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.     

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.     

African 1, an Epidemiologically Important Clonal Complex of Mycobacterium bovis Dominant in Mali,Nigeria, Cameroon,and Chad

We have identified a clonal complex of Mycobacterium bovis present at high frequency in cattle in population samples from several sub-Saharan west-central African countries. This closely related group of bacteria is defined by a specific chromosomal deletion (RDAf1) and can be identified by the absence of spacer 30 in the standard spoligotype typing scheme. We have named this group of strains the African 1 (Af1) clonal complex and have defined the spoligotype signature of this clonal complex as being the same as the M. bovis BCG vaccine strain but with the deletion of spacer 30. Strains of the Af1 clonal complex were found at high frequency in population samples of M. bovis from cattle in Mali, Cameroon, Nigeria, and Chad, and using a combination of variable-number tandem repeat typing and spoligotyping, we show that the population of M. bovis in each of these countries is distinct, suggesting that the recent mixing of strains between countries is not common in this area of Africa. Strains with the Af1-specific deletion (RDAf1) were not identified in M. bovis isolates from Algeria, Burundi, Ethiopia, Madagascar, Mozambique, South Africa, Tanzania, and Uganda. Furthermore, the spoligotype signature of the Af1 clonal complex has not been identified in population samples of bovine tuberculosis from Europe, Iran, and South America. These observations suggest that the Af1 clonal complex is geographically localized, albeit to several African countries, and we suggest that the dominance of the clonal complex in this region is the result of an original introduction into cows naïve to bovine tuberculosis.Mycobacterium bovis causes bovine tuberculosis (TB), an important disease of domesticated cattle that has a major economic and health impact throughout the world (61, 64, 65). The pathogen is a member of the Mycobacterium tuberculosis complex, which includes many species and subspecies that cause similar pathologies in a variety of mammalian hosts. The most notable member of the complex is M. tuberculosis, the most important bacterial pathogen of humans. In contrast to M. tuberculosis, which is largely host restricted to humans, M. bovis is primarily maintained in bovids, in particular, domesticated cattle, although the pathogen can frequently be recovered from other mammals, including humans (61). Bovine TB is found in cattle throughout the world and has been reported on every continent where cattle are farmed (3).Bovine TB has been reduced or eliminated from domestic cattle in many developed countries by the application of a test-and-cull policy that removes infected cattle (3, 8, 16, 17, 61, 64, 65). However, in Africa, although bovine TB is known to be common in both cattle and wildlife, control policies have not been enforced in many countries due to cost implications, lack of capacity, and infrastructure limitations (8, 16, 17, 57). In 1998, Cosivi et al. reported of bovine TB, “Of all nations in Africa, only seven apply disease control measures as part of a test-and-slaughter policy and consider bovine TB a notifiable disease; the remaining 48 control the disease inadequately or not at all” (16). In the intervening years, the situation is not thought to have improved (8); however, preliminary surveys of bovine TB have been carried out in some African countries (4, 7, 12, 37, 44, 49, 53, 54, 56).The most common epidemiological molecular-typing method applied to strains of M. bovis is