Effects of Swine Manure on Macrolide,Lincosamide, and Streptogramin B Antimicrobial Resistance in Soils

Current agricultural practices involve inclusion of antimicrobials in animal feed and result in manure containing antimicrobials and antimicrobial-resistant microorganisms. This work evaluated the effects of land application of swine manure on the levels of tetracycline, macrolide, and lincosamide antimicrobials and on macrolide, lincosamide, and streptogramin B (MLS_B) resistance in field soil samples and laboratory soil batch tests. MLS_B and tetracycline antimicrobials were quantified after solid-phase extraction using liquid chromatography-tandem mass spectrometry. The prevalence of the ribosomal modification responsible for MLS_B resistance in the same samples was quantified using fluorescence in situ hybridization. Macrolide antimicrobials were not detected in soil samples, while tetracyclines were detected, suggesting that the latter compounds persist in soil. No significant differences in ribosomal methylation or presumed MLS_B resistance were observed when amended and unamended field soils were compared, although a transient (<20-day) increase was observed in most batch tests. Clostridium cluster XIVa accounted for the largest fraction of resistant bacteria identified in amended soils. Overall, this study did not detect a persistent increase in the prevalence of MLS_B resistance due to land application of treated swine manure.Treated swine manure contains substantial levels of both antimicrobial-resistant microorganisms (10, 26) and antimicrobials (7, 18, 33). Land application of manure could therefore contribute to public health risks associated with the increasing prevalence of antimicrobial resistance in pathogens both directly, through the dissemination of antimicrobial-resistant pathogens, and indirectly, through the introduction of and selection for antimicrobial resistance genes. Because limited data are available, this connection is largely a theoretical connection, particularly for the indirect effects. However, a recent retrospective study of antimicrobial resistance in soil did support the hypothesis that there is an environmental connection by documenting that there was an increase in the abundance of antibiotic resistance genes in samples collected from 1940 to 2008, during which time antimicrobial production increased dramatically (12).The fate of antimicrobials in amended soils is a function of their sorptive properties, the soil characteristics, and the potential for abiotic and biotic degradation of the antimicrobials. Tetracyclines tend to adsorb to soil (21, 23), which leads to persistence in amended soils (3, 7, 11), although they are also susceptible to degradation (3, 4). The macrolide tylosin frequently is not detected (3, 4, 7, 11, 33) and is likely rapidly degraded in manure and soils (8, 16, 24). However, persistence of tylosin for several months in amended soil has also been reported (6). The differences in degradation rates may be caused by differences in soil characteristics, manure-to-soil ratios, and/or microbial communities (15, 16, 21).Addition of both antimicrobials and antimicrobial-resistant microorganisms might be expected to result in an increase in the levels of resistance. However, most studies have not shown that there is a long-term increase in antimicrobial resistance due to land application of manure at agronomically prescribed rates (5, 9, 26). Transient (i.e., <45-day) increases have been reported (9, 26), as have elevated levels of resistance at sites near manure piles (5). In contrast, another report showed that there were significantly higher levels of tylosin resistance in soils that received animal manure from operations that used subtherapeutic levels of antimicrobials than in soils at sites where there was no use of subtherapeutic levels of antimicrobials (19). One limitation of these studies was their use of culture-based methods to quantify resistance; the results may not be representative of the entire microbial community. The molecular methods that have been used to quantify resistance also have limitations, and the most serious limitation is the inability of these methods to examine the full diversity of known and unknown resistance genes. The previous molecular studies of the impact of land application on resistance were largely restricted to qualitative analyses (10, 25), although quantitative PCR methods for analysis of tetracycline resistance genes have recently been used for cattle and swine lagoons (14, 20). In a retrospective soil study, Knapp et al. (12), who also used quantitative PCR, found multiple site differences, which made it difficult to evaluate the impact of manure application. However, the site with the highest manure application rate did not show the highest levels of antimicrobial resistance, suggesting that there are other factors that have a greater influence on the prevalence of resistance.In the present study, a variation of the fluorescence in situ hybridization (FISH) technique was used to assess the impact of land application of swine manure on the levels of macrolide-lincosamide-streptogramin B (MLS_B) resistance. Although the MLS_B antimicrobials are chemically distinct, methylation or mutation of a single base of the 23S rRNA prevents binding and results in cross-resistance to all three classes (29). The prevalence of MLS_B antimicrobial resistance in the microbial community can therefore be quantified indirectly by hybridization of an oligonucleotide probe to unmethylated, MLS_B-sensitive ribosomes, using either membrane hybridization (1, 10) or FISH (31). These methods do not require culturing or a comprehensive knowledge of the diversity of resistance gene sequences, but they do not detect resistance to specific antimicrobials that results from other mechanisms, such as macrolide efflux.This study focused on evaluating the impact of land application of swine manure on the levels of antimicrobials and the prevalence of antimicrobial resistance in the soil environment. The concentrations of tetracycline, macrolide, and lincosamide antimicrobials and the prevalence of MLS_B resistance were compared for field soils that received no manure, swine manure from farms that did not use antimicrobials (referred to below as organic farms), and swine manure from conventional farms to determine whether land application affects the levels of antimicrobials and MLS_B resistance. The effects of addition of manure, antimicrobials (lincomycin and chlortetracycline), and MLS_B-resistant microorganisms on the prevalence of MLS_B resistance were also compared using soil batch tests.     

