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.     

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.     

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.     

Fluorescence In Situ Hybridization Method Using a Peptide Nucleic Acid Probe for Identification of Salmonella spp. in a Broad Spectrum of Samples

A fluorescence in situ hybridization (FISH) method for the rapid detection of Salmonella spp. using a novel peptide nucleic acid (PNA) probe was developed. The probe theoretical specificity and sensitivity were both 100%. The PNA-FISH method was optimized, and laboratory testing on representative strains from the Salmonella genus subspecies and several related bacterial species confirmed the predicted theoretical values of specificity and sensitivity. The PNA-FISH method has been successfully adapted to detect cells in suspension and is hence able to be employed for the detection of this bacterium in blood, feces, water, and powdered infant formula (PIF). The blood and PIF samples were artificially contaminated with decreasing pathogen concentrations. After the use of an enrichment step, the PNA-FISH method was able to detect 1 CFU per 10 ml of blood (5 × 10⁹ ± 5 × 10⁸ CFU/ml after an overnight enrichment step) and also 1 CFU per 10 g of PIF (2 × 10⁷ ± 5 × 10⁶ CFU/ml after an 8-h enrichment step). The feces and water samples were also enriched according to the corresponding International Organization for Standardization methods, and results showed that the PNA-FISH method was able to detect Salmonella immediately after the first enrichment step was conducted. Moreover, the probe was able to discriminate the bacterium in a mixed microbial population in feces and water by counter-staining with 4′,6-diamidino-2-phenylindole (DAPI). This new method is applicable to a broad spectrum of samples and takes less than 20 h to obtain a diagnosis, except for PIF samples, where the analysis takes less than 12 h. This procedure may be used for food processing and municipal water control and also in clinical settings, representing an improved alternative to culture-based techniques and to the existing Salmonella PNA probe, Sal23S10, which presents a lower specificity.Salmonella spp. are enteropathogenic bacteria that cause diseases that range from a mild gastroenteritis to systemic infections (5, 18) The disease severity is determined by the virulence characteristics of the Salmonella strain, host species, and host health condition. Phylogenetic analysis has demonstrated that the genus Salmonella includes two species: Salmonella bongori and Salmonella enterica. Salmonella strains are conventionally identified and classified according to the Kauffmann-White serotyping scheme, which is based on antigenic variation in the outer membrane (23). To date, more than 2,500 Salmonella serovars have been identified, and most of them are capable of infecting a wide variety of animal species and humans (33). Salmonella can be transmitted directly by person to person via the fecal-oral route or by contact with external reservoirs if fecal contamination of soil, water, and foods occurs. It is therefore necessary to develop robust detection methods for all of these sample types.The diagnostic method currently used for Salmonella detection is bacterial culture (International Organization for Standardization [ISO] method 6579:2002), a time-consuming and laborious process (40). A rapid and reliable tool to assist disease control management should aim to reduce salmonellosis in both people and animals. For this purpose a number of assays, such as the enzyme-linked immunosorbent assay (ELISA), PCR, and fluorescence in situ hybridization (FISH), have been developed to decrease the time required to identify Salmonella in food, feces, water, and other clinical samples (8, 10, 14, 15, 25, 26, 31, 41).Several authors have compared some of these approaches, especially culture-based, ELISA, and PCR methods, for Salmonella detection. Some authors found that PCR and ELISA-based methods failed to detect some samples that were positive by culture method (12, 13, 36, 39, 40). Even so, PCR-based methods have proved to be more accurate. Other work showed that when a selective enrichment step was performed before PCR, all Salmonella samples recovered by the culture method were detected. Moreover, the presence of Salmonella that was not recovered by the culture method could be detected by PCR (13, 35). These studies revealed that the enrichment step could increase the molecular assay sensitivity by eliminating problems such as the low numbers of bacteria and the presence of inhibitory substances in certain types of samples, such as food and fecal matter (11, 28, 36). However, PCR-based methods usually require a DNA extraction step, and none of the methods referred to above allows a direct, in situ visualization of the bacterium within the sample.FISH is a molecular assay widely applied for bacterial identification and localization within samples (2, 3). The method is usually based on the specific binding of nucleic acid probes to particular RNAs, due to their higher numbers of copies in the cells. There are already some studies reporting Salmonella detection by FISH using DNA probes (21, 29). A recently developed synthetic DNA analogue, named peptide nucleic acid (PNA), capable of hybridizing to complementary nucleic acid targets, has made FISH procedures easier and more efficient (38, 42). PNA-FISH methods have been successfully applied to the detection of several pathogenic microorganisms (6, 16, 17, 19, 22, 30, 34, 37, 42). For Salmonella, a PNA probe, designated Sal23S10, that targets the 23S rRNA of both Salmonella species has been already developed (31). However, the probe is also complementary to Actinobacillus actinomycetemcomitans, Buchnera aphidicola, and Haemophilus influenzae 23S rRNAs.In this paper, we identify and describe the design of a new fluorescently labeled PNA probe for the specific identification of the Salmonella genus. A novel, rapid, and reliable PNA-FISH method that can be easily applied to a great variety of sample types, either clinical or environmental, has consequently been developed and optimized.     