spoligotyping. This method identifies polymorphism in the presence of spacer units in the direct-repeat (DR) region in strains of the M. tuberculosis complex (36, 67). The DR is composed of multiple, virtually identical 36-bp regions interspersed with unique DNA spacer sequences of similar size (direct variant repeat [DVR] units). Spacer sequences are unique to the DR region, and copies are not located elsewhere in the chromosome (68). The DR region may contain over 60 DVR units; however, 43 of the spacer units were selected from the spacer sequences of the M. tuberculosis reference strain H37Rv and M. bovis BCG strain P3 and are used in the standard application of spoligotyping to strains of the M. tuberculosis complex (29, 36). The DR region is polymorphic because of the loss (deletion) of single or multiple spacers, and each spoligotype pattern from strains of M. bovis is given an identifier (http://www.Mbovis.org).Several studies of the DR regions in closely related strains of M. tuberculosis have concluded that the evolutionary trend for this region is primarily loss of single DVRs or multiple contiguous DVRs (22, 29, 68); duplication of DVR units or point mutations in spacer sequences were found to be rare. The loss of discrete units observed by Groenen et al. (29) led them to suggest that the mechanism for spacer loss was homologous recombination between repeat units. However, a study by Warren et al. (69) suggested that for strains of M. tuberculosis, insertion of IS6110 sequences into the DR region and recombination between adjacent IS6110 elements were more important mechanisms for the loss of spacer units.The population structure of the M. tuberculosis group of organisms is apparently highly clonal, without any transfer and recombination of chromosomal sequences between strains (15, 30, 60, 61). In a strictly clonal population, the loss by deletion of unique chromosomal DNA cannot be replaced by recombination from another strain, and the deleted region will act as a molecular marker for the strain and all its descendants. Deletions of specific chromosomal regions (regions of difference [RDs] or large sequence polymorphisms) have been very successful at identifying phylogenetic relationships in the M. tuberculosis complex (11, 25, 26, 35, 48, 50, 61, 62, 66). However, because the loss of spoligotype spacer sequences is so frequent, identical spoligotype patterns can occur independently in unrelated lineages (homoplasy), and therefore, the deletion of spoligotype spacers may be an unreliable indicator of phylogenetic relationship (61, 69).In samples of M. bovis strains from Cameroon, Nigeria, Chad, and Mali, spoligotyping was used to show that many of the strains had similar spoligotype patterns that lacked spacer 30, and it has been suggested that strains from these four countries are phylogenetically related (12, 18, 49, 53). We have extended the previous observations of spoligotype similarities between strains from these countries and confirmed the existence of a unique clonal complex of M. bovis, all descended from a single strain in which a specific deletion of chromosomal DNA occurred. We have named this clonal complex of M. bovis strains African 1 (Af1), and we show that this clonal complex is dominant in these four west-central African countries but rare in eastern and southern Africa. Extended genotyping, using variable-number tandem repeats (VNTR), of strains with the most common spoligotype patterns suggests that each of these four west-central African countries has a unique population structure. Evolutionary scenarios that may have led to the present day distribution of the Af1 clonal complex are discussed.     

