Field-aged rice hull biochar stimulated the methylation of mercury and altered the microbial community in a paddy soil under controlled redox condition changes.

Mercury (Hg) contaminated paddy soils are hot spots for methylmercury (MeHg) which can enter the food chain via rice plants causing high risks for human health. Biochar can immobilize Hg and reduce plant uptake of MeHg. However, the effects of biochar on the microbial community and Hg (de)methylation under dynamic redox conditions in paddy soils are unclear. Therefore, we determined the microbial community in an Hg contaminated paddy soil non-treated and treated with rice hull biochar under controlled redox conditions (< 0 mV to 600 mV) using a biogeochemical microcosm system. Hg methylation exceeded demethylation in the biochar-treated soil. The aromatic hydrocarbon degraders Phenylobacterium and Novosphingobium provided electron donors stimulating Hg methylation. MeHg demethylation exceeded methylation in the non-treated soil and was associated with lower available organic matter. Actinobacteria were involved in MeHg demethylation and interlinked with nitrifying bacteria and nitrogen-fixing genus Hyphomicrobium. Microbial assemblages seem more important than single species in Hg transformation. For future directions, the demethylation potential of Hyphomicrobium assemblages and other nitrogen-fixing bacteria should be elucidated. Additionally, different organic matter inputs on paddy soils under constant and dynamic redox conditions could unravel the relationship between Hg (de)methylation, microbial carbon utilization and nitrogen cycling.

Microbial Response to Phytostabilization in Mining Impacted Soils Using Maize in Conjunction with Biochar and Compost.

Even after remediation, mining impacted soils can leave behind a landscape inhospitable to plant growth and containing residual heavy metals. While phytostabilization can be used to restore such sites by limiting heavy metal spread, it is reliant on soil capable of supporting plant growth. Manure-based biochars, coupled with compost, have demonstrated the ability to improve soil growth conditions in mine impacted soils, however there is a paucity of information regarding their influence on resident microbial populations. The objective of this study was to elucidate the impact of these soil amendments on microbial community structure and function in mine impacted soils placed under phytostabilization management with maize. To this aim, a combination of phospholipid fatty acid (PLFA) and enzymatic analyses were performed. Results indicate that microbial biomass is significantly increased upon addition of biochar and compost, with maximal microbial biomass achieved with 5% poultry litter biochar and compost (62.82 nmol g-1 dry soil). Microbial community structure was impacted by biochar type, rate of application, and compost addition, and influenced by pH (r2 = 0.778), EC (r2 = 0.467), and Mg soil concentrations (r2 = 0.453). In three of the four enzymes analyzed, poultry litter biochar treatments were observed with increased activity rates that were often significantly greater than the unamended control. Overall, enzyme activities rates were influenced by biochar type and rate, and addition of compost. These results suggest that using a combination of biochar and compost can be utilized as a management tool to support phytostabilization strategies in mining impacted soils.

Long-term biosolids land application influences soil health.

Soil health assessments associated with organic amendment applications have primarily focused attention on manure or composts. Yet, quantifying specific changes in soil health associated with biosolids land applications has yet to be determined. Our objectives were to evaluate the changes in various soil indicators, and utilizing the Soil Management Assessment Framework (SMAF), quantify changes in soil indicator scores and soil health indices as affected by either increasing inorganic N fertilizer (0 up to 112 kg N ha-1) or biosolids (0 up to 11.2 dry Mg ha-1) applied every other year over 22 years. Soils were sampled (0 to 20 cm depth) following 22 years of N fertilizer or biosolids inputs to a dryland wheat-fallow (Triticum aestivum L.) rotation, 11 soil health indicators were monitored under SMAF guidelines, and indicators, indicator scores, and soil health indices were analyzed statistically. In general, increasing N fertilizer application rates had little effect on soil indicators, SMAF indicator scores or soil health indices. Increasing biosolids application rates increased soil organic C (SOC) and potentially mineralizable N (PMN). The SMAF indicator scores showed upward trends for soil pH, SOC, PMN, and microbial biomass C (MBC) associated with increasing biosolids application rates; discussing trends are important as these indicator scores are combined to provide soil health indices. Indeed, increasing biosolids application rates increased soil chemical and biological health indices, leading to an improvement in the overall soil health index. When comparing the overall N fertilizer to biosolids effect, biosolids applications significantly improved the soil biological health index. Results indicate that long-term biosolids land application to semi-arid, dryland wheat fallow rotations, similar to those studied, improve various aspects of soil health. These findings suggest that biosolids may play a pivotal role in dryland agroecosystem sustainability.

Diversity of Plasmids and Genes Encoding Resistance to Extended-Spectrum ß-Lactamase in Escherichia coli from Different Animal Sources.

Antimicrobial resistance associated with the spread of plasmid-encoded extended-spectrum ß-lactamase (ESBL) genes conferring resistance to third generation cephalosporins is increasing worldwide. However, data on the population of ESBL producing E. coli in different animal sources and their antimicrobial characteristics are limited. The purpose of this study was to investigate potential reservoirs of ESBL-encoded genes in E. coli isolated from swine, beef, dairy, and poultry collected from different regions of the United States using whole-genome sequencing (WGS). Three hundred isolates were typed into different phylogroups, characterized by BOX AIR-1 PCR and tested for resistance to antimicrobials. Of the 300 isolates, 59.7% were resistant to sulfisoxazole, 49.3% to tetracycline, 32.3% to cephalothin, 22.3% to ampicillin, 20% to streptomycin, 16% to ticarcillin; resistance to the remaining 12 antimicrobials was less than 10%. Phylogroups A and B1 were most prevalent with A (n = 92, 30%) and B1 (87 = 29%). A total of nine E. coli isolates were confirmed as ESBL producers by double-disk synergy testing and multidrug resistant (MDR) to at least three antimicrobial drug classes. Using WGS, significantly higher numbers of ESBL-E. coli were detected in swine and dairy manure than from any other animal sources, suggesting that these may be the primary animal sources for ESBL producing E. coli. These isolates carry plasmids, such as IncFIA(B), IncFII, IncX1, IncX4, IncQ1, CollRNAI, Col440I, and acquired ARGs aph(6)-Id, aph(3″)-Ib, aadA5, aph(3')-Ia, blaCTX-M-15, blaTEM-1B, mphA, ermB, catA1, sul1, sul2, tetB, dfrA17. One of the E. coli isolates from swine with ST 410 was resistant to nine antibiotics and carried more than 28 virulence factors, and this ST has been shown to belong to an international high-risk clone. Our data suggests that ESBL producing E. coli are widely distributed in different animal sources, but swine and dairy cattle may be their main reservoir.