Antibiotic Resistance Patterns and Detection of blaDHA-1 in Salmonella Species Isolates from Chicken Farms in South Korea

Fifteen nonrepetitive ampicillin-resistant Salmonella spp. were identified among 91 Salmonella sp. isolates during nationwide surveillance of Salmonella in waste from 131 chicken farms during 2006 and 2007. Additional phenotyping and genetic characterization of these 15 isolates by using indicator cephalosporins demonstrated that resistance to ampicillin and reduced susceptibility to cefoxitin in three isolates was caused by TEM-1 and DHA-1 β-lactamases. Plasmid profiling and Southern blot analysis of these three DHA-1-positive Salmonella serovar Indiana isolates and previously reported unrelated clinical isolates of DHA-1-positive Salmonella serovar Montevideo, Klebsiella pneumoniae, and Escherichia coli from humans and swine indicated the involvement of the large-size plasmid. Restriction enzyme digestion of the plasmids from the transconjugants showed variable restriction patterns except for the two Salmonella serovar Indiana isolates identified in this study. To the best of our knowledge, this is the first report of the presence of the DHA-1 gene among Salmonella spp. of animal origin.Nontyphoidal Salmonella (NTS) strains are a significant cause of gastrointestinal infections of food origin. These microbes are a heterogeneous group of medically important Gram-negative bacteria and can infect a wide range of animals, including humans (3, 6, 9-11, 25).Currently, no antimicrobial therapies are recommended for the treatment of NTS infection unless a patient is of extreme age, has an underlying disease, or is infected with an invasive Salmonella sp. However, the use of antibiotics in treatment of clinical enteric infection has been heavily compromised by emerging multidrug-resistant microbes (4, 17, 18, 23). In particular, resistance due to extended-spectrum β-lactamases (ESBLs) and AmpC β-lactamases is of special concern as these enzymes confer resistance to some of the front-line antibiotics used to treat enteric infection in humans and animals (4, 13, 14, 19).Four classes of β-lactamases are known to confer resistance to β-lactam antibiotics. Among these, plasmid-mediated class A and class C β-lactamases have been frequently reported, whereas class B and class D β-lactamases are relatively rare (4). TEM and SHV enzymes of class A β-lactamases are generally found in Gram-negative bacteria and are derived by one or more amino acid substitutions around the active site of the enzyme that is responsible for the ESBL phenotype (4). Recently, the CTX-M enzyme of class A β-lactamases has been increasingly reported from enteric microbes, like Salmonella and Escherichia coli (4, 5, 9, 15). These have greater activity against cefotaxime than do other oxyimino-β-lactam substrates, like ceftazidime, ceftriaxone, or cefepime (4, 5). Plasmid-mediated AmpC β-lactamases, like DHA and CMY, are not inhibited by clavulanic acid and have been isolated from a wide variety of clinical and community-acquired microbes (2, 4, 13, 14, 16). These β-lactamases are native to the chromosomes of many Gram-negative bacilli but are missing in some genera, like Salmonella (4). The majority of β-lactamases reported in Salmonella to date have been derived from human clinical isolates, and only limited information is available regarding Salmonella spp. derived from farm animals, although isolates from both humans and animals are of clinical and epidemiological importance (4, 15, 25).In light of this knowledge gap, our study focused on assessing the distribution of Salmonella serovars in poultry farms in South Korea. Subsequently, isolates were analyzed for resistance to antibiotics commonly used in farms. Phenotypic and genetic characteristics of ampicillin-resistant Salmonella isolates were tested to gain insight into what β-lactamases were prevalent among these strains. We also characterized DHA-1-associated plasmids in these Salmonella spp. and compared them with clinical isolates of Salmonella, Klebsiella pneumoniae, and Escherichia coli from humans and from swine.     

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.     

Use of a Mixture of Bacteriophages for Biological Control of Salmonella enterica Strains in Compost

Bacteriophages specific to Salmonella strains were isolated from sewage effluent and characterized. A five-strain bacteriophage mixture was applied to dairy manure compost inoculated with Salmonella enterica serotype Typhimurium. Bacteriophage treatment resulted in a greater than 2-log-unit reduction of Salmonella within 4 h at all moisture levels compared to the controls.Composting is a complex process designed to mitigate the risk of pathogen contamination while producing a nutrient-rich substrate, suitable for land application (19). When performed properly, pathogenic enteric microorganisms, such as Salmonella and Escherichia coli O157:H7, are reduced to undetectable levels in most cases (18). Some studies, however, have revealed that Salmonella strains are able to survive if composting is performed improperly (11). Furthermore, the surfaces of compost heaps have been shown to reach insufficient temperatures for the complete inactivation of pathogenic bacteria (27) and may result in pathogen regrowth (12).The growing demand for organically grown fruits and vegetables emphasizes the need for safe soil amendments and organic fertilizers. Despite increased awareness of the potential risk of pathogen contamination of crops, multiple outbreaks of food-borne illnesses associated with fresh produce have occurred (3, 21). The persistence of human pathogens in compost has led researchers to explore different approaches for pathogen reduction, such as irradiation or ammonia supplementation (20, 24). To date, there are no reports on the potential for using bacteriophage to reduce pathogen contamination of compost. Recent bacteriophage studies have evaluated their effectiveness in live animals (2, 26, 28), on fresh produce (15, 23, 25), and on meat products (7, 30). Application of bacteriophages may therefore be a preventive step in the preharvest stages of food production.The objectives of this study were to isolate and characterize bacteriophages specific to Salmonella serovars and to develop a bacteriophage mixture effective in reducing pathogen contamination in compost under different environmental conditions.     