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.     

Expression of a Synthesized Gene Encoding Cationic Peptide Cecropin B in Transgenic Tomato Plants Protects against Bacterial Diseases

The cationic lytic peptide cecropin B (CB), isolated from the giant silk moth (Hyalophora cecropia), has been shown to effectively eliminate Gram-negative and some Gram-positive bacteria. In this study, the effects of chemically synthesized CB on plant pathogens were investigated. The S₅₀s (the peptide concentrations causing 50% survival of a pathogenic bacterium) of CB against two major pathogens of the tomato, Ralstonia solanacearum and Xanthomonas campestris pv. vesicatoria, were 529.6 μg/ml and 0.29 μg/ml, respectively. The CB gene was then fused to the secretory signal peptide (sp) sequence from the barley α-amylase gene, and the new construct, pBI121-spCB, was used for the transformation of tomato plants. Integration of the CB gene into the tomato genome was confirmed by PCR, and its expression was confirmed by Western blot analyses. In vivo studies of the transgenic tomato plant demonstrated significant resistance to bacterial wilt and bacterial spot. The levels of CB expressed in transgenic tomato plants (∼0.05 μg in 50 mg of leaves) were far lower than the S₅₀ determined in vitro. CB transgenic tomatoes could therefore be a new mode of bioprotection against these two plant diseases with significant agricultural applications.Bacterial plant diseases are a source of great losses in the annual yields of most crops (5). The agrochemical methods and conventional breeding commonly used to control these bacterially induced diseases have many drawbacks. Indiscriminate use of agrochemicals has a negative impact on human, as well as animal, health and contributes to environmental pollution. Conventional plant-breeding strategies have limited scope due to the paucity of genes with these traits in the usable gene pools and their time-consuming nature. Consequently, genetic engineering and transformation technology offer better tools to test the efficacies of genes for crop improvement and to provide a better understanding of their mechanisms. One advance is the possibility of creating transgenic plants that overexpress recombinant DNA or novel genes with resistance to pathogens (36). In particular, strengthening the biological defenses of a crop by the production of antibacterial proteins with other origins (not from plants) offers a novel strategy to increase the resistance of crops to diseases (35, 39, 41). These antimicrobial peptides (AMPs) include such peptides as cecropins (2, 15, 20, 23-24, 27, 31, 42, 50), magainins (1, 9, 14, 29, 47), sarcotoxin IA (35, 40), and tachyplesin I (3). The genes encoding these small AMPs in plants have been used in practice to enhance their resistance to bacterial and fungal pathogens (8, 22, 40). The expression of AMPs in vivo (mostly cecropins and a synthetic analog of cecropin and magainin) with either specific or broad-spectrum disease resistance in tobacco (14, 24, 27), potato (17, 42), rice (46), banana (9), and hybrid poplar (32) have been reported. The transgenic plants showed considerably greater resistance to certain pathogens than the wild types (4, 13, 24, 27, 42, 46, 50). However, detailed studies of transgenic tomatoes expressing natural cecropin have not yet been reported.The tomato (Solanum lycopersicum) is one of the most commonly consumed vegetables worldwide. The annual yield of tomatoes, however, is severely affected by two common bacterial diseases, bacterial wilt and bacterial spot, which are caused by infection with the Gram-negative bacteria Ralstonia solanacearum and Xanthomonas campestris pv. vesicatoria, respectively. Currently available pesticides are ineffective against R. solanacearum, and thus bacterial wilt is a serious problem.Cecropins, one of the natural lytic peptides found in the giant silk moth, Hyalophora cecropia (25), are synthesized in lipid bodies as proteins consisting of 31 to 39 amino acid residues. They adopt an α-helical structure on interaction with bacterial membranes, resulting in the formation of ion channels (12). At low concentrations (0.1 μM to 5 μM), cecropins exhibit lytic antibacterial activity against a number of Gram-negative and some Gram-positive bacteria, but not against eukaryotic cells (11, 26, 33), thus making them potentially powerful tools for engineering bacterial resistance in crops. Moreover, cecropin B (CB) shows the strongest activity against Gram-negative bacteria within the cecropin family and therefore has been considered an excellent candidate for transformation into plants to improve their resistance against bacterial diseases.The introduction of genes encoding cecropins and their analogs into tobacco has been reported to have contradictory results regarding resistance against pathogens (20). However, subsequent investigations of these tobacco plants showed that the expression of CB in the plants did not result in accumulation of detectable levels of CB, presumably due to degradation of the peptide by host peptidases (20, 34). Therefore, protection of CB from cellular degradation is considered to be vital for the exploitation of its antibacterial activity in transgenic plants. The secretory sequences of several genes are helpful, because they cooperate with the desired genes to enhance extracellular secretion (24, 40, 46). In the present study, a natural CB gene was successfully transferred into tomatoes. The transgenic plants showed significant resistance to the tomato diseases bacterial wilt and bacterial spot, as well as with a chemically synthesized CB peptide.     