Pig Manure Contamination Marker Selection Based on the Influence of Biological Treatment on the Dominant Fecal Microbial Groups

The objective of this study was to identify a microbial marker for pig manure contamination. We quantified the persistence of four dominant bacterial groups from the pig intestinal tract throughout manure handling at 10 livestock operations (including aerobic digestion) by using molecular typing. The partial 16S rRNA genes of Bacteroides-Prevotella, Eubacterium-Clostridiaceae, Bacillus-Streptococcus-Lactobacillus (BSL), and Bifidobacterium group isolates were amplified and analyzed by capillary electrophoresis single-strand conformation polymorphism. The most dominant bacterial populations were identified by cloning and sequencing their 16S rRNA genes. The results showed that Bifidobacterium spp. and, to a lesser extent, members of the BSL group, were less affected by the aerobic treatment than either Eubacterium-Clostridiaceae or Bacteroides-Prevotella. Two Bifidobacterium species found in raw manure were still present in manure during land application, suggesting that they can survive outside the pig intestinal tract and also survive aerobic treatment. The 16S-23S rRNA internal transcribed spacer of one species, Bifidobacterium thermacidophilum subsp. porcinum, was sequenced, and a specific pair of primers was designed for its detection in the environment. With this nested PCR assay, this potential marker was not detected in samples from 30 bovine, 30 poultry, and 28 human fecal samples or in 15 urban wastewater effluents. As it was detected in runoff waters after spreading of pig manure, we propose this marker as a suitable microbial indicator of pig manure contamination.Brittany represents only 7% of France but is the main pig production area and hosts approximately 14 million fatteners per year. This high concentration of confined pig feeding has led to the overapplication of manure to soil, which contributes to water pollution. Physical and biological manure treatment processes have been developed to limit nitrogen and phosphorus pollution (5). As these treatments were not designed to eliminate microbial pollution, even treated manure can contain pathogenic microorganisms (27) and agricultural soils and water systems can thus potentially still be contaminated through surface runoff and seepage. As manure application can increase the number of pathogens in the soil (18), pig feces may represent a significant risk to human health in Brittany. Currently, the monitoring of bacteria to assess fecal contamination (Escherichia coli, fecal coliforms, and enterococci) does not differentiate contamination from pig slurry from pollution by other animals or humans. It is thus important to develop analytic tools to specifically detect this source of pollution.Many studies have already proposed potential markers for the detection of host-specific fecal pollution (2, 3, 8, 12-15, 20, 37, 38, 48, 49). Much of this research has concentrated on distinguishing human and animal sources of contamination (3, 8, 20, 30, 38). Some studies have focused on identifying individual sources of animal pollution and have described molecular markers for feces from ducks (13), chickens (37), bovines (2, 3, 49), or cervids (6). Biomarkers have been proposed for porcine fecal contamination but rarely for porcine manure, the bacterial composition of which differs from that of porcine feces (9). Molecular markers have been developed to target the 16S rRNA gene sequences of dominant Eubacteria (2, 14, 43, 48) or methanogenic Archaebacteria (54) of the pig intestinal tract, whereas Khatib et al. (29) targeted the STII toxin gene from enterotoxigenic E. coli. Among the dominant groups of pig fecal Eubacteria, which include Bacteroides-Prevotella, Eubacterium-Clostridiacea, Lactobacillus-Streptococcus (34, 45, 51, 58), and to a lesser extent Bifidobacterium (40), the Bacteroides-Prevotella group has been particularly well studied (14, 22, 44). This marker of pig feces was described by Okabe et al. (44), but their work was based on feces sampled from only two farms and the number of clones analyzed was low. Gourmelon et al. (22) also detected the presence of a specific marker of pig feces belonging to the Bacteroides-Prevotella group in five stored manure samples. Although these studies revealed the presence of specific markers in fecal samples and in the subsequent pig manure samples, they did not address the possible disappearance of these anaerobic bacteria during the storage or biological treatment of the manure.Due to the lack of data concerning the bacterial flora of manure, the aims of this study were (i) to compare the monitoring of the Bacteroides-Prevotella group with that of Eubacterium-Clostridiaceae, Bacillus-Streptococcus-Lactobacillus (BSL), and Bifidobacterium throughout the biological manure treatment process and (ii) to search for a molecular marker among these groups of bacteria that was consistently present in the manure intended for land application. In the first part of this study, the persistence of the dominant bacteria throughout treatment was studied by using molecular typing, capillary electrophoresis-single-strand conformation polymorphism (CE-SSCP) (45) based on the analysis of the 16S rRNA genes. CE-SSCP is a fingerprinting technique in which single-stranded DNA fragments of the same length are separated based on the conformation of their secondary structure (23). The major advantages of this technique are its reproducibility between runs and its high resolution power with fewer false results than with denaturing gradient gel electrophoresis (25, 26).The second part of this article describes the relevance of the potential marker of pig manure (Bifidobacterium thermacidophilum subsp. porcinum) selected according to the results of the CE-SSCP profiles and the subsequent identification of dominant peaks of the CE-SSCP profiles. The specificity of this pig marker was then tested by assessing the host distribution in a selection of fecal, manure, and wastewater samples.     

Cultivation of Fastidious Bacteria by Viability Staining and Micromanipulation in a Soil Substrate Membrane System

Soil substrate membrane systems allow for microcultivation of fastidious soil bacteria as mixed microbial communities. We isolated established microcolonies from these membranes by using fluorescence viability staining and micromanipulation. This approach facilitated the recovery of diverse, novel isolates, including the recalcitrant bacterium Leifsonia xyli, a plant pathogen that has never been isolated outside the host.The majority of bacterial species have never been recovered in the laboratory (1, 14, 19, 24). In the last decade, novel cultivation approaches have successfully been used to recover “unculturables” from a diverse range of divisions (23, 25, 29). Most strategies have targeted marine environments (4, 23, 25, 32), but soil offers the potential for the investigation of vast numbers of undescribed species (20, 29). Rapid advances have been made toward culturing soil bacteria by reformulating and diluting traditional media, extending incubation times, and using alternative gelling agents (8, 21, 29).The soil substrate membrane system (SSMS) is a diffusion chamber approach that uses extracts from the soil of interest as the growth substrate, thereby mimicking the environment under investigation (12). The SSMS enriches for slow-growing oligophiles, a proportion of which are subsequently capable of growing on complex media (23, 25, 27, 30, 32). However, the SSMS results in mixed microbial communities, with the consequent difficulty in isolation of individual microcolonies for further characterization (10).Micromanipulation has been widely used for the isolation of specific cell morphotypes for downstream applications in molecular diagnostics or proteomics (5, 15). This simple technology offers the opportunity to select established microcolonies of a specific morphotype from the SSMS when combined with fluorescence visualization (3, 11). Here, we have combined the SSMS, fluorescence viability staining, and advanced micromanipulation for targeted isolation of viable, microcolony-forming soil bacteria.     