Role of Geobacter sulfurreducens Outer Surface c-Type Cytochromes in Reduction of Soil Humic Acid and Anthraquinone-2,6-Disulfonate

Deleting individual genes for outer surface c-type cytochromes in Geobacter sulfurreducens partially inhibited the reduction of humic substances and anthraquinone-2,6,-disulfonate. Complete inhibition was obtained only when five of these genes were simultaneously deleted, suggesting that diverse outer surface cytochromes can contribute to the reduction of humic substances and other extracellular quinones.Humic substances can play an important role in the reduction of Fe(III), and possibly other metals, in sedimentary environments (6, 34). Diverse dissimilatory Fe(III)-reducing microorganisms (3, 5, 7, 9, 11, 19-22, 25) can transfer electrons onto the quinone moieties of humic substances (38) or the model compound anthraquinone-2,6-disulfonate (AQDS). Reduced humic substances or AQDS abiotically reduces Fe(III) to Fe(II), regenerating the quinone. Electron shuttling in this manner can greatly increase the rate of electron transfer to insoluble Fe(III) oxides, presumably because soluble quinone-containing molecules are more accessible for microbial reduction than insoluble Fe(III) oxides (19, 22). Thus, catalytic amounts of humic substances have the potential to dramatically influence rates of Fe(III) reduction in soils and sediments and can promote more rapid degradation of organic contaminants coupled to Fe(III) reduction (1, 2, 4, 10, 24).To our knowledge, the mechanisms by which Fe(III)-reducing microorganisms transfer electrons to humic substances have not been investigated previously for any microorganism. However, reduction of AQDS has been studied using Shewanella oneidensis (17, 40). Disruption of the gene for MtrB, an outer membrane protein required for proper localization of outer membrane cytochromes (31), inhibited reduction of AQDS, as did disruption of the gene for the outer membrane c-type cytochrome, MtrC (17). However, in each case inhibition was incomplete, and it was suggested that there was a possibility of some periplasmic reduction (17), which would be consistent with the ability of AQDS to enter the cell (40).The mechanisms for electron transfer to humic substances in Geobacter species are of interest because molecular studies have frequently demonstrated that Geobacter species are the predominant Fe(III)-reducing microorganisms in sedimentary environments in which Fe(III) reduction is an important process (references 20, 32, and 42 and references therein). Geobacter sulfurreducens has routinely been used for investigations of the physiology of Geobacter species because of the availability of its genome sequence (29), a genetic system (8), and a genome-scale metabolic model (26) has made it possible to take a systems biology approach to understanding the growth of this organism in sedimentary environments (23).     

Characterization of Environmentally Persistent Escherichia coli Isolates Leached from an Irish Soil