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.     

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.     

A Bacillus anthracis S-Layer Homology Protein That Binds Heme and Mediates Heme Delivery to IsdC

The sequestration of iron by mammalian hosts represents a significant obstacle to the establishment of a bacterial infection. In response, pathogenic bacteria have evolved mechanisms to acquire iron from host heme. Bacillus anthracis, the causative agent of anthrax, utilizes secreted hemophores to scavenge heme from host hemoglobin, thereby facilitating iron acquisition from extracellular heme pools and delivery to iron-regulated surface determinant (Isd) proteins covalently attached to the cell wall. However, several Gram-positive pathogens, including B. anthracis, contain genes that encode near iron transporter (NEAT) proteins that are genomically distant from the genetically linked Isd locus. NEAT domains are protein modules that partake in several functions related to heme transport, including binding heme and hemoglobin. This finding raises interesting questions concerning the relative role of these NEAT proteins, relative to hemophores and the Isd system, in iron uptake. Here, we present evidence that a B. anthracis S-layer homology (SLH) protein harboring a NEAT domain binds and directionally transfers heme to the Isd system via the cell wall protein IsdC. This finding suggests that the Isd system can receive heme from multiple inputs and may reflect an adaptation of B. anthracis to changing iron reservoirs during an infection. Understanding the mechanism of heme uptake in pathogenic bacteria is important for the development of novel therapeutics to prevent and treat bacterial infections.Pathogenic bacteria need to acquire iron to survive in mammalian hosts (12). However, the host sequesters most iron in the porphyrin heme, and heme itself is often bound to proteins such as hemoglobin (14, 28, 85). Circulating hemoglobin can serve as a source of heme-iron for replicating bacteria in infected hosts, but the precise mechanisms of heme extraction, transport, and assimilation remain unclear (25, 46, 79, 86). An understanding of how bacterial pathogens import heme will lead to the development of new anti-infectives that inhibit heme uptake, thereby preventing or treating infections caused by these bacteria (47, 68).The mechanisms of transport of biological molecules into a bacterial cell are influenced by the compositional, structural, and topological makeup of the cell envelope. Gram-negative bacteria utilize specific proteins to transport heme through the outer membrane, periplasm, and inner membrane (83, 84). Instead of an outer membrane and periplasm, Gram-positive bacteria contain a thick cell wall (59, 60). Proteins covalently anchored to the cell wall provide a functional link between extracellular heme reservoirs and intracellular iron utilization pathways (46). In addition, several Gram-positive and Gram-negative bacterial genera also contain an outermost structure termed the S (surface)-layer (75). The S-layer is a crystalline array of protein that surrounds the bacterial cell and may serve a multitude of functions, including maintenance of cell architecture and protection from host immune components (6, 7, 18, 19, 56). In bacterial pathogens that manifest an S-layer, the “force field” function of this structure raises questions concerning how small molecules such as heme can be successfully passed from the extracellular milieu to cell wall proteins for delivery into the cell cytoplasm.Bacillus anthracis is a Gram-positive, spore-forming bacterium that is the etiological agent of anthrax disease (30, 33). The life cycle of B. anthracis begins after a phagocytosed spore germinates into a vegetative cell inside a mammalian host (2, 40, 69, 78). Virulence determinants produced by the vegetative cells facilitate bacterial growth, dissemination to major organ systems, and eventually host death (76-78). The release of aerosolized spores into areas with large concentrations of people is a serious public health concern (30).Heme acquisition in B. anthracis is mediated by the action of IsdX1 and IsdX2, two extracellular hemophores that extract heme from host hemoglobin and deliver the iron-porphyrin to cell wall-localized IsdC (21, 45). Both IsdX1 and IsdX2 harbor near iron transporter domains (NEATs), a conserved protein module found in Gram-positive bacteria that mediates heme uptake from hemoglobin and contributes to bacterial pathogenesis upon infection (3, 8, 21, 31, 44, 46, 49, 50, 67, 81, 86). Hypothesizing that B. anthracis may contain additional mechanisms for heme transport, we provide evidence that B. anthracis S-layer protein K (BslK), an S-layer homology (SLH) and NEAT protein (32, 43), is surface localized and binds and transfers heme to IsdC in a rapid, contact-dependent manner. These results suggest that the Isd system is not a self-contained conduit for heme trafficking and imply that there is functional cross talk between differentially localized NEAT proteins to promote heme uptake during infection.     

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).     

Genetic Diversity and Multihost Pathogenicity of Clinical and Environmental Strains of Burkholderia cenocepacia

