independent molecular techniques. We developed a SYBR green I quantitative PCR (qPCR) assay to quantify total numbers of methyl coenzyme M reductase α-subunit (mcrA) genes. TaqMan probes were also designed to target nine different phylogenetic groups of methanogens in qPCR assays. Total mcrA and mcrA levels of different methanogen phylogenetic groups were determined from six samples: four samples from anaerobic digesters used to treat either primarily cow or pig manure and two aliquots from an acidic peat sample stored at 4°C or 20°C. Only members of the Methanosaetaceae, Methanosarcina, Methanobacteriaceae, and Methanocorpusculaceae and Fen cluster were detected in the environmental samples. The three samples obtained from cow manure digesters were dominated by members of the genus Methanosarcina, whereas the sample from the pig manure digester contained detectable levels of only members of the Methanobacteriaceae. The acidic peat samples were dominated by both Methanosarcina spp. and members of the Fen cluster. In two of the manure digester samples only one methanogen group was detected, but in both of the acidic peat samples and two of the manure digester samples, multiple methanogen groups were detected. The TaqMan qPCR assays were successfully able to determine the environmental abundance of different phylogenetic groups of methanogens, including several groups with few or no cultivated members.Methanogens are integral to carbon cycling, catalyzing the production of methane and carbon dioxide, both potent greenhouse gases, during organic matter degradation in anaerobic soils and sediment (8). Methanogens are widespread in anaerobic environments, including tundra (36), freshwater lake and wetland sediments (9, 12), estuarine and marine sediments (2), acidic peatlands (4, 14), rice field soil (10, 16), animal guts (41), landfills (30), and anaerobic digesters treating animal manure (1), food processing wastewater (27), and municipal wastewater and solid waste (37, 57). Methane produced in anaerobic digesters may be captured and used for energy production, thus offsetting some or all of the cost of operation and reducing the global warming potential of methane release to the atmosphere.Methanogens are difficult to study through culture-based methods, and therefore many researchers have instead used culture-independent techniques to study methanogen populations. The 16S rRNA gene is the most widely used target for gene surveys, and a number of primers and probes have been developed to target methanogen groups (9, 11, 31, 36, 38, 40, 46, 48, 57). To eliminate potential problems with nonspecific amplification, some researchers have developed primers for the gene sequence of the α-subunit of the methyl coenzyme M reductase (mcrA) (17, 30, 49). The Mcr is exclusive to the methanogens with the exception of the methane-oxidizing Archaea (18) and shows mostly congruent phylogeny to the 16S rRNA gene, allowing mcrA analysis to be used in conjunction with, or independently of, that of the 16S rRNA gene (3, 30, 49). A number of researchers have examined methanogen communities with mcrA and have found uncultured clades quite different in sequence from cultured methanogen representatives (9, 10, 12, 14, 17, 22, 28, 47).Previous studies described methanogen communities by quantitation of different clades through the use of rRNA-targeted or rRNA gene-targeted probes with techniques such as dot blot hybridization (1, 27, 37, 38, 48) and fluorescent in situ hybridization (11, 40, 44, 57). Real-time quantitative PCR (qPCR) is an alternate technique capable of determining the copy number of a particular gene present in the DNA extracted from an environmental sample. Only a few studies have used qPCR to quantitatively examine different clades within methanogen communities, and most of these studies have exclusively targeted the 16S rRNA gene (19, 41, 42, 54-56). Far fewer researchers have used qPCR to quantify methanogen clades by targeting the mcrA (21, 34, 45), and these studies were limited to only a few phylogenetic groups.In this paper we present a methodology for determining methanogen gene copy numbers through the use of qPCR targeting the mcrA. Methanogens were quantified in total using methanogen-specific primers in SYBR green assays and also as members of nine different phylogenetic groups using TaqMan probes targeting specific subsets of methanogens.     