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.     

Diversity and Abundance of Zoonotic Pathogens and Indicators in Manures of Feedlot Cattle in Australia

The occurrence of 10 pathogens and three fecal indicators was assessed by quantitative PCR in manures of Australian feedlot cattle. Most samples tested positive for one or more pathogens. For the dominant pathogens Campylobacter jejuni, Listeria monocytogenes, Giardia spp., Cryptosporidium spp., and eaeA-positive Escherichia coli, 10² to 10⁷ genome copies g⁻¹ (dry weight) manure were recovered.More than 600,000 tons of feedlot cattle manure are generated each year in Australia, which raises concern for potential water, air, and soil contamination (21, 27). Hence, better monitoring and knowledge of the resulting risks are needed (5, 26). Most zoonotic pathogens associated with cattle are well described in the literature, especially those of major health significance, including the bacterial pathogens Campylobacter spp., Listeria monocytogenes, pathogenic Escherichia coli (particularly serotypes O157 and O111), Salmonella enterica, Yersinia spp., Leptospira spp., Coxiella burnetii, Mycobacterium avium subsp. paratuberculosis, and the parasitic protozoa Giardia lamblia and Cryptosporidium parvum (2, 21, 27). While studies of pathogen occurrence in manure are numerous, data suited to quantitatively estimating end user risks are still limited. Few surveys quantify multiple pathogens (11, 12, 14, 28), and none have concurrently measured all 10 above in cattle manure. A further constraint on risk assessment is that most data were generated in North America or Europe, where cli-mate and environment can differ markedly from Australian conditions.Addressing this knowledge gap now appears feasible, as real-time quantitative PCR (qPCR) can be used as an alternative to culture-based methods for quantifying environmental pathogens (7, 23, 29). Improvements in sample preparation and nucleic acid cleanup methods have largely overcome problems associated with the molecular biology-based analysis of fecal matter (22). Further, qPCR can detect stressed, damaged, and otherwise nonculturable cells persisting in a state of dormancy or indeed dead (15, 17, 29). The aim of this paper is to report on a quantitative survey of zoonotic pathogens and indicators in manures from Australian feedlot beef cattle.A total of 128 composited samples (five subsamples each) representing fresh feces (n = 32), pen manure (n = 32), harvested pen manure (n = 28), stockpiled manure (n = 23), composted manure (n = 6), and carcass compost (n = 7) were collected from five cattle feedlots in eastern Australia in the winter/summer of 2009 (13). All samples were assayed for the 10 key pathogens listed above and also fecal indicators (total coliforms, E. coli, and enterococci).     

Abundance of Novel and Diverse tfdA-Like Genes,Encoding Putative Phenoxyalkanoic Acid Herbicide-Degrading Dioxygenases,in Soil

Phenoxyalkanoic acid (PAA) herbicides are widely used in agriculture. Biotic degradation of such herbicides occurs in soils and is initiated by α-ketoglutarate- and Fe²⁺-dependent dioxygenases encoded by tfdA-like genes (i.e., tfdA and tfdAα). Novel primers and quantitative kinetic PCR (qPCR) assays were developed to analyze the diversity and abundance of tfdA-like genes in soil. Five primer sets targeting tfdA-like genes were designed and evaluated. Primer sets 3 to 5 specifically amplified tfdA-like genes from soil, and a total of 437 sequences were retrieved. Coverages of gene libraries were 62 to 100%, up to 122 genotypes were detected, and up to 389 genotypes were predicted to occur in the gene libraries as indicated by the richness estimator Chao1. Phylogenetic analysis of in silico-translated tfdA-like genes indicated that soil tfdA-like genes were related to those of group 2 and 3 Bradyrhizobium spp., Sphingomonas spp., and uncultured soil bacteria. Soil-derived tfdA-like genes were assigned to 11 clusters, 4 of which were composed of novel sequences from this study, indicating that soil harbors novel and diverse tfdA-like genes. Correlation analysis of 16S rRNA and tfdA-like gene similarity indicated that any two bacteria with D > 20% of group 2 tfdA-like gene-derived protein sequences belong to different species. Thus, data indicate that the soil analyzed harbors at least 48 novel bacterial species containing group 2 tfdA-like genes. Novel qPCR assays were established to quantify such new tfdA-like genes. Copy numbers of tfdA-like genes were 1.0 × 10⁶ to 65 × 10⁶ per gram (dry weight) soil in four different soils, indicating that hitherto-unknown, diverse tfdA-like genes are abundant in soils.Phenoxyalkanoic acid (PAA) herbicides such as MCPA (4-chloro-2-methyl-phenoxyacetic acid) and 2,4-D (2,4-dichlorophenoxyacetic acid) are widely used to control broad-leaf weeds in agricultural as well as nonagricultural areas (19, 77). Degradation occurs primarily under oxic conditions in soil, and microorganisms play a key role in the degradation of such herbicides in soil (62, 64). Although relatively rapidly degraded in soil (32, 45), both MCPA and 2,4-D are potential groundwater contaminants (10, 56, 70), accentuating the importance of bacterial PAA herbicide-degrading bacteria in soils (e.g., references 3, 5, 6, 20, 41, 59, and 78).Degradation can occur cometabolically or be associated with energy conservation (15, 54). The first step in the degradation of 2,4-D and MCPA is initiated by the product of cadAB or tfdA-like genes (29, 30, 35, 67), which constitutes an α-ketoglutarate (α-KG)- and Fe²⁺-dependent dioxygenase. TfdA removes the acetate side chain of 2,4-D and MCPA to produce 2,4-dichlorophenol and 4-chloro-2-methylphenol, respectively, and glyoxylate while oxidizing α-ketoglutarate to CO₂ and succinate (16, 17).Organisms capable of PAA herbicide degradation are phylogenetically diverse and belong to the Alpha-, Beta-, and Gammproteobacteria and the Bacteroidetes/Chlorobi group (e.g., references 2, 14, 29-34, 39, 60, 68, and 71). These bacteria harbor tfdA-like genes (i.e., tfdA or tfdAα) and are categorized into three groups on an evolutionary and physiological basis (34). The first group consists of beta- and gammaproteobacteria and can be further divided into three distinct classes based on their tfdA genes (30, 46). Class I tfdA genes are closely related to those of Cupriavidus necator JMP134 (formerly Ralstonia eutropha). Class II tfdA genes consist of those of Burkholderia sp. strain RASC and a few strains that are 76% identical to class I tfdA genes. Class III tfdA genes are 77% identical to class I and 80% identical to class II tfdA genes and linked to MCPA degradation in soil (3). The second group consists of alphaproteobacteria, which are closely related to Bradyrhizobium spp. with tfdAα genes having 60% identity to tfdA of group 1 (18, 29, 34). The third group also harbors the tfdAα genes and consists of Sphingomonas spp. within the alphaproteobacteria (30).Diverse PAA herbicide degraders of all three groups were identified in soil by cultivation-dependent studies (32, 34, 41, 78). Besides CadAB, TfdA and certain TfdAα proteins catalyze the conversion of PAA herbicides (29, 30, 35). All groups of tfdA-like genes are potentially linked to the degradation of PAA herbicides, although alternative primary functions of group 2 and 3 TfdAs have been proposed (30, 35). However, recent cultivation-independent studies focused on 16S rRNA genes or solely on group 1 tfdA sequences in soil (e.g., references 3-5, 13, and 41). Whether group 2 and 3 tfdA-like genes are also quantitatively linked to the degradation of PAA herbicides in soils is unknown. Thus, tools to target a broad range of tfdA-like genes are needed to resolve such an issue. Primers used to assess the diversity of tfdA-like sequences used in previous studies were based on the alignment of approximately 50% or less of available sequences to date (3, 20, 29, 32, 39, 47, 58, 73). Primers specifically targeting all major groups of tfdA-like genes to assess and quantify a broad diversity of potential PAA degraders in soil are unavailable. Thus, the objectives of this study were (i) to develop primers specific for all three groups of tfdA-like genes, (ii) to establish quantitative kinetic PCR (qPCR) assays based on such primers for different soil samples, and (iii) to assess the diversity and abundance of tfdA-like genes in soil.     