A collection of 54 clinical and agricultural isolates of Burkholderia cenocepacia was analyzed for genetic relatedness by using multilocus sequence typing (MLST), pathogenicity by using onion and nematode infection models, antifungal activity, and the distribution of three marker genes associated with virulence. The majority of clinical isolates were obtained from cystic fibrosis (CF) patients in Michigan, and the agricultural isolates were predominantly from Michigan onion fields. MLST analysis resolved 23 distinct sequence types (STs), 11 of which were novel. Twenty-six of 27 clinical isolates from Michigan were genotyped as ST-40, previously identified as the Midwest B. cenocepacia lineage. In contrast, the 12 agricultural isolates represented eight STs, including ST-122, that were identical to clinical isolates of the PHDC lineage. In general, pathogenicity to onions and the presence of the pehA endopolygalacturonase gene were detected only in one cluster of related strains consisting of agricultural isolates and the PHDC lineage. Surprisingly, these strains were highly pathogenic in the nematode Caenorhabditis elegans infection model, killing nematodes faster than the CF pathogen Pseudomonas aeruginosa PA14 on slow-kill medium. The other strains displayed a wide range of pathogenicity to C. elegans, notably the Midwest clonal lineage which displayed high, moderate, and low virulence. Most strains displayed moderate antifungal activity, although strains with high and low activities were also detected. We conclude that pathogenicity to multiple hosts may be a key factor contributing to the potential of B. cenocepacia to opportunistically infect humans both by increasing the prevalence of the organism in the environment, thereby increasing exposure to vulnerable hosts, and by the selection of virulence factors that function in multiple hosts.The betaproteobacterium Burkholderia cenocepacia, 1 of now 17 classified species belonging to the Burkholderia cepacia complex (BCC), is ubiquitous and extremely versatile in its metabolic capabilities and interactions with other organisms (38, 40, 57, 58). Strains of B. cenocepacia are pathogens of onion and banana plants, opportunistic pathogens of humans, symbionts of numerous plant rhizospheres, contaminants of pharmaceutical and industrial products, and inhabitants of soil and surface waters (14, 29, 33, 34, 37, 45). Originally described as a pathogen of onions (8), organisms of the BCC emerged in the past 3 decades as serious human pathogens, capable of causing devastating chronic lung infections in persons with cystic fibrosis (CF) or chronic granulomatous disease (21, 24, 28). Infections due to BCC are a serious concern to CF patients due to their inherent antibiotic resistance and high potential for patient-to-patient transmission (23). Although 16 of the BCC species have been recovered from respiratory secretions of CF patients in many countries (46, 58), B. cenocepacia has been the most common species isolated in North America, detected in 50% of 606, 83% of 447, and 45.6% of 1,218 patients in recent studies (35, 46, 52).The epidemiology of infectious disease caused by B. cenocepacia appears to involve patient-to-patient spread of genetically distinct lineages. B. cenocepacia lineages, such as ET12, Midwest, and PHDC, have been identified from large numbers of individuals in disease outbreaks in North America and Europe (11, 32, 54). A recently developed multilocus sequence typing (MLST) scheme has been shown to be a reliable epidemiologic tool for differentiating between the five subgroups (IIIA to IIIE) of B. cenocepacia, and strains representing three of these subgroups (IIIA, IIIB, and IIID) have been recovered from CF patients (2). Outside of the patient-to-patient transmission of clonal lineages, the mode of acquisition of strains causing sporadic cases of B. cenocepacia in CF patients remains unclear, although environmental sources are a logical reservoir for infection. Previously, an isolate of B. cenocepacia indistinguishable from the PHDC epidemic clonal lineage by using standard typing methods (e.g., repetitive-sequence-based PCR, randomly amplified polymorphic DNA, pulsed-field gel electrophoresis) was detected in an agricultural soil sample (34). Similarly, three distinct MLST sequence types containing both clinical and environmental (plant and soil) B. cenocepacia isolates were identified (1). These findings suggest that natural populations of B. cenocepacia in soil or associated with plants are a potential reservoir for the emergence of new human pathogenic lineages.Experimental models for the study of virulence potential and traits of B. cenocepacia include mouse and rat models with genetic defects allowing chronic lung infections to be established (e.g., see reference 48). Nematode (Caenorhabditis elegans), alfalfa (Medicago sativa), and onion (Allium cepa) models have also been routinely utilized for the identification of virulence factors (5, 29, 31). C. elegans has been extensively used to study the pathogenesis and virulence factors of a wide variety of bacterial and fungal pathogens (9, 15, 42, 51, 56). In several pathogens, including Pseudomonas (56) and Burkholderia (20), putative virulence factors important for the pathogenesis in mammalian systems (15, 51) have been identified using the C. elegans model. The C. elegans model might be limited in the detection of host-specific virulence factors; however, several attributes, such as small size and rapid development, make it an excellent whole animal model for pathogenesis research (16, 51).The evidence that individual strains of B. cenocepacia can be pathogenic to both plants and humans and are prevalent in various environmental niches has provoked particular interest in elucidating the clinical pathogenic potential of environmental isolates. The basis of this study was to examine whether genetically related B. cenocepacia strains exhibit shared characteristics that contribute to their pathogenicity in multiple hosts and to examine the potential for circulating environmental isolates to emerge as new clinical pathogens. Here, we tested the degree of virulence in animal (nematode) and plant (onion) infection models, the production of antifungal activity, and the genetic relatedness of clinical and environmental B. cenocepacia subgroup IIIB strains predominantly isolated from Michigan.     

Cell-to-Cell Spread of the RNA Interference Response Suppresses Semliki Forest Virus (SFV) Infection of Mosquito Cell Cultures and Cannot Be Antagonized by SFV