Vertical Distribution of Ammonia-Oxidizing Crenarchaeota and Methanogens in the Epipelagic Waters of Lake Kivu (Rwanda-Democratic Republic of the Congo)

Four stratified basins in Lake Kivu (Rwanda-Democratic Republic of the Congo) were sampled in March 2007 to investigate the abundance, distribution, and potential biogeochemical role of planktonic archaea. We used fluorescence in situ hybridization with catalyzed-reported deposition microscopic counts (CARD-FISH), denaturing gradient gel electrophoresis (DGGE) fingerprinting, and quantitative PCR (qPCR) of signature genes for ammonia-oxidizing archaea (16S rRNA for marine Crenarchaeota group 1.1a [MCG1] and ammonia monooxygenase subunit A [amoA]). Abundance of archaea ranged from 1 to 4.5% of total DAPI (4′,6-diamidino-2-phenylindole) counts with maximal concentrations at the oxic-anoxic transition zone (∼50-m depth). Phylogenetic analysis of the archaeal planktonic community revealed a higher level of richness of crenarchaeal 16S rRNA gene sequences (21 of the 28 operational taxonomic units [OTUs] identified [75%]) over euryarchaeotal ones (7 OTUs). Sequences affiliated with the kingdom Euryarchaeota were mainly recovered from the anoxic water compartment and mostly grouped into methanogenic lineages (Methanosarcinales and Methanocellales). In turn, crenarchaeal phylotypes were recovered throughout the sampled epipelagic waters (0- to 100-m depth), with clear phylogenetic segregation along the transition from oxic to anoxic water masses. Thus, whereas in the anoxic hypolimnion crenarchaeotal OTUs were mainly assigned to the miscellaneous crenarchaeotic group, the OTUs from the oxic-anoxic transition and above belonged to Crenarchaeota groups 1.1a and 1.1b, two lineages containing most of the ammonia-oxidizing representatives known so far. The concomitant vertical distribution of both nitrite and nitrate maxima and the copy numbers of both MCG1 16S rRNA and amoA genes suggest the potential implication of Crenarchaeota in nitrification processes occurring in the epilimnetic waters of the lake.Lake Kivu is a meromictic lake located in the volcanic region between Rwanda and the Democratic Republic of the Congo and is the smallest of the African Great Rift Lakes. The monimolimnion of the lake contains a large amount of dissolved CO₂ and methane (300 km³ and 60 km³, respectively) as a result of geological and biological activity (24, 73, 85). This massive accumulation converts Lake Kivu into one of the largest methane reservoirs in the world and into a unique ecosystem for geomicrobiologists interested in the methane cycle and in risk assessment and management (34, 71, 72, 85). Comprehensive studies on the diversity and activity of planktonic populations of both large and small eukaryotes and their trophic interplay operating in the epilimnetic waters of the lake are available (33, 39, 49). Recent surveys have also provided a deeper insight into the seasonal variations of photosynthetic and heterotrophic picoplankton (67, 68), although very few data exist on the composition, diversity, and spatial distribution of bacterial and archaeal communities. In this regard, the studies conducted so far of the bacterial/archaeal ecology in Lake Kivu have been mostly focused on the implications on the methane cycle (34, 73), but none have addressed the presence and distribution of additional archaeal populations in the lake.During the last few years, microbial ecology studies carried out in a wide variety of habitats have provided compelling evidence of the ubiquity and abundance of mesophilic archaea (4, 10, 13, 19). Moreover, the discovery of genes encoding enzymes related to nitrification and denitrification in archaeal metagenomes from soil and marine waters (29, 86, 88) and the isolation of the first autotrophic archaeal nitrifier (40) demonstrated that some archaeal groups actively participate in the carbon and nitrogen cycles (56, 64, 69). In relation to aquatic environments, genetic markers of ammonia-oxidizing archaea (AOA) of the marine Crenarchaeota group 1.1a (MCG1) have consistently been found in water masses of several oceanic regions (6, 14, 17, 26, 28, 30, 37, 42, 51, 52, 89), estuaries (5, 9, 26, 53), coastal aquifers (26, 66), and stratified marine basins (15, 41, 44). Although less information is available for freshwater habitats, recent studies carried out in oligotrophic high-mountain and arctic lakes showed an important contribution of AOA in both the planktonic and the neustonic microbial assemblages (4, 61, 89).The oligotrophic nature of Lake Kivu and the presence of a well-defined redoxcline may provide an optimal niche for the development of autotrophic AOA populations. Unfortunately, no studies of the involvement of microbial planktonic populations in cycling nitrogen in the lake exist, and only data on the distribution of dissolved inorganic nitrogen species in relation to phytoplankton ecology (67, 68) and nutrient loading are available (54, 58). Our goals here were to ascertain whether or not archaeal populations other than methane-related lineages were relevant components of the planktonic microbial community and to determine whether the redox gradient imposed by the oxic-anoxic interphase acts as a threshold for their vertical distribution in epipelagic waters (0- to 100-m depth). To further explore the presence and potential activity of nitrifying archaeal populations in Lake Kivu, samples were analyzed for the abundance and vertical distribution of signature genes for these microorganisms, i.e., the 16S rRNA of MCG1 and the ammonia monooxygenase subunit A (amoA) gene by quantitative PCR.     