Transport and Distribution of Salmonella enterica Serovar Typhimurium in Loamy and Sandy Soil Monoliths with Applied Liquid Manure

A leaching experiment, where liquid manure spiked with Salmonella enterica serovar Typhimurium (Tet⁺) DSM554 was applied to soil surfaces, was conducted on intact soil monoliths (60 cm in diameter and 100 cm long). A total of 6.5 × 10¹⁰ CFU was applied to each column. We found that Salmonella serovar Typhimurium could be transported to a 1-m depth in loamy soil at concentrations reaching 1.3 × 10⁵ CFU/ml of leachate. The test strain was found in concentrations ranging from 300 to 1.3⁵ cells/ml in loamy soil throughout the 27 days of the experiment, while concentrations below 20 cells/ml were sporadically detected in the leachates from sandy monoliths. Real-time PCR targeting invA DNA showed a clear correspondence between the total and culturable numbers of cells in the leachate, indicating that most cells leached were viable. On day 28, distribution of Salmonella serovar Typhimurium at five depths in the four monoliths was determined. The highest recovery rate, ranging from 1.5% to 3.8% of the total applied inoculum, was found in the top 0.2 m.The spreading of liquid manure on agricultural land is an economic and practical solution for improving soil quality. However, animal manure frequently contains zoonotic pathogenic bacteria such as certain Escherichia coli, Salmonella spp., and Campylobacter spp. (8, 9, 24). Salmonellosis is one of the most frequently reported food-borne diseases in Europe, accounting for 67.5% of reported food-borne outbreaks (32). Salmonella enterica serovar Typhimurium, which accounts for 23% of nonhuman isolates (from animal, food, feed, and environmental sources) and for 18% of human isolates, was the second-most-common serotype found worldwide from 2000 to 2004 (38). Human salmonellosis has been related to the consumption of water or foods contaminated with animal manure (9, 15), which frequently contains a variety of different enteric pathogenic microorganisms (27).Manure disposed of on agricultural land may create a risk for microbial contamination of surface water and groundwater. Field scale studies have reported manure bacteria in drainage water (5, 25, 36, 37). In addition to contamination originating from manure, pathogens in irrigation water may contaminate soil, water, crops, and, subsequently, animals and humans (7, 15). Disease outbreaks have been associated with water and food directly or indirectly contaminated with animal manure (9). Recent data from 1990 to 2004 showed that compared to beef, poultry, seafood, and eggs, fresh produce caused the second-highest number of food-borne disease outbreaks as well as the highest number of reported illnesses per outbreak (4).When microorganisms enter the soil environment, their survival and distribution is affected by various factors, including soil texture, pH, temperature, water saturation, and the intensity of rain events (17, 28). Preferential water movement in macropores is probably the primary route by which bacteria move down through the soil (1, 2, 23). Studies of bacterial transport have been made mostly on homogenized natural soils in laboratory soil columns (2, 18, 30, 33). The purpose of our study was to perform a large-scale experiment using intact 100-cm-deep soil monoliths to compare the leaching of Salmonella serovar Typhimurium in two structurally different soils: a loamy soil with high absorptive properties and macropores and a coarse sandy soil with less-absorptive properties and less preferential flow. The monoliths were initially exposed to normal precipitation events followed by intensive precipitation, where the leaching of Salmonella serovar Typhimurium cells was observed. After the leaching experiment, the concentrations of Salmonella serovar Typhimurium cells at five depths were determined. Furthermore, we validated bacterial enumerations based on cultures on agar plates with real-time PCR to determine if a proportion of the test strain entered a nonculturable state.     