Soils are typically considered to be suboptimal environments for enteric organisms, but there is increasing evidence that Escherichia coli populations can become resident in soil under favorable conditions. Previous work reported the growth of autochthonous E. coli in a maritime temperate Luvic Stagnosol soil, and this study aimed to characterize, by molecular and physiological means, the genetic diversity and physiology of environmentally persistent E. coli isolates leached from the soil. Molecular analysis (16S rRNA sequencing, enterobacterial repetitive intergenic consensus PCR, pulsed-field gel electrophoresis, and a multiplex PCR method) established the genetic diversity of the isolates (n = 7), while physiological methods determined the metabolic capability and environmental fitness of the isolates, relative to those of laboratory strains, under the conditions tested. Genotypic analysis indicated that the leached isolates do not form a single genetic grouping but that multiple genotypic groups are capable of surviving and proliferating in this environment. In physiological studies, environmental isolates grew well across a broad range of temperatures and media, in comparison with the growth of laboratory strains. These findings suggest that certain E. coli strains may have the ability to colonize and adapt to soil conditions. The resulting lack of fecal specificity has implications for the use of E. coli as an indicator of fecal pollution in the environment.Escherichia coli is a well-established indicator of fecal contamination in the environment. The organism''s validity as an indicator of water pollution is dependent, among other factors, on its fecal specificity and its inability to multiply outside the primary host, the gastrointestinal tracts of humans and warm-blooded animals (9). While many pathogens and indicator organisms are considered to be poorly adapted for long-term survival, or proliferation, outside their primary hosts (24), there is increasing evidence that this view needs to be reconsidered with respect to E. coli (17, 38). In particular, questions remain about its fate and survival capacity in environmental matrices, such as soil. While the habitat within the primary host is characterized by constant warm temperature conditions and a ready availability of nutrients and carbon, that of soil is often characterized by oligotrophic and highly dynamic conditions, temperature and pH variation, predatory populations, and competition with environmentally adapted indigenous microflora (39). Soils are thus typically considered to be suboptimal environments for enteric organisms, and growth is thought to be negligible, with die-off of organisms at rates reported to be a function of the interaction of numerous factors, including the type and physiological state of the microorganism, the physical, chemical, and biological properties of the soil, atmospheric conditions (including sunlight, moisture, and temperature), and organism application method (10).In recent years, the growth of E. coli in soils, sediments, and water in tropical and subtropical regions has been widely documented, and the organism is considered to be an established part of the soil biota within these regions (4, 5, 7, 12, 14, 19, 25, 32). The integration of E. coli as a component of the indigenous microflora in soils of tropical and subtropical regions may be attributable to the nutrient-rich nature and warm temperatures of these habitats (21, 39), combined with the metabolic versatility of the organism and its simple nutritional requirements (21). In addition to tropical and subtropical regions, the presence of autochthonous E. coli populations in the cooler soils of temperate and northern temperate regions has also been reported (6, 20, 22, 37), with one report on an alpine soil (34) and, most recently, a report on a maritime temperate grassland soil (3). The growth of E. coli within soils can act as a reservoir for the further contamination of bodies of water (20, 31, 32), compromising the indicator status of E. coli within these regions. As such, an understanding of the ecological characteristics of E. coli in soil is critical to its validation as an indicator organism. With respect to the input of pathogenic E. coli into the environment, this knowledge becomes essential for assessing the potential health risk to human and animal hosts from agricultural activities such as landspreading of manures and slurries (24).It has been suggested that E. coli can sustain autochthonous populations within soils in temperate regions, wherever favorable conditions exist (21). The phenotypic traits of the organism (including its metabolic diversity and its ability to grow both aerobically and anaerobically in a broad temperature range) may assist the persistence, colonization, and growth of E. coli when conditions permit. The challenging nature of the soil environment and the disparity of conditions between the primary host and the secondary habitat raises the question of how these E. coli populations survive and compete for niche space among the highly competitive and diverse coexisting populations of the indigenous microflora (15, 21). There is some evidence that naturalized E. coli may form genetically distinct populations in the environment (17, 20, 34, 36). This suggests that autochthonous E. coli populations in soil may have increased environmental fitness, facilitating their residence in soil (20, 34, 38). Little is known, however, of the physiology of these organisms, and their capacity for survival in soil remains poorly understood (21).Previous work (3) recorded continuous low-level leaching of viable E. coli from lysimeters of a poorly drained Luvic Stagnosol soil type, more than 9 years after the last application of fecal material. This finding was indicative of the growth of E. coli within the soil and suggested the presence of autochthonous E. coli populations within the soil that could be leached subsequently. To our knowledge, prior to this report, naturalized autochthonous E. coli populations persisting under the relatively oligotrophic, low-temperature conditions of maritime temperate soil environments had not been described previously. Growth within this soil was attributed chiefly to favorable characteristics of the soil, which include high clay and moisture contents, nutrient retention, and the presence of anaerobic zones. The objective of this work was to characterize, by molecular and physiological means, the genetic diversity and physiology of environmentally persistent E. coli isolates leached. In particular, we were interested in determining if the isolates possessed phenotypic characteristics that may enhance their capacity to survive and occupy niche space within the soil. This study tested the hypothesis that E. coli clones persisting in lysimeters of this soil form a genetically distinct grouping and possess a physiology tailored to the soil environment.     

Assessment of Bias Associated with Incomplete Extraction of Microbial DNA from Soil

DNA extraction bias is a frequently cited but poorly understood limitation of molecular characterizations of environmental microbial communities. To assess the bias of a commonly used soil DNA extraction kit, we varied the cell lysis protocol and conducted multiple extractions on subsamples of clay, sand, and organic soils. DNA, as well as bacterial and fungal ribosomal gene copies as measured by quantitative PCR, continued to be isolated in successive extractions. When terminal restriction fragment length polymorphism was used, a significant shift in community composition due to extraction bias was detected for bacteria but not for fungi. Pyrosequencing indicated that the relative abundances of sequences from rarely cultivated groups such as Acidobacteria, Gemmatimonades, and Verrucomicrobia were higher in the first extraction than in the sixth but that the reverse was true for Proteobacteria and Actinobacteria. This suggests that the well-known phylum-level bacterial cultivation bias may be partially exaggerated by DNA extraction bias. We conclude that bias can be adequately reduced in many situations by pooling three successive extractions, and additional measures should be considered when divergent soil types are compared or when comprehensive community analysis is necessary.The vast majority of soil bacteria (1, 7, 27) and fungi (13, 29) cannot be cultured via traditional laboratory techniques and must be identified using molecular methods. Successful characterization of microbial communities is therefore often dependent on DNA that is extracted from the environment. However, extraction of high-quality DNA from soil can be problematic (8, 11, 22, 26). Commercial DNA extraction kits are now commonly used in the assessment of taxonomic and functional diversity, community composition, and population abundance (e.g., references 19, 21, 23, 25, and 31). Studies comparing various kits (18, 32) or comparing commercial kits to other methods (2, 10, 24) have shown that DNA yield and purity vary depending on methodology and soil type. While these comparative studies are valuable, it is still unclear to what extent these protocols yield genomic DNA representative of the microbial community found within soil.Our objective in this study was to optimize and assess the bias of a widely used commercial soil DNA extraction kit. We hypothesized that cell lysis would be enhanced and DNA would be removed from adsorption sites by conducting multiple extractions on a single sample, thereby increasing genomic DNA yield and obtaining a more complete survey of microbial taxa. This hypothesis was tested by (i) varying the extraction protocol and measuring DNA yield for three soils with differing characteristics and (ii) examining extraction bias in the genomic DNA obtained from successive extractions by using an improved method. Analytical replicates rather than biological replicates were used in order to focus strictly on variation and bias introduced through methodology, although multiple soil types were analyzed to determine whether biases detected were consistent.     