Temporal Variation of Bacterial Respiration and Growth Efficiency in Tropical Coastal Waters

We investigated the temporal variation of bacterial production, respiration, and growth efficiency in the tropical coastal waters of Peninsular Malaysia. We selected five stations including two estuaries and three coastal water stations. The temperature was relatively stable (averaging around 29.5°C), whereas salinity was more variable in the estuaries. We also measured dissolved organic carbon and nitrogen (DOC and DON, respectively) concentrations. DOC generally ranged from 100 to 900 μM, whereas DON ranged from 0 to 32 μM. Bacterial respiration ranged from 0.5 to 3.2 μM O₂ h⁻¹, whereas bacterial production ranged from 0.05 to 0.51 μM C h⁻¹. Bacterial growth efficiency was calculated as bacterial production/(bacterial production + respiration), and ranged from 0.02 to 0.40. Multiple correlation analyses revealed that bacterial production was dependent upon primary production (r² = 0.169, df = 31, and P < 0.02) whereas bacterial respiration was dependent upon both substrate quality (i.e., DOC/DON ratio) (r² = 0.137, df = 32, and P = 0.03) and temperature (r² = 0.113, df = 36, and P = 0.04). Substrate quality was the most important factor (r² = 0.119, df = 33, and P = 0.04) for the regulation of bacterial growth efficiency. Using bacterial growth efficiency values, the average bacterial carbon demand calculated was from 5.30 to 11.28 μM C h⁻¹. When the bacterial carbon demand was compared with primary productivity, we found that net heterotrophy was established at only two stations. The ratio of bacterial carbon demand to net primary production correlated significantly with bacterial growth efficiency (r² = 0.341, df = 35, and P < 0.001). From nonlinear regression analysis, we found that net heterotrophy was established when bacterial growth efficiency was <0.08. Our study showed the extent of net heterotrophy in these waters and illustrated the importance of heterotrophic microbial processes in coastal aquatic food webs.As our understanding of the marine food web evolves, we recognize the importance of microorganisms in aquatic ecosystems. Bacteria are the main respirers and recycle a large pool of dissolved organic matter to higher trophic levels (6, 13). Therefore, bacterial production is a key process in dissolved organic matter flux. However, the transfer of dissolved organic matter to bacteria is more accurately reflected by bacterial carbon demand (BCD) or carbon consumption (23). One way to obtain BCD from bacterial production is through bacterial growth efficiency (BGE) or growth yield. BGE is an important parameter to evaluate the fate of organic carbon inputs and to determine whether bacteria act as a link (recyclers) or sink (mineralizers). Therefore, understanding the patterns of variation in BGE is fundamental for our knowledge of carbon cycling (14).BGE is essentially the ratio of carbon converted to biomass relative to all the carbon consumed, where carbon consumption is either measured as the sum of bacterial production and respiration (5, 24), dissolved organic matter utilization (3), or both (12). Although bacterial production is frequently measured, bacterial respiration measurements are still scarce (23) and are often derived from production rates assuming constant growth efficiency (7, 30). The use of a constant growth efficiency is, however, not valid in some situations as studies have shown that BGE varies over both time and space (8, 27, 28, 32).From cross-system compilations (15, 38, 49) and a comprehensive study in a temperate salt marsh estuary (4, 5), we begin to understand the factors that affect bacterial growth efficiency. Although substrate quantity and quality affect growth efficiency (4, 15), temperature is also an important factor (49). However, the effect of temperature differs for both bacterial production and bacterial respiration (5) and is distorted by substrate limitation (38).Most of the above studies are from temperate regions where there is marked seasonality in temperature. In temperate regions, the effects of temperature are usually more