Strategy for Extracting DNA from Clay Soil and Detecting a Specific Target Sequence via Selective Enrichment and Real-Time (Quantitative) PCR Amplification

We present a simple strategy for isolating and accurately enumerating target DNA from high-clay-content soils: desorption with buffers, an optional magnetic capture hybridization step, and quantitation via real-time PCR. With the developed technique, μg quantities of DNA were extracted from mg samples of pure kaolinite and a field clay soil.Isolating and characterizing DNA sequences for use in molecular methods are integral to evaluating microbial community diversity in soil (6, 21, 22, 24, 37). Any isolation protocol should maximize nucleic acid isolation while minimizing copurification of enzymatic inhibitors. Although several methods that focus on extraction of total community DNA from environmental soil and water samples have been published (7, 21, 26, 34), the lack of a standard nucleic acid isolation protocol (32) reflects the difficulty in accomplishing these goals, most likely due to the complex nature of the soil environment.DNA extraction is especially difficult for soils containing clay (3, 5), given the tight binding of DNA strands to clay soil particles (7, 10, 20). Additionally, extracellular DNA binds to and is copurified with soil humic substances (10), which inhibit the activity of enzymes such as restriction endonucleases and DNA polymerase (6, 13, 23). Although clay-bound DNA can be PCR amplified in the absence of inhibitors (1), it is often the case that inhibitors are present in the soil environment, among them bilirubin, bile salts, urobilinogens, and polysaccharides (40). Of these inhibitors, humic substances have been found to be the most recalcitrant (36).A promising technique for isolating specific target sequences from soil particles and enzymatic inhibitors is the magnetic capture hybridization-PCR technique (MCH-PCR) presented by Jacobsen (19) and used to obtain high detection sensitivities (11, 38).We have found no evidence in the published literature of the use of MCH-PCR on soils that have high clay contents and here present a three-step strategy for isolating specific DNA sequences from the most difficult soil environment—clay that contains humic substances—and enumerating a specific target sequence from the crude extract.     

Alignment of the c-Type Cytochrome OmcS along Pili of Geobacter sulfurreducens

Immunogold localization revealed that OmcS, a cytochrome that is required for Fe(III) oxide reduction by Geobacter sulfurreducens, was localized along the pili. The apparent spacing between OmcS molecules suggests that OmcS facilitates electron transfer from pili to Fe(III) oxides rather than promoting electron conduction along the length of the pili.There are multiple competing/complementary models for extracellular electron transfer in Fe(III)- and electrode-reducing microorganisms (8, 18, 20, 44). Which mechanisms prevail in different microorganisms or environmental conditions may greatly influence which microorganisms compete most successfully in sedimentary environments or on the surfaces of electrodes and can impact practical decisions on the best strategies to promote Fe(III) reduction for bioremediation applications (18, 19) or to enhance the power output of microbial fuel cells (18, 21).The three most commonly considered mechanisms for electron transfer to extracellular electron acceptors are (i) direct contact between redox-active proteins on the outer surfaces of the cells and the electron acceptor, (ii) electron transfer via soluble electron shuttling molecules, and (iii) the conduction of electrons along pili or other filamentous structures. Evidence for the first mechanism includes the necessity for direct cell-Fe(III) oxide contact in Geobacter species (34) and the finding that intensively studied Fe(III)- and electrode-reducing microorganisms, such as Geobacter sulfurreducens and Shewanella oneidensis MR-1, display redox-active proteins on their outer cell surfaces that could have access to extracellular electron acceptors (1, 2, 12, 15, 27, 28, 31-33). Deletion of the genes for these proteins often inhibits Fe(III) reduction (1, 4, 7, 15, 17, 28, 40) and electron transfer to electrodes (5, 7, 11, 33). In some instances, these proteins have been purified and shown to have the capacity to reduce Fe(III) and other potential electron acceptors in vitro (10, 13, 29, 38, 42, 43, 48, 49).Evidence for the second mechanism includes the ability of some microorganisms to reduce Fe(III) that they cannot directly contact, which can be associated with the accumulation of soluble substances that can promote electron shuttling (17, 22, 26, 35, 36, 47). In microbial fuel cell studies, an abundance of planktonic cells and/or the loss of current-producing capacity when the medium is replaced is consistent with the presence of an electron shuttle (3, 14, 26). Furthermore, a soluble electron shuttle is the most likely explanation for the electrochemical signatures of some microorganisms growing on an electrode surface (26, 46).Evidence for the third mechanism is more circumstantial (19). Filaments that have conductive properties have been identified in Shewanella (7) and Geobacter (41) species. To date, conductance has been measured only across the diameter of the filaments, not along the length. The evidence that the conductive filaments were involved in extracellular electron transfer in Shewanella was the finding that deletion of the genes for the c-type cytochromes OmcA and MtrC, which are necessary for extracellular electron transfer, resulted in nonconductive filaments, suggesting that the cytochromes were associated with the filaments (7). However, subsequent studies specifically designed to localize these cytochromes revealed that, although the cytochromes were extracellular, they were attached to the cells or in the exopolymeric matrix and not aligned along the pili (24, 25, 30, 40, 43). Subsequent reviews of electron transfer to Fe(III) in Shewanella oneidensis (44, 45) appear to have dropped the nanowire concept and focused on the first and second mechanisms.Geobacter sulfurreducens has a number of c-type cytochromes (15, 28) and multicopper proteins (12, 27) that have been demonstrated or proposed to be on the outer cell surface and are essential for extracellular electron transfer. Immunolocalization and proteolysis studies demonstrated that the cytochrome OmcB, which is essential for optimal Fe(III) reduction (15) and highly expressed during growth on electrodes (33), is embedded in the outer membrane (39), whereas the multicopper protein OmpB, which is also required for Fe(III) oxide reduction (27), is exposed on the outer cell surface (39).OmcS is one of the most abundant cytochromes that can readily be sheared from the outer surfaces of G. sulfurreducens cells (28). It is essential for the reduction of Fe(III) oxide (28) and for electron transfer to electrodes under some conditions (11). Therefore, the localization of this important protein was further investigated.     

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).     