In their vertebrate hosts, arboviruses such as Semliki Forest virus (SFV) (Togaviridae) generally counteract innate defenses and trigger cell death. In contrast, in mosquito cells, following an early phase of efficient virus production, a persistent infection with low levels of virus production is established. Whether arboviruses counteract RNA interference (RNAi), which provides an important antiviral defense system in mosquitoes, is an important question. Here we show that in Aedes albopictus-derived mosquito cells, SFV cannot prevent the establishment of an antiviral RNAi response or prevent the spread of protective antiviral double-stranded RNA/small interfering RNA (siRNA) from cell to cell, which can inhibit the replication of incoming virus. The expression of tombusvirus siRNA-binding protein p19 by SFV strongly enhanced virus spread between cultured cells rather than virus replication in initially infected cells. Our results indicate that the spread of the RNAi signal contributes to limiting virus dissemination.In animals, RNA interference (RNAi) was first described for Caenorhabditis elegans (27). The production or introduction of double-stranded RNA (dsRNA) in cells leads to the degradation of mRNAs containing homologous sequences by sequence-specific cleavage of mRNAs. Central to RNAi is the production of 21- to 26-nucleotide small interfering RNAs (siRNAs) from dsRNA and the assembly of an RNA-induced silencing complex (RISC), followed by the degradation of the target mRNA (23, 84). RNAi is a known antiviral strategy of plants (3, 53) and insects (21, 39, 51). Study of Drosophila melanogaster in particular has given important insights into RNAi responses against pathogenic viruses and viral RNAi inhibitors (31, 54, 83, 86, 91). RNAi is well characterized for Drosophila, and orthologs of antiviral RNAi genes have been found in Aedes and Culex spp. (13, 63).Arboviruses, or arthropod-borne viruses, are RNA viruses mainly of the families Bunyaviridae, Flaviviridae, and Togaviridae. The genus Alphavirus within the family Togaviridae contains several mosquito-borne pathogens: arboviruses such as Chikungunya virus (16) and equine encephalitis viruses (88). Replication of the prototype Sindbis virus and Semliki Forest virus (SFV) is well understood (44, 71, 74, 79). Their genome consists of a positive-stranded RNA with a 5′ cap and a 3′ poly(A) tail. The 5′ two-thirds encodes the nonstructural polyprotein P1234, which is cleaved into four replicase proteins, nsP1 to nsP4 (47, 58, 60). The structural polyprotein is encoded in the 3′ one-third of the genome and cleaved into capsid and glycoproteins after translation from a subgenomic mRNA (79). Cytoplasmic replication complexes are associated with cellular membranes (71). Viruses mature by budding at the plasma membrane (35).In nature, arboviruses are spread by arthropod vectors (predominantly mosquitoes, ticks, flies, and midges) to vertebrate hosts (87). Little is known about how arthropod cells react to arbovirus infection. In mosquito cell cultures, an acute phase with efficient virus production is generally followed by the establishment of a persistent infection with low levels of virus production (9). This is fundamentally different from the cytolytic events following arbovirus interactions with mammalian cells and pathogenic insect viruses with insect cells. Alphaviruses encode host response antagonists for mammalian cells (2, 7, 34, 38).RNAi has been described for mosquitoes (56) and, when induced before infection, antagonizes arboviruses and their replicons (1, 4, 14, 15, 29, 30, 32, 42, 64, 65). RNAi is also functional in various mosquito cell lines (1, 8, 43, 49, 52). In the absence of RNAi, alphavirus and flavivirus replication and/or dissemination is enhanced in both mosquitoes and Drosophila (14, 17, 31, 45, 72). RNAi inhibitors weakly enhance SFV replicon replication in tick and mosquito cells (5, 33), posing the questions of how, when, and where RNAi interferes with alphavirus infection in mosquito cells.Here we use an A. albopictus-derived mosquito cell line to study RNAi responses to SFV. Using reporter-based assays, we demonstrate that SFV cannot avoid or efficiently inhibit the establishment of an RNAi response. We also demonstrate that the RNAi signal can spread between mosquito cells. SFV cannot inhibit cell-to-cell spread of the RNAi signal, and spread of the virus-induced RNAi signal (dsRNA/siRNA) can inhibit the replication of incoming SFV in neighboring cells. Furthermore, we show that SFV expression of a siRNA-binding protein increases levels of virus replication mainly by enhancing virus spread between cells rather than replication in initially infected cells. Taken together, these findings suggest a novel mechanism, cell-to-cell spread of antiviral dsRNA/siRNA, by which RNAi limits SFV dissemination in mosquito cells.     