apparent (5, 32) and can sometimes distort the effects of other factors (4). Although temperature plays a major role in controlling heterotrophic activity in temperate regions, it plays a lesser role in the tropics, where temperatures are more stable and relatively higher. Tropical oceans cover about 40% of the global ocean (37), and yet knowledge of the structure and function of this ecosystem remains limited, especially in the region of Southeast Asia (29). Only a few related studies are available, and those are from tropical coastal waters in Goa, India (45), and mangrove and estuarine waters in Peninsular Malaysia (27, 28). Substrate quality is often suggested as a more important factor than temperature (27, 28, 45).In this paper, we addressed the following question: What is the effect of substrate quality and temperature toward BGE in tropical coastal waters? We measured bacterial respiration, production, and growth efficiency in tropical coastal waters and related their variation to changes in temperature and dissolved organic nutrient concentrations. Here, we show that although both temperature and substrate quality affected respiration, BGE was related to substrate quality only.     

Selection of a Bacillus pumilus Strain Highly Active against Ceratitis capitata (Wiedemann) Larvae

Ceratitis capitata (Wiedemann) (Diptera: Tephritidae), the Mediterranean fruit fly (medfly), is one of the most important fruit pests worldwide. The medfly is a polyphagous species that causes losses in many crops, which leads to huge economic losses. Entomopathogenic bacteria belonging to the genus Bacillus have been proven to be safe, environmentally friendly, and cost-effective tools to control pest populations. As no control method for C. capitata based on these bacteria has been developed, isolation of novel strains is needed. Here, we report the isolation of 115 bacterial strains and the results of toxicity screening with adults and larvae of C. capitata. As a result of this analysis, we obtained a novel Bacillus pumilus strain, strain 15.1, that is highly toxic to C. capitata larvae. The toxicity of this strain for C. capitata was related to the sporulation process and was observed only when cultures were incubated at low temperatures before they were used in a bioassay. The mortality rate for C. capitata larvae ranged from 68 to 94% depending on the conditions under which the culture was kept before the bioassay. Toxicity was proven to be a special characteristic of the newly isolated strain, since other B. pumilus strains did not have a toxic effect on C. capitata larvae. The results of the present study suggest that B. pumilus 15.1 could be considered a strong candidate for developing strategies for biological control of C. capitata.The Mediterranean fruit fly (medfly), Ceratitis capitata, is considered a highly invasive agricultural and economically important pest throughout the world. In less than 200 years the range of this species has expanded from its native habitat in sub-Saharan Africa, and it has become a cosmopolitan species (26) that is present on five continents (14, 46). The wide distribution of the medfly is attributed, among other things, to its remarkably polyphagous behavior (more than 300 host plants have been reported) (43), to its resistance to cold climates (65), and to successful establishment after multiple introductions (30, 49) as a result of the increasing frequency of global trade (46).Medfly infestations cause serious economic losses and sometimes result in complete loss of crops (76). Numerous methods have been tried to control medfly populations, including chemical products, such as malathion and other organophosphate insecticides (4, 8), classic biological control programs based on the release of some of parasitoids and predators (38, 41, 44), toxic baits (2, 13, 31, 32, 35, 56), mass trapping systems (24, 51), the sterile insect technique (7, 34, 61, 63, 72, 73), and development of integrated strategies of management (71). In spite of all of these attempts, control of Mediterranean fruit fly populations has been ineffective, and losses associated with this