The Prevalence of Multidrug Resistance Is Higher among Bovine than Human Salmonella enterica Serotype Newport,Typhimurium, and 4,5,12:i:? Isolates in the United States but Differs by Serotype and Geographic Region

Salmonella represents an important zoonotic pathogen worldwide, but the transmission dynamics between humans and animals as well as within animal populations are incompletely understood. We characterized Salmonella isolates from cattle and humans in two geographic regions of the United States, the Pacific Northwest and the Northeast, using three common subtyping methods (pulsed-field gel electrophoresis [PFGE], multilocus variable number of tandem repeat analysis [MLVA], and multilocus sequence typing [MLST]). In addition, we analyzed the distribution of antimicrobial resistance among human and cattle Salmonella isolates from the two study areas and characterized Salmonella persistence on individual dairy farms. For both Salmonella enterica subsp. enterica serotypes Newport and Typhimurium, we found multidrug resistance to be significantly associated with bovine origin of isolates, with the odds of multidrug resistance for Newport isolates from cattle approximately 18 times higher than for Newport isolates from humans. Isolates from the Northwest were significantly more likely to be multidrug resistant than those from the Northeast, and susceptible and resistant isolates appeared to represent distinct Salmonella subtypes. We detected evidence for strain diversification during Salmonella persistence on farms, which included changes in antimicrobial resistance as well as genetic changes manifested in PFGE and MLVA pattern shifts. While discriminatory power was serotype dependent, the combination of PFGE data with either MLVA or resistance typing data consistently allowed for improved subtype discrimination. Our results are consistent with the idea that cattle are an important reservoir of multidrug-resistant Salmonella infections in humans. In addition, the study provides evidence for the value of including antimicrobial resistance data in epidemiological investigations and highlights the benefits and potential problems of combining subtyping methods.Salmonella is an important human and animal pathogen worldwide. In the United States, Salmonella causes an estimated 1.4 million human cases, 15,000 hospitalizations, and more than 400 deaths each year (44, 75). Human infections can be acquired through contact with animals or humans shedding Salmonella or through contaminated environments, but the majority of human infections are food-borne, and a large number of human outbreaks have been linked to foods of animal origin (20). Beef represents one well-recognized source of human infection (71). In addition, a number of human cases have been linked to dairy products or cattle contact, for instance at state fairs or on dairy farms (for example, see references 25, 35, and 61).Salmonella enterica subsp. enterica serotypes Typhimurium and Newport are commonly isolated from human cases, including those linked to cattle (20, 61). In 2006, Salmonella serotypes Typhimurium and Newport were isolated from 17 and 8% of reported human salmonellosis cases in the United States, respectively, making them the first and third most common human disease-associated serotypes in the United States (15). S. enterica serotype 4,5,12:i:− is both genetically and antigenically closely related to Salmonella serotype Typhimurium, of which it represents a monophasic variant (62). Salmonella enterica serotype 4,5,12:i:− is characterized by a deletion of flagellar genes fliA and fliB, which prevents expression of the phase 2 flagellar antigen (60). In the United States, the prevalence of Salmonella serotype 4,5,12:i:− has increased considerably over the past 10 years, and in 2006, Salmonella serotype 4,5,12:i:− represented the sixth most commonly isolated serotype from humans in the United States (15, 60).Salmonella serotype Newport represents two distinct clonal groups or lineages—one predominantly associated with isolates from cattle (i.e., Newport lineage A) and one associated with isolates from birds (i.e., Newport lineage B) (1, 33). Members of both lineages cause human infections (1, 33). The two Newport lineages can be clearly distinguished by multilocus sequence typing (MLST) and pulsed-field gel electrophoresis (PFGE), and some correlation between genetic lineage and antimicrobial resistance profile seems to exist (1, 33). In general, Newport lineage B isolates are pansusceptible or resistant to only a few antimicrobial drugs. In contrast, lineage A is strongly associated with multidrug resistance and includes a Newport subtype commonly referred to as Newport MDR-AmpC (1, 33).The prevalence of antimicrobial resistance among Salmonella serotype Newport and Typhimurium isolates has increased worldwide during the last 2 decades, predominantly as a result of emerging multidrug-resistant (MDR) strains (14, 52, 65). During the 1990s, Salmonella serotype Typhimurium phage type DT104 with pentaresistance to ampicillin, chloramphenicol, streptomycin, sulfonamides, and tetracycline (ACSSuT) increased considerably in prevalence around the world, and some isolates acquired resistance to additional antimicrobial agents, including trimethoprim or ciprofloxacin (52). MDR Salmonella serotype Typhimurium DT104 has been isolated from a wide variety of host species and caused numerous large human outbreaks around the world (65). Salmonella serotype Newport MDR-AmpC, characterized by resistance to ACSSuT and carrying a plasmid encoding resistance to amoxicillin-clavulanic acid, cefoxitin, ceftiofur, and cephalothin emerged in the United States during the late 1990s, where it quickly became widespread among humans and cattle, leading to several large human outbreaks (14).Whether antimicrobial drug use in animals facilitates the emergence of MDR human pathogens is still subject to debate. Some studies report a temporal association between the introduction of new antimicrobial agents in veterinary medicine and the emergence of antimicrobial resistance (for instance, see references 22 and 58), but questions regarding the underlying evolutionary mechanisms, the origin and distribution of naturally occurring resistance genes, and the role of antimicrobial usage among humans remain (for example, see references 2 and 66 for reviews on this topic). Moreover, some studies report a higher prevalence of antimicrobial resistance among Salmonella isolates from farm animals than humans. Gebreyes et al. (26), for instance, found a higher prevalence of antimicrobial resistance among Salmonella isolates from pigs than humans, but potential effects attributable to differences in serotype distribution are difficult to assess in this study. In recent years, risk factors for MDR have received considerable attention. Infections with MDR Salmonella strains can lead to treatment failures, may be of longer duration, and may result in more severe clinical disease. Hence, such infections lead more often to hospitalization or death than infections with susceptible Salmonella strains, but serotype or subtype differences between resistant and susceptible Salmonella strains complicate the interpretation of clinical data (34, 41, 68).Subtyping methods allow characterization of Salmonella isolates and include phenotypic methods (e.g., serotyping or phage typing) as well as molecular subtyping methods, such as pulsed-field gel electrophoresis (PFGE), ribotyping, multilocus variable number of tandem repeat analysis (MLVA), and multilocus sequence typing (MLST) (5). PFGE is widely used and robust, and rigorous standardization allows comparison between laboratories (5). However, the method is time-intensive and laborious, requires careful standardization and analysis, does not allow phylogenetic inference, and can in rare cases be affected by endogenous nucleases or DNA methylation (for a review of this topic, see reference 5). MLVA and MLST are rapid, allow for easy data exchange between laboratories, and provide some phylogenetic information (5). MLVA is highly discriminatory but subject to rapid diversification and therefore most appropriate for the analysis of closely related isolates. While MLST lacks discriminatory power within Salmonella serotypes, it is highly reproducible and allows for phylogenetic analysis of more distantly related isolates (1, 5, 33). PFGE and MLST can be performed regardless of serotype, but MLVA protocols are serotype specific and have so far only been validated for a limited number of Salmonella serotypes. Moreover, MLVA can be complicated by inaccurate sizing of DNA fragments, and the degree of reliability can be considerably influenced by nucleotide composition and fragment length (5). Overall, these subtyping methods differ considerably in discriminatory power and sometimes yield conflicting results, and the most appropriate subtyping method or combination thereof strongly depends on serotype and chosen application (19, 56, 72, 76). Other genetic or phenotypic characteristics, such as antimicrobial resistance patterns or the presence of specific plasmids, have also been used successfully for subtyping in outbreak investigations and other epidemiological studies and can provide valuable additional information (7, 8, 40, 63, 64).Here we describe the distribution and subtype diversity of Salmonella serotypes Newport, 4,5,12:i:−, and Typhimurium among cattle and humans in two geographic regions of the United States, and we assess common risk factors for multidrug resistance. In addition, we utilize three Salmonella subtyping methods (PFGE, MLVA, and MLST), analyze their usefulness for characterizing isolates representing three common human-associated Salmonella serotypes, and compare the combined discriminatory power of PFGE and MLVA to that of PFGE and antimicrobial resistance patterns.     