Chickens and Cattle as Sources of Sporadic Domestically Acquired Campylobacter jejuni Infections in Finland

A substantial sampling among domestic human campylobacter cases, chicken process lots, and cattle at slaughter was performed during the seasonal peak of human infections. Campylobacter jejuni isolates (n = 419) were subtyped using pulsed-field gel electrophoresis with SmaI, and isolates representing overlapping types (n = 212) were further subtyped using KpnI for restriction. The SmaI/KpnI profiles of 55.4% (97/175) of the human isolates were indistinguishable from those of the chicken or cattle isolates. The overlapping SmaI/KpnI subtypes accounted for 69.8% (30/43) and 15.9% (32/201) of the chicken and cattle isolates, respectively. The occurrence of identical SmaI/KpnI subtypes with human C. jejuni isolates was significantly associated with animal host species (P < 0.001). A temporal association of isolates from chickens and patients was possible in 31.4% (55/175) of the human infections. Besides chickens as sources of C. jejuni in the sporadic infections, the role of cattle appears notable. New approaches to restrict the occurrence of campylobacters in other farm animals may be needed in addition to hygienic measures in chicken production. However, only about half of the human infections were attributable to these sources.The incidence of human enteric infections caused by campylobacters is highest in the summer months, showing a consistent peak at the end of July in Finland (www.ktl.fi/attachments/suomi/julkaisut/julkaisusarja_b/2008/2008b09.pdf), as well as in other Nordic countries (16, 33). Almost 70% of campylobacter infections detected in July and August in Finland are domestically acquired, whereas the annual average proportion of domestic cases is about 30%, and most of them are caused by Campylobacter jejuni (30). The prevalence of campylobacters in Finnish broiler flocks peaks simultaneously with the human cases (7), and similar sero- and genotypes have been reported among human and poultry strains isolated in Finland and in other countries (5, 8, 21-23). Several epidemiological studies have identified the handling and consumption of raw or undercooked poultry meat as a major risk factor for campylobacter enteritis (for example, see references 18, 20, and 41), whereas opposite conclusions about the significance of the consumption of chicken meat were drawn from the Swedish case-control study among young children (2) and an extensive Danish register-based study (6).Data derived from the genotyping studies of C. jejuni isolates from human infections and animals support the current suggestion that poultry is the most important single source of sporadic campylobacteriosis (12, 22, 29). However, several reports on genotype comparisons suggest that poultry may be a less significant source of campylobacters than generally thought, and other animal reservoirs should also be considered notable sources of campylobacters pathogenic to humans (3, 8, 17, 27, 31). Studies of the temporal occurrence of campylobacters in human infections and poultry flocks have revealed that the peak in prevalence, as well as some of the overlapping sero- and genotypes, is detected in humans prior to being detected in poultry (21, 28).Although cattle are well-known carriers of campylobacters, the survival of these fragile organisms in beef is poor (39, 42). In recent years, some authors (1, 4, 10) have raised the question of an indirect association between cattle and human cases. In a Finnish study combining data from the multilocus sequence typing of campylobacters isolated from production animals and from epidemiological studies of human cases, significant associations emerged between certain sequence-type complexes from human infections and contact with cattle, the consumption of unpasteurized milk, or the tasting or consumption of raw minced meat (23).The aim of this study was to investigate the contributions of poultry and cattle as sources of human C. jejuni infections in Finland by comparing over a limited time frame the macrorestriction profiles obtained from pulsed-field gel electrophoresis (PFGE) analysis of a geographically representative collection of C. jejuni isolates from domestically acquired sporadic human infections, chicken process lots, and cattle.     

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.     

Roles of Minor Pilin Subunits Spy0125 and Spy0130 in the Serotype M1 Streptococcus pyogenes Strain SF370