pest worldwide are constantly increasing (21, 46).Insecticides based on microbial agents (bacteria, fungi, and viruses) are a promising alternative that has received a great deal of attention for control of C. capitata (5, 13, 18, 40, 55), but so far no such insecticide has reached a commercial stage. Among the microbial insecticides, bacteria are very successful agents in biological control programs (17, 29). The entomopathogenic bacteria belonging to the genus Bacillus are natural agents used for biological control of invertebrate pests and are the basis of many commercial insecticides. Three species of the genus Bacillus have been mass produced and commercialized: Bacillus sphaericus, Bacillus thuringiensis, and Paenibacillus popilliae (formerly Bacillus popilliae) (29, 54). These organisms have different spectra and levels of activity that are correlated with the nature of the toxins, which are very frequently produced during sporulation (16, 17). B. thuringiensis was the first Bacillus species used in biological control programs for pests and human vector disease insects (17, 62). During its stationary phase, this Gram-positive, aerobic, ubiquitous, endospore-forming bacterium produces parasporal crystalline inclusions composed mainly of two types of insecticidal proteins (Cry and Cyt toxins) (62) that are toxic to a variety of insects, in some cases at the species level.There have been some reports of B. thuringiensis strains active against other fruit flies (3, 37, 58, 59, 67), but there has been no report of any Bacillus strain with activity against C. capitata.The aim of this study was to search for novel bacteria belonging to the genus Bacillus, specifically B. thuringiensis, with activity against adults and larvae of C. capitata that could be used as biological control agents. Isolation of 115 bacterial strains, evaluation of the insecticidal activities of these strains, and identification of a novel strain of Bacillus pumilus that is highly toxic to C. capitata larvae are reported here. In addition, we found that toxicity was observed only when cultures of B. pumilus strain 15.1 were exposed to low temperatures. The isolation of this novel pathogenic strain could be important for future development of biotechnological strategies aimed at reducing the economic losses caused by C. capitata.     

Influence of Environmental Gradients on the Abundance and Distribution of Mycobacterium spp. in a Coastal Lagoon Estuary

Environmental mycobacteria are of increasing concern in terms of the diseases they cause in both humans and animals. Although they are considered to be ubiquitous in aquatic environments, few studies have examined their ecology, and no ecological studies of coastal marine systems have been conducted. This study uses indirect gradient analysis to illustrate the strong relationships that exists between coastal water quality and the abundance of Mycobacterium spp. within a U.S. mid-Atlantic embayment. Mycobacterium species abundance and water quality conditions (based on 16 physical and chemical variables) were examined simultaneously in monthly samples obtained at 18 Maryland and Virginia coastal bay stations from August 2005 to November 2006 (n = 212). A quantitative molecular assay for Mycobacterium spp. was evaluated and applied, allowing for rapid, direct enumeration. By using indirect gradient analysis (environmental principal-components analysis), a strong linkage between eutrophic conditions, characterized by low dissolved-oxygen levels and elevated nutrient concentrations, and mycobacteria was determined. More specifically, a strong nutrient response was noted, with all nitrogen components and turbidity measurements correlating positively with abundance (r values of >0.30; P values of <0.001), while dissolved oxygen showed a strong negative relationship (r = −0.38; P = 0.01). Logistic regression models developed using salinity, dissolved oxygen, and total nitrogen showed a high degree of concordance (83%). These results suggest that coastal restoration and management strategies designed to reduce eutrophication may also reduce total mycobacteria in