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.     

Percolation and Survival of Escherichia coli O157:H7 and Salmonella enterica Serovar Typhimurium in Soil Amended with Contaminated Dairy Manure or Slurry

The effect of cattle manure and slurry application on percolation and survival of Escherichia coli O157:H7 and Salmonella enterica serovar Typhimurium was investigated for different soil depths after the addition of water. Four treatments were chosen for the first set of experiments: (i) addition of inoculated farmyard manure on the soil surface, (ii) mixing of inoculated farmyard manure with the top 10 cm of soil, (iii) addition of inoculated slurry on the soil surface, and (iv) injection of inoculated slurry into the top 10 cm of the soil. Homogeneity of water distribution in the soil profile was confirmed by a nondestructive nuclear magnetic resonance method. Survival data were fitted to a modified logistic model, and estimated survival times were compared. In the second set of experiments, pathogen-inoculated farmyard manure or slurry was applied to soil columns with 1-month-old lettuce plants. More pathogen cells percolated to greater depths after slurry than after manure application. Survival of E. coli O157:H7 was significantly longer in soil with slurry than in that with manure, while survival of Salmonella serovar Typhimurium was equally high with manure and slurry. The densities of the pathogens were not different in the rhizosphere compared to the bulk soil with manure, while the densities were higher by 0.88 ± 0.11 and 0.71 ± 0.23 log CFU per g (dry weight), respectively, in the rhizosphere than in bulk soil after slurry application. Our results suggest that surface application of manure may decrease the risk of contamination of groundwater and lettuce roots compared to injection of slurry.In the last 10 years food-borne disease outbreaks have increasingly been associated with the consumption of fresh vegetables and fruits contaminated with human pathogenic bacteria (3, 31). A significant number of the outbreaks were attributed to Escherichia coli O157:H7 and Salmonella enterica serovar Typhimurium. Bovine manure and slurry are the main environmental sources of these pathogens, with average concentrations between 10³ and 10⁴ CFU per g (dry weight) (gdw) of manure or slurry (24), but the density can be as high as 10⁷ CFU gdw⁻¹ of manure (10).Utilization of organic manures such as farmyard manure and slurry is the most economic and practical option for improving soil quality while providing as well an additional source of nutrients for growing plants. This is especially true for organic farms, where synthetic fertilizers cannot be used. Both organic and conventional soils can be fertilized with liquid slurry and/or farmyard manure. However, farmyard manure is more frequently used at organic farms.The survival of E. coli O157:H7 and Salmonella serovar Typhimurium is thought to be better in slurry than in farmyard manure (24; also A. V. Semenov, L. van Overbeek, N. Hidayah, A. J. Termorshuizen, and A. H. C. van Bruggen, submitted for publication) but is also dependent on the way manure or slurry is applied to agricultural fields (24). Survival of the pathogens may range from several days (turned composted manure) to more than a year (nonaerated manure) (9, 19). This broad difference in survival times is caused by various abiotic factors such as temperature (30, 38), presence of oxygen (A. V. Semenov, et al., submitted), and chemical composition (5) as well as by biological factors (e.g., microbial community composition) (5, 16, 30). The presence of plant roots is often neglected in controlled experiments although root exudates may support survival of human pathogens by providing a supply of easily available nutrients (18). Moreover, it has been shown that E. coli O157:H7 and Salmonella serovar Typhimurium may become associated with the surface of plants growing in soil amended with contaminated manure (15, 23) and may even be internalized by the plants (7, 18, 20, 32).When microorganisms are introduced on or in soil, their movement is mainly determined by the flow of percolating water (13). Water flow and the ultimate distribution of bacteria in soil are affected by soil texture, pH, temperature, and the structure of the root system in soil (17). Like other bacteria, E. coli O157:H7 and Salmonella serovar Typhimurium are able to move through the soil profile with water after rainfall or irrigation and can even reach the groundwater (2, 21). In field experiments, 20% of E. coli cells applied with contaminated slurry to the field were found in drain water (37). This water can contaminate plants when it is used for irrigation. Since E. coli O157:H7 can survive in well water up to 65 days (1), there is a high risk that private water supplies could be contaminated with enteric pathogens.Laboratory transport studies can mimic bacterial transport in field conditions only to a certain extent. The natural heterogeneity in field soil leads to the appearance of cracks and macropores through which water flow may occur while relatively homogeneous soil is commonly used in laboratory experiments. This may lead to underestimations of the movement of enteropathogens through the homogenized and possibly compacted soil. On the other hand, the presence of artificial boundaries (the so-called “wall effect”) and unexpected cracks may lead to overestimations of the movement of water and bacteria through the soil in mesocosms. The wall effect can be minimized by inserting sandpaper against the inner wall of soil columns while cracks can be minimized by careful packing of the soil. Nuclear magnetic resonance (NMR) can be used to check the homogeneity of water distribution in a soil column. NMR is a nondestructive and noninvasive spectroscopic method to measure static and dynamic water behavior in heterogeneous substrates (35). The data received from magnetic resonance images can give information about the spin density and spin relaxation values that reflect the interaction of water with the soil. These measurements have been proven to be highly correlated with water content in soils (35).While it was shown that water is the most important dispersal factor for percolation of bacteria in different types of soil (13, 36) as well as for percolation of enteropathogens under various management practices (11), the movement and distribution of E. coli O157:H7 and Salmonella serovar Typhimurium in soil after application of manure and slurry are still unclear. It is also not clear if and how survival of enteric pathogens is influenced by the depth of the soil where they end up after transport through the soil.The objectives of our study were the following: (i) to determine the extent of percolation of water and E. coli O157:H7 and Salmonella serovar Typhimurium from contaminated manure or slurry through a soil column, (ii) to determine the influence of application methods of manure and slurry on percolation and survival of these pathogens at different depths in a soil column, and (iii) to determine the influence of plant roots on percolation and survival of the pathogens, applied with manure or slurry, at different depths in bulk soil and the rhizosphere.     