Prevalence and Impact of Bacteriophages on the Presence of Escherichia coli O157:H7 in Feedlot Cattle and Their Environment

The relationship between endemic bacteriophages infecting E. coli O157:H7 (referred to as “phage”) and levels of shedding of E. coli O157:H7 by cattle was investigated in two commercial feedlots in southern Alberta, Canada. Between May and November 2007, 10 pens of cattle were monitored by collection of pooled fecal pats, water with sediment from troughs, manure slurry from the pen floor, and rectal fecal samples from individual animals (20 per pen) at two separate times. Bacteriophages infecting E. coli O157:H7 were detected more frequently (P < 0.001) after 18 to 20 h enrichment than by initial screening and were recovered in 239 of 855 samples (26.5% of 411 pooled fecal pats, 23.8% of 320 fecal grab samples, 21.8% of 87 water trough samples, and 94.6% of 37 pen floor slurry samples). Overall, prevalence of phage was highest (P < 0.001) in slurry. Recovery of phage from pooled fecal pats was highest (P < 0.05) in May. Overall recovery did not differ (P > 0.10) between fecal grab samples and pooled fecal pats. A higher prevalence of phage in fecal pats or water trough samples was associated (P < 0.01) with reduced prevalence of E. coli O157:H7 in rectal fecal samples. There was a weak but significant negative correlation between isolation of phage and E. coli O157:H7 in fecal grab samples (r = −0.11; P < 0.05). These data demonstrate that the prevalence of phage fluctuates in a manner similar to that described for E. coli O157:H7. Phage were more prevalent in manure slurry than other environmental sources. The likelihood of fecal shedding of E. coli O157:H7 was reduced if cattle in the pen harbored phage.Bacteriophages are the most abundant biological entities on earth. An estimated 10³⁰ marine bacteriophages are harbored in the ocean, and they significantly influence microbial communities and function (27). As resistance is an increasing challenge in antimicrobial therapy, the antimicrobial nature of bacteriophages is being more intensively studied (13, 15). Bacteriophages naturally inhabit the mammalian gastrointestinal tract (1, 8), and Escherichia coli-infecting bacteriophages are commonly isolated from sewage, hospital wastewater, and fecal samples from humans and animals (3). Ruminants have been shown to shed up to 10⁷ bacteriophage per gram of feces (6), and in humans multiple types of bacteriophage exhibiting activity against E. coli have been isolated from a single fecal sample (7).E. coli O157:H7 is an important zoonotic bacterium carried asymptomatically by cattle and readily isolated from manure, manure slurry, and drinking water in dairies and feedlots (11, 24, 30). Additionally, E. coli O157:H7 shedding by cattle has a seasonal pattern, peaking in the summer months (2, 25). Bacteriophage strains that infect E. coli O157:H7 have also been isolated from animal feces and have shown lytic activity against this bacterium in vivo and in vitro (5, 23, 28, 31). In recent studies, such phages were shown to be widely distributed in cattle and in feces on the pen floor within feedlots (4, 18). However, the relationships between the presence of E. coli O157:H7-infecting bacteriophage in cattle and their environment and the shedding of this bacterium by cattle are largely undefined. Consequently, the aims of the present study were (i) to determine the prevalence of endemic E. coli O157:H7-infecting bacteriophage (referred to as “phage”) in feedlots over a 7-month period and (ii) to compare the presence of phage to the occurrence of E. coli O157:H7 in cattle and their environment.     

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.     

In Situ Phylogenetic Structure and Diversity of Wild Bradyrhizobium Communities

Bacteria often infect their hosts from environmental sources, but little is known about how environmental and host-infecting populations are related. Here, phylogenetic clustering and diversity were investigated in a natural community of rhizobial bacteria from the genus Bradyrhizobium. These bacteria live in the soil and also form beneficial root nodule symbioses with legumes, including those in the genus Lotus. Two hundred eighty pure cultures of Bradyrhizobium bacteria were isolated and genotyped from wild hosts, including Lotus angustissimus, Lotus heermannii, Lotus micranthus, and Lotus strigosus. Bacteria were cultured directly from symbiotic nodules and from two microenvironments on the soil-root interface: root tips and mature (old) root surfaces. Bayesian phylogenies of Bradyrhizobium isolates were reconstructed using the internal transcribed spacer (ITS), and the structure of phylogenetic relatedness among bacteria was examined by host species and microenvironment. Inoculation assays were performed to confirm the nodulation status of a subset of isolates. Most recovered rhizobial genotypes were unique and found only in root surface communities, where little bacterial population genetic structure was detected among hosts. Conversely, most nodule isolates could be classified into several related, hyper-abundant genotypes that were phylogenetically clustered within host species. This pattern suggests that host infection provides ample rewards to symbiotic bacteria but that host specificity can strongly structure only a small subset of the rhizobial community.Symbiotic bacteria often encounter hosts from environmental sources (32, 48, 60), which leads to multipartite life histories including host-inhabiting and environmental stages. Research on host-associated bacteria, including pathogens and beneficial symbionts, has focused primarily on infection and proliferation in hosts, and key questions about the ecology and evolution of the free-living stages have remained unanswered. For instance, is host association ubiquitous within a bacterial lineage, or if not, do host-infecting genotypes represent a phylogenetically nonrandom subset? Assuming that host infection and free-living existence exert different selective pressures, do bacterial lineages diverge into specialists for these different lifestyles? Another set of questions addresses the degree to which bacteria associate with specific host partners. Do bacterial genotypes invariably associate with specific host lineages, and is such specificity controlled by one or both partners? Alternatively, is specificity simply a by-product of ecological cooccurrence among bacteria and hosts?Rhizobial bacteria comprise several distantly related proteobacterial lineages, most notably the genera Azorhizobium, Bradyrhizobium, Mesorhizobium, Rhizobium, and Sinorhizobium (52), that have acquired the ability to form nodules on legumes and symbiotically fix nitrogen. Acquisition of nodulation and nitrogen fixation loci has likely occurred through repeated lateral transfer of symbiotic loci (13, 74). Thus, the term “rhizobia” identifies a suite of symbiotic traits in multiple genomic backgrounds rather than a taxonomic classification. When rhizobia infect legume hosts, they differentiate into specialized endosymbiotic cells called bacteroids, which reduce atmospheric nitrogen in exchange for photosynthates from the plant (35, 60). Rhizobial transmission among legume hosts is infectious. Rhizobia can spread among hosts through the soil (60), and maternal inheritance (through seeds) is unknown (11, 43, 55). Nodule formation on hosts is guided by reciprocal molecular signaling between bacteria and plant (5, 46, 58), and successful infection requires a compatible pairing of legume and rhizobial genotypes. While both host and symbiont genotypes can alter the outcome of rhizobial competition for adsorption (34) and nodulation (33, 39, 65) of legume roots, little is known about how this competition plays out in nature.Rhizobia can achieve reproductive success via multiple lifestyles (12), including living free in the soil (14, 44, 53, 62), on or near root surfaces (12, 18, 19, 51), or in legume nodules (60). Least is known about rhizobia in bulk soil (not penetrated by plant roots). While rhizobia can persist for years in soil without host legumes (12, 30, 61), it appears that growth is often negligible in bulk soil (4, 10, 14, 22, 25). Rhizobia can also proliferate in the rhizosphere (soil near the root zone) of legumes (4, 10, 18, 19, 22, 25, 51). Some rhizobia might specialize in rhizosphere growth and infect hosts only rarely (12, 14, 51), whereas other genotypes are clearly nonsymbiotic because they lack key genes (62) and must therefore persist in the soil. The best-understood rhizobial lifestyle is the root nodule symbiosis with legumes, which is thought to offer fitness rewards that are superior to life in the soil (12). After the initial infection, nodules grow and harbor increasing populations of bacteria until the nodules senesce and the rhizobia are released into the soil (11, 12, 38, 40, 55). However, rhizobial fitness in nodules is not guaranteed. Host species differ in the type of nodules they form, and this can determine the degree to which differentiated bacteroids can repopulate the soil (11, 12, 38, 59). Furthermore, some legumes can hinder the growth of nodules with ineffective rhizobia, thus punishing uncooperative symbionts (11, 27, 28, 56, 71).Here, we investigated the relationships between environmental and host-infecting populations of rhizobia. A main objective was to test the hypothesis that rhizobia exhibit specificity among host species as well as among host microenvironments, specifically symbiotic nodules, root surfaces, and root tips. We predicted that host infection and environmental existence exert different selective pressures on rhizobia, leading to divergent patterns of clustering, diversity, and abundance of rhizobial genotypes.     