coastal waters.Environmental mycobacteria, or nontuberculous mycobacteria (NTM), include all species of mycobacteria other than those in the Mycobacterium tuberculosis complex and M. leprae. In general, NTM are aerobic, acid-fast, gram-positive, non-spore-forming, nonmotile organisms found as free-living saprophytes in soil and water (12, 14, 20, 21, 35). However, several members of this group can cause serious disease in humans, including pulmonary infections, cervical lymphadenitis, ulcerative necrosis, skin infections, and disseminated infections associated primarily with autoimmune disorders (12, 29). For example, disseminated infection with the Mycobacterium avium complex can occur in up to 40% of late-stage AIDS patients in developed countries (43). NTM can also have costly and problematic effects on wild and domesticated animals (17, 23). Thus, understanding the sources and reservoirs of these bacteria has become a priority in recent years (12, 34).While the mode of infection has been poorly established for many cases involving NTM, water is commonly implicated as either a source or a vector (12, 43). NTM are considered to be ubiquitous in the environment and have been cultured globally from samples obtained from freshwaters and marine natural waters (12), swimming pools and hot tubs (11, 25), and drinking water supplies (12, 13), among others. However, only a limited number of attempts have been made to examine the association of their distribution and abundance with environmental parameters (1, 21, 24). The abundance of the M. avium complex was found to correlate positively with water temperature and levels of zinc and humic and fulvic acids and negatively with the dissolved-oxygen content and pH in brown-water swamps in the southeastern United States (24). In a study of Finnish brook waters, acidic conditions, along with the presence of peatlands, chemical oxygen demand, increased precipitation, water color, and concentrations of several metals, were found to favor total NTM (20, 21). However, recent efforts with samples from the Rio Grande River in the United States found positive correlations with the presence of coliforms and Escherichia coli counts and negative correlations with chemical toxicity and water temperature in this alkaline, oligotrophic system (1). Although system-specific differences may be apparent, no attempts to examine mycobacterial ecology in marine and estuarine systems have been reported to date.Historically, researchers have relied on culture-based techniques for detection and enumeration of mycobacteria from environmental samples (1, 20, 21, 43). Because of the slow growth of many mycobacteria, culture from environmental samples requires decontamination, which can severely impact both the quantity and diversity of species recovered (18, 19). Recently, quantitative PCR (qPCR) has gained favor as a means of rapidly enumerating organisms or genes in environmental samples (5, 15, 38, 40). This method allows for the continuous monitoring of the reaction through the use of fluorescent reporter molecules or DNA stains. Because of this strategy, the reaction can be evaluated at the peak of the exponential phase, reducing errors of reagent depletion and assay efficiency associated with end point reads. Quantification is based on the principle that the amount of the starting template is directly proportional to the number of cycles required to reach the peak of the exponential phase, and is evaluated through the preparation of standards.Like many coastal lagoon estuaries, the shallow embayments bordering the Maryland and Virginia seaboard are highly susceptible to anthropogenic influence, as they are visited by millions of people annually for vacation and water-related recreation (44). While eutrophication and degraded environmental conditions have been generally linked to factors or organisms which can ultimately influence human health, little attention has been given to the response of bacteria (16, 45). In this paper, we describe our efforts to examine environmental influences on the abundance and distribution of NTM in a dynamic estuarine system.     