Adhesive pili on the surface of the serotype M1 Streptococcus pyogenes strain SF370 are composed of a major backbone subunit (Spy0128) and two minor subunits (Spy0125 and Spy0130), joined covalently by a pilin polymerase (Spy0129). Previous studies using recombinant proteins showed that both minor subunits bind to human pharyngeal (Detroit) cells (A. G. Manetti et al., Mol. Microbiol. 64:968-983, 2007), suggesting both may act as pilus-presented adhesins. While confirming these binding properties, studies described here indicate that Spy0125 is the pilus-presented adhesin and that Spy0130 has a distinct role as a wall linker. Pili were localized predominantly to cell wall fractions of the wild-type S. pyogenes parent strain and a spy0125 deletion mutant. In contrast, they were found almost exclusively in culture supernatants in both spy0130 and srtA deletion mutants, indicating that the housekeeping sortase (SrtA) attaches pili to the cell wall by using Spy0130 as a linker protein. Adhesion assays with antisera specific for individual subunits showed that only anti-rSpy0125 serum inhibited adhesion of wild-type S. pyogenes to human keratinocytes and tonsil epithelium to a significant extent. Spy0125 was localized to the tip of pili, based on a combination of mutant analysis and liquid chromatography-tandem mass spectrometry analysis of purified pili. Assays comparing parent and mutant strains confirmed its role as the adhesin. Unexpectedly, apparent spontaneous cleavage of a labile, proline-rich (8 of 14 residues) sequence separating the N-terminal ∼1/3 and C-terminal ∼2/3 of Spy0125 leads to loss of the N-terminal region, but analysis of internal spy0125 deletion mutants confirmed that this has no significant effect on adhesion.The group A Streptococcus (S. pyogenes) is an exclusively human pathogen that commonly colonizes either the pharynx or skin, where local spread can give rise to various inflammatory conditions such as pharyngitis, tonsillitis, sinusitis, or erysipelas. Although often mild and self-limiting, GAS infections are occasionally very severe and sometimes lead to life-threatening diseases, such as necrotizing fasciitis or streptococcal toxic shock syndrome. A wide variety of cell surface components and extracellular products have been shown or suggested to play important roles in S. pyogenes virulence, including cell surface pili (1, 6, 32). Pili expressed by the serotype M1 S. pyogenes strain SF370 mediate specific adhesion to intact human tonsil epithelia and to primary human keratinocytes, as well as cultured keratinocyte-derived HaCaT cells, but not to Hep-2 or A549 cells (1). They also contribute to adhesion to a human pharyngeal cell line (Detroit cells) and to biofilm formation (29).Over the past 5 years, pili have been discovered on an increasing number of important Gram-positive bacterial pathogens, including Bacillus cereus (4), Bacillus anthracis (4, 5), Corynebacterium diphtheriae (13, 14, 19, 26, 27, 44, 46, 47), Streptococcus agalactiae (7, 23, 38), and Streptococcus pneumoniae (2, 3, 24, 25, 34), as well as S. pyogenes (1, 29, 32). All these species produce pili that are composed of a single major subunit plus either one or two minor subunits. During assembly, the individual subunits are covalently linked to each other via intermolecular isopeptide bonds, catalyzed by specialized membrane-associated transpeptidases that may be described as pilin polymerases (4, 7, 25, 41, 44, 46). These are related to the classical housekeeping sortase (usually, but not always, designated SrtA) that is responsible for anchoring many proteins to Gram-positive bacterial cell walls (30, 31, 33). The C-terminal ends of sortase target proteins include a cell wall sorting (CWS) motif consisting, in most cases, of Leu-Pro-X-Thr-Gly (LPXTG, where X can be any amino acid) (11, 40). Sortases cleave this substrate between the Thr and Gly residues and produce an intermolecular isopeptide bond linking the Thr to a free amino group provided by a specific target. In attaching proteins to the cell wall, the target amino group is provided by the lipid II peptidoglycan precursor (30, 36, 40). In joining pilus subunits, the target is the ɛ-amino group in the side chain of a specific Lys residue in the second subunit (14, 18, 19). Current models of pilus biogenesis envisage repeated transpeptidation reactions adding additional subunits to the base of the growing pilus, until the terminal subunit is eventually linked covalently via an intermolecular isopeptide bond to the cell wall (28, 41, 45).The major subunit (sometimes called the backbone or shaft subunit) extends along the length of the pilus and appears to play a structural role, while minor subunits have been detected either at the tip, the base, and/or at occasional intervals along the shaft, depending on the species (4, 23, 24, 32, 47). In S. pneumoniae and S. agalactiae one of the minor subunits acts as an adhesin, while the second appears to act as a linker between the base of the assembled pilus and the cell wall (7, 15, 22, 34, 35). It was originally suggested that both minor subunits of C. diphtheriae pili could act as adhesins (27). However, recent data showed one of these has a wall linker role (26, 44) and may therefore not function as an adhesin.S. pyogenes strain SF370 pili are composed of a major (backbone) subunit, termed Spy0128, plus two minor subunits, called Spy0125 and Spy0130 (1, 32). All three are required for efficient adhesion to target cells (1). Studies employing purified recombinant proteins have shown that both of the minor subunits, but not the major subunit, bind to Detroit cells (29), suggesting both might act as pilus-presented adhesins. Here we report studies employing a combination of recombinant proteins, specific antisera, and allelic replacement mutants which show that only Spy0125 is the pilus-presented adhesin and that Spy0130 has a distinct role in linking pili to the cell wall.     

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).