Social Interactions and Distribution of Bacillus subtilis Pherotypes at Microscale

Bacillus subtilis strains communicate through the comQXPA quorum sensing (QS) system, which regulates genes expressed during early stationary phase. A high polymorphism of comQXP′ loci was found in closely related strains isolated from desert soil samples separated by distances ranging from meters to kilometers. The observed polymorphism comprised four communication groups (pherotypes), such that strains belonging to the same pherotype exchanged information efficiently but strains from different pherotypes failed to communicate. To determine whether the same level of polymorphism in the comQXP′ QS system could be detected at microscale, B. subtilis isolates were obtained from two separate 1-cm³ soil samples, which were progressively divided into smaller sections. Cross-activation studies using pherotype-responsive reporter strains indicated the same number of communication pherotypes at microscale as previously determined at macroscale. Sequencing of the housekeeping gene gyrA and the QS comQ gene confirmed different evolutionary rates of these genes. Furthermore, an asymmetric communication response was detected inside the two pherotype clusters, suggesting continuous evolution of the QS system and possible development of new languages. To our knowledge, this is the first microscale study demonstrating the presence of different QS languages among isolates of one species, and the implications of this microscale diversity for microbial interactions are discussed.Quorum sensing (QS), a widespread phenomenon in the bacterial world, controls a wide range of cell density-dependent behaviors. Bacillus subtilis uses QS to control production of antimicrobial peptides, bacteriocins, and antibiotics (20) but also to alternate between two cell types during stationary phase: competent cells, able to take in DNA from the environment, and dormant spores, able to survive harsh environmental conditions (9, 12, 24). Development of genetic competence in B. subtilis is controlled by a QS system encoded by the comQXPA operon (2, 53, 54). This involves the ComX pheromone that accumulates during exponential growth (25, 46, 47) and is initially synthesized as a 55-residue protein that is processed, modified, and released into the extracellular medium as a 5- to 10-amino-acid peptide. The isoprenoidal modification on the tryptophan residue of this peptide is catalyzed by the ComQ protein (2, 25, 34, 35, 42, 52). Upon reaching the threshold concentration, processed and modified ComX binds to the membrane-associated, histidine protein kinase ComP and triggers the QS response, linking autophosphorylation of ComP and transfer of phosphate to the response regulator ComA (59). The level of phosphorylated ComA is also controlled by dephosphorylation, which is dependent on a separate QS system involving competence sporulation factor (CSF) and the RapC phosphatase (3, 59). Phosphorylated ComA directly controls expression of various genes (6, 33), including the srfAB operon that contains the comS gene (15, 41), required for development of competence (55).Previous studies of environmental B. subtilis strains indicate a high polymorphism (approximately 56% identity at the nucleotide level) in the QS locus, which is restricted to comQ, comX, and the N-terminal region of the comP gene. Sequences surrounding this locus, downstream gene comA, a C-terminal region of comP, and the upstream degQ gene, are highly conserved (2, 53, 54). Sequence analysis of the comQXP loci of 13 strains indicated clustering into four distinct similarity groups (2). These groups were congruent for comQ, comX, and the N-terminal region of comP, indicating coevolution of the three genes. In addition, the similarity groups correlated with four pherotypes, able to communicate efficiently within but not between groups. Similar variation has been reported for the agr QS system in staphylococci (19, 56) and in the competence QS system of Streptococcus pneumoniae (17, 19, 37, 38, 60).B. subtilis is often referred to as a soil-dwelling organism, its spores persisting in soil until encountering conditions suitable for germination and growth (10). The basic structural unit of soil ecosystems is the soil aggregate, in which biogeochemical processes occur at scales relevant to microorganisms. Approximately 50% of the volume of a soil aggregate represents open pores, while the remainder consists of mineral particles (sand, silt, and clay) held together by organic material (48), with which B. subtilis may be preferentially associated (16, 43). Soil aggregates can be classified as macroaggregates (diameter, >250 μm) and microaggregates (diameter, 2 to 250 μm) (39), but little is known about the distribution of bacteria within aggregates. Structural organization of the soil creates a mosaic of microenvironments, within which water movement and diffusion of nutrients and other molecules play key roles in functioning of the soil microbiota (7, 13, 39). These roles may vary with the scale at which they operate. Tisdall and Oades (51) suggest that scales at which microorganisms are important in the soil aggregation process range between 2 and 2,000 μm, depending on the specific system being investigated (13). Although the microscale distribution of microorganisms and their associated functions have rarely been studied, it is becoming recognized that greater knowledge of spatial organization at the scale of a soil aggregate (microscale) is essential for a better understanding of soil ecosystem function and of the mechanisms that generate and maintain diversity, including speciation, extinction, dispersal, and interactions within and between species (7, 13, 26).The aim of this study was to assess the potential role of QS in generating and maintaining microscale diversity within the soil. This was achieved by determining the genomic and functional diversification of the B. subtilis QS system with regard to geographical distance and ecological characteristics. Isolates were obtained from two 1-cm³ sandy, riverbank soil samples separated by approximately 5 m, allowing assessment of macroscale diversity. In addition, each riverbank soil sample was treated as a separate macroaggregate that was progressively sectioned to obtain subsamples of different sizes, allowing assessment of microscale diversity. The riverbank soil B. subtilis isolates were compared with Bacillus isolates previously obtained from desert soil samples separated by distances of meters to kilometers (2, 40), representing macroscale distribution. The Bacillus isolates were used to (i) correlate geographical distance (microscale/macroscale) with genomic distance of the QS comQ gene and the housekeeping gyrA gene, (ii) investigate and compare the specificity of the QS response of microscale and macroscale isolates, and (iii) explore dominance of pherotypes inside soil aggregates. To our knowledge, this is the first investigation of a QS system that addresses the genomic and functional diversification of bacterial populations at microscale.     