Girdling Affects Ectomycorrhizal Fungal (EMF) Diversity and Reveals Functional Differences in EMF Community Composition in a Beech Forest

The relationships between plant carbon resources, soil carbon and nitrogen content, and ectomycorrhizal fungal (EMF) diversity in a monospecific, old-growth beech (Fagus sylvatica) forest were investigated by manipulating carbon flux by girdling. We hypothesized that disruption of the carbon supply would not affect diversity and EMF species numbers if EM fungi can be supplied by plant internal carbohydrate resources or would result in selective disappearance of EMF taxa because of differences in carbon demand of different fungi. Tree carbohydrate status, root demography, EMF colonization, and EMF taxon abundance were measured repeatedly during 1 year after girdling. Girdling did not affect root colonization but decreased EMF species richness of an estimated 79 to 90 taxa to about 40 taxa. Cenococcum geophilum, Lactarius blennius, and Tomentella lapida were dominant, colonizing about 70% of the root tips, and remained unaffected by girdling. Mainly cryptic EMF species disappeared. Therefore, the Shannon-Wiener index (H′) decreased but evenness was unaffected. H′ was positively correlated with glucose, fructose, and starch concentrations of fine roots and also with the ratio of dissolved organic carbon to dissolved organic nitrogen (DOC/DON), suggesting that both H′ and DOC/DON were governed by changes in belowground carbon allocation. Our results suggest that beech maintains numerous rare EMF species by recent photosynthate. These EM fungi may constitute biological insurance for adaptation to changing environmental conditions. The preservation of taxa previously not known to colonize beech may, thus, form an important reservoir for future forest development.In temperate and boreal forest ecosystems, most tree species form ectomycorrhizal fungal (EMF) associations. EM fungi ensheathe the root tip, forming characteristic mantlelike structures (1). The presence and lengths of hyphae emanating from the mantle are characteristic of different EMF species and establish different soil exploration types (2). It has been assumed that EMF communities are adapted specifically to mobilize sparse soil nutrient resources in boreal and temperate forests (11, 50). Current estimates indicate that about 80% of all nitrogen and phosphorus present in plants has been taken up via mycorrhizas (30, 41, 63).Unlike free-living soil microbes, EM fungi have direct access to reduced carbon from their host plants. More than 50 years ago, Melin and Nilsson (46) showed that ¹⁴C applied to leaves was recovered within one day in EM fungi, suggesting a strong dependence of fungal metabolism on host photosynthesis. Subsequent isotopic studies corroborated tight connections between current photosynthate and EM fungi (28, 42). EMF hyphae constitute the main path of plant-derived carbon into the soil (24, 29). Furthermore, EMF hyphae contribute substantially to soil respiration (25% from hyphae and 15% from roots) (27). As hyphal respiration decreases strongly in response to girdling of trees, a tight metabolic link between extramatrical mycelia and host photosynthetic activity must exist (5, 9, 32). In addition, fruiting body formation of EMF species was strongly dependent on host photosynthetic capacity (32, 40). In contrast, the significance of the current assimilate supply for EMF colonization of root tips and for community composition is not yet well understood. Since trees contain substantial stores of carbohydrates in the roots and stem (7), it may be expected that EM fungi can be maintained if this carbon resource is available. For example, defoliation experiments with conifers, which restricted but did not eliminate current photosynthate transfer to roots, showed no effects on root EMF colonization. Massive defoliation that negatively affected aboveground biomass production suppressed morphotypes with thick mantles compared to those with thin mantles, suggesting a shift to less-carbon-demanding EMF species (14, 40, 44, 54, 56). Earlier studies also reported decreased EMF colonization of root tips (21, 52).In a common garden experiment with young beech trees, strong shading over several years, which severely limited plant growth, suppressed EMF colonization and resulted in low EMF diversity (20). EMF community composition was affected strongly by shading and slightly by short-term girdling, suggesting that EMF taxa are sensitive to changes in plant internal carbohydrate resources (20). However, the overall EMF diversity was low, probably because the young trees were grown in nutrient-rich compost soil (20). The significance of photoassimilates for EMF abundance, diversity, and community composition, therefore, remains to be shown for adult forest trees, which usually have high EMF diversity and low nitrogen availability (10, 26, 53, 61).The aim of this work was to test the hypothesis that EMF abundance and diversity are independent of the current photoassimilate supply and can be maintained by internal resources. To investigate this concept, old-growth beech trees (Fagus sylvatica L.) were girdled to suppress carbon allocation to roots. Since disruption of the current assimilate flux affects the carbohydrate source strength, we hypothesized that changes in EMF taxon composition would occur if EMF species had different carbon demands. Tree carbohydrate status, root demography, EMF colonization, and EMF taxon abundance were measured repeatedly during 1 year after girdling. Since girdling also affects carbon release into and probably nutrient uptake from soil, the influence of possible feedback by changes in the ratio of dissolved organic carbon to dissolved organic nitrogen (DOC/DON) in the soil on EMF diversity was also assessed.