Flagellated but Not Hyperfimbriated Salmonella enterica Serovar Typhimurium Attaches to and Forms Biofilms on Cholesterol-Coated Surfaces

The asymptomatic, chronic carrier state of Salmonella enterica serovar Typhi occurs in the bile-rich gallbladder and is frequently associated with the presence of cholesterol gallstones. We have previously demonstrated that salmonellae form biofilms on human gallstones and cholesterol-coated surfaces in vitro and that bile-induced biofilm formation on cholesterol gallstones promotes gallbladder colonization and maintenance of the carrier state. Random transposon mutants of S. enterica serovar Typhimurium were screened for impaired adherence to and biofilm formation on cholesterol-coated Eppendorf tubes but not on glass and plastic surfaces. We identified 49 mutants with this phenotype. The results indicate that genes involved in flagellum biosynthesis and structure primarily mediated attachment to cholesterol. Subsequent analysis suggested that the presence of the flagellar filament enhanced binding and biofilm formation in the presence of bile, while flagellar motility and expression of type 1 fimbriae were unimportant. Purified Salmonella flagellar proteins used in a modified enzyme-linked immunosorbent assay (ELISA) showed that FliC was the critical subunit mediating binding to cholesterol. These studies provide a better understanding of early events during biofilm development, specifically how salmonellae bind to cholesterol, and suggest a target for therapies that may alleviate biofilm formation on cholesterol gallstones and the chronic carrier state.The serovars of Salmonella enterica are diverse, infect a broad array of hosts, and cause significant morbidity and mortality in impoverished and industrialized nations worldwide. S. enterica serovar Typhi is the etiologic agent of typhoid fever, a severe illness characterized by sustained bacteremia and a delayed onset of symptoms that afflicts approximately 20 million people each year (14, 19). Serovar Typhi can establish a chronic infection of the human gallbladder, suggesting that this bacterium utilizes novel mechanisms to mediate enhanced colonization and persistence in a bile-rich environment.There is a strong correlation between gallbladder abnormalities, particularly gallstones, and development of the asymptomatic Salmonella carrier state (47). Antibiotic regimens are typically ineffective in carriers with gallstones (47), and these patients have an 8.47-fold-higher risk of developing hepatobiliary carcinomas (28, 46, 91). Elimination of chronic infections usually requires gallbladder removal (47), but surgical intervention is cost-prohibitive in developing countries where serovar Typhi is prevalent. Thus, understanding the progression of infection to the carrier state and developing alternative treatment options are of critical importance to human health.The formation of biofilms on gallstones has been hypothesized to facilitate enhanced colonization of and persistence in the gallbladder. Over the past 2 decades, bacterial biofilms have been increasingly implicated as burdens for food and public safety worldwide, and they are broadly defined as heterogeneous communities of microorganisms that adhere to each other and to inert or live surfaces (17, 22, 67, 89, 102). A sessile environment provides selective advantages in natural, medical, and industrial ecosystems for diverse species of commensal and pathogenic bacteria, including Streptococcus mutans (40, 92, 104), Staphylococcus aureus (15, 35, 100), Escherichia coli (21, 74), Vibrio cholerae (39, 52, 107), and Pseudomonas aeruginosa (23, 58, 73, 105). Bacterial biofilms are increasingly associated with many chronic infections in humans and exhibit heightened resistance to commonly administered antibiotics and to engulfment by professional phagocytes (54, 55, 59). The bacterial gene expression profiles for planktonic and biofilm phenotypes differ (42, 90), and the changes are likely regulated by external stimuli, including nutrient availability, the presence of antimicrobials, and the composition of the binding substrate.Biofilm formation occurs in sequential, highly ordered stages and begins with attachment of free-swimming, planktonic bacteria to a surface. Subsequent biofilm maturation is characterized by the production of a self-initiated extracellular matrix (ECM) composed of nucleic acid, proteins, or exopolysaccharides (EPS) that encase the community of microorganisms. Planktonic cells are continuously shed from the sessile, matrix-bound population, which can result in reattachment and fortification of the biofilm or systemic infection and release of the organism into the environment. Shedding of serovar Typhi by asymptomatic carriers can contaminate food and water and account for much of the person-to-person transmission in underdeveloped countries.Our laboratory has previously reported that bile is required for formation of mature biofilms with characteristic EPS production by S. enterica serovars Typhimurium, Enteritidis, and Typhi on human gallstones and cholesterol-coated Eppendorf tubes (18, 78). Cholesterol is the primary constituent of human cholesterol gallstones, and use of cholesterol-coated tubes creates an in vitro uniform surface that mimics human gallstones (18). It was also demonstrated that Salmonella biofilms that formed on different surfaces had unique phenotypes and required expression of specific EPS (18, 77), yet the factors mediating Salmonella binding to gallstones and cholesterol-coated surfaces during the initiation of biofilm formation remain unknown. Here, we show that the presence of serovar Typhimurium flagella promotes binding specifically to cholesterol in the early stages of biofilm development and that the FliC subunit is a critical component. Bound salmonellae expressing intact flagella provided a scaffold for other cells to bind to during later stages of biofilm growth. Elucidation of key mechanisms that mediate adherence to cholesterol during Salmonella bile-induced biofilm formation on gallstone surfaces promises to reveal novel drug targets for alleviating biofilm formation in chronic cases.     

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).