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.     

Population Structure of the Lyme Borreliosis Spirochete Borrelia burgdorferi in the Western Black-Legged Tick (Ixodes pacificus) in Northern California

Factors potentially contributing to the lower incidence of Lyme borreliosis (LB) in the far-western than in the northeastern United States include tick host-seeking behavior resulting in fewer human tick encounters, lower densities of Borrelia burgdorferi-infected vector ticks in peridomestic environments, and genetic variation among B. burgdorferi spirochetes to which humans are exposed. We determined the population structure of B. burgdorferi in over 200 infected nymphs of the primary bridging vector to humans, Ixodes pacificus, collected in Mendocino County, CA. This was accomplished by sequence typing the spirochete lipoprotein ospC and the 16S-23S rRNA intergenic spacer (IGS). Thirteen ospC alleles belonging to 12 genotypes were found in California, and the two most abundant, ospC genotypes H3 and E3, have not been detected in ticks in the Northeast. The most prevalent ospC and IGS biallelic profile in the population, found in about 22% of ticks, was a new B. burgdorferi strain defined by ospC genotype H3. Eight of the most common ospC genotypes in the northeastern United States, including genotypes I and K that are associated with disseminated human infections, were absent in Mendocino County nymphs. ospC H3 was associated with hardwood-dominated habitats where western gray squirrels, the reservoir host, are commonly infected with LB spirochetes. The differences in B. burgdorferi population structure in California ticks compared to the Northeast emphasize the need for a greater understanding of the genetic diversity of spirochetes infecting California LB patients.In the United States, Lyme borreliosis (LB) is the most commonly reported vector-borne illness and is caused by infection with the spirochete Borrelia burgdorferi (3, 9, 52). The signs and symptoms of LB can include a rash, erythema migrans, fever, fatigue, arthritis, carditis, and neurological manifestations (50, 51). The black-legged tick, Ixodes scapularis, and the western black-legged tick, Ixodes pacificus, are the primary vectors of B. burgdorferi to humans in the United States, with the former in the northeastern and north-central parts of the country and the latter in the Far West (9, 10). These ticks perpetuate enzootic transmission cycles together with a vertebrate reservoir host such as the white-footed mouse, Peromyscus leucopus, in the Northeast and Midwest (24, 35), or the western gray squirrel, Sciurus griseus, in California (31, 46).B. burgdorferi is a spirochete species with a largely clonal population structure (14, 16) comprising several different strains or lineages (8). The polymorphic ospC gene of B. burgdorferi encodes a surface lipoprotein that increases expression within the tick during blood feeding (47) and is required for initial infection of mammalian hosts (25, 55). To date, approximately 20 North American ospC genotypes have been described (40, 45, 49, 56). At least four, and possibly up to nine, of these genotypes are associated with B. burgdorferi invasiveness in humans (1, 15, 17, 49, 57). Restriction fragment length polymorphism (RFLP) and, subsequently, sequence analysis of the 16S-23S rRNA intergenic spacer (IGS) are used as molecular typing tools to investigate genotypic variation in B. burgdorferi (2, 36, 38, 44, 44, 57). The locus maintains a high level of variation between related species, and this variation reflects the heterogeneity found at the genomic level of the organism (37). The IGS and ospC loci appear to be linked (2, 8, 26, 45, 57), but the studies to date have not been representative of the full range of diversity of B. burgdorferi in North America.Previous studies in the northeastern and midwestern United States have utilized IGS and ospC genotyping to elucidate B. burgdorferi evolution, host strain specificity, vector-reservoir associations, and disease risk to humans. In California, only six ospC and five IGS genotypes have been described heretofore in samples from LB patients or I. pacificus ticks (40, 49, 56) compared to approximately 20 ospC and IGS genotypes identified in ticks, vertebrate hosts, or humans from the Northeast and Midwest (8, 40, 45, 49, 56). Here, we employ sequence analysis of both the ospC gene and IGS region to describe the population structure of B. burgdorferi in more than 200 infected I. pacificus nymphs from Mendocino County, CA, where the incidence of LB is among the highest in the state (11). Further, we compare the Mendocino County spirochete population to populations found in the Northeast.