Microbial response to designer biochar and compost treatments for mining impacted soils.

The Oronogo-Duenweg mining belt is a designated United States Environmental Protection Agency Superfund site due to lead-contaminated soil and groundwater by former mining and smelting operations. Sites that have undergone remediation - in which the O, A, and B horizons have been removed alongside the lead contamination - have an exposed C horizon and are incalcitrant to revegetation efforts. Soils also continue to contain quantifiable Cd and Zn concentrations. In order to improve soil conditions and encourage successful site revegetation, our study employed three biochars, sourced from different feedstocks (poultry litter, beef lot manure, and lodge pole pine), at two rates of application (2.5%, and 5%), coupled with compost (0%, 2.5% and 5% application rates). Two plant species - switchgrass (Panicum virgatum) and buffalograss (Bouteloua dactyloides) - were grown in the amended soils. Amendment of soils with poultry litter biochar applied at 5% resulted in the greatest reduction of soil bioavailable Cd and Zn. Above ground biomass yields were greatest with beef lot manure biochar applied at 2.5% with 5% compost, or with 5% biochar at 2.5% and 5% compost rates. Maximal microbial biomass was achieved with 5% poultry litter biochar and 5% compost, and microbial communities in soils amended with poultry litter biochar distinctly clustered away from all other soil treatments. Additionally, poultry litter biochar amended soils had the highest enzyme activity rates for ß-glucosidase, N-acetyl-ß-D-glucosaminidase, and esterase. These results suggest that soil reclamation using biochar and compost can improve mine-impacted soil biogeophysical characteristics, and potentially improve future remediation efforts.

Phytostabilization of acidic mine tailings with biochar, biosolids, lime, and locally-effective microbes: Do amendment mixtures influence plant growth, tailing chemistry, and microbial composition?

Abandoned mine lands present persistent environmental challenges to ecosystems and economies; reclamation an important step for overcoming these challenges. Phytostabilization is an elegant and cost-effective reclamation strategy, however, establishing plants on severely degraded soils is problematic, often requiring soil amendment additions. We evaluated whether amendment mixtures composed of lime, biochar, biosolids, and locally effective microbes (LEM) could alleviate the constraints that hinder phytostabilization success. We hypothesized that 1) plants grown in tailings amended with lime, biochar, and biosolids (LBB) would establish faster and grow larger than plants grown in tailings amended with lime only, and 2) the LEM source would influence microbial community function and structure in amended mine tailings. We conducted a greenhouse study that simulated in situ conditions to measure the influence of LBB-LEM amendment blends on plant growth, plant nutrients, metal concentrations, microbial function, and microbial community structure. Blue wildrye [Elymus glaucus Buckley ssp. Jepsonii (Burtt Davy) Gould] was grown in tailings collected from the Formosa mine site amended with various combinations of LBB-LEM. The above and below ground biomass of plants grown in mine tailings amended with LBB was 3 to 4 times larger than the biomass of plants grown in tailings amended only with lime. Although the LEM addition did not influence immediate plant growth, it did affect nutrient content and altered the rhizosphere community membership. As such, it is not yet clear if LEM-driven alterations in microbial membership will advance mine reclamation strategies by improving long-term growth.

A newly developed Escherichia coli isolate panel from a cross section of U.S. animal production systems reveals geographic and commodity-based differences in antibiotic resistance gene carriage.

There are limited numbers of Escherichia coli isolate panels that represent United States food animal production. The majority of existing Escherichia coli isolate panels are typically designed: (i) to optimize genetic and/or phenotypic diversity; or (ii) focus on human isolates. To address this shortfall in agriculturally-related resources, we have assembled a publicly-available isolate panel (AgEc) from the four major animal production commodities in the United States, including beef, dairy, poultry, and swine, as well as isolates from agriculturally-impacted environments, and other commodity groups. Diversity analyses by phylotyping and Pulsed-field Gel Electrophoresis revealed a highly diverse composition, with the 300 isolates clustered into 71 PFGE sub-types based upon an 80% similarity cutoff. To demonstrate the panel's utility, tetracycline and sulfonamide resistance genes were assayed, which identified 131 isolates harboring genes involved in tetracycline resistance, and 41 isolates containing sulfonamide resistance genes. There was strong overlap in the two pools of isolates, 38 of the 41 isolates harboring sulfonamide resistance genes also contained tetracycline resistance genes. Analysis of antimicrobial resistance gene patterns revealed significant differences along commodity and geographical lines. This panel therefore provides the research community an E. coli isolate panel for study of issues pertinent to U.S. food animal production.

Biochar compost blends facilitate switchgrass growth in mine soils by reducing Cd and Zn bioavailability.

Biochars have the potential to reclaim mine-impacted soils; however, their variable physico-chemical properties incite speculation about their successful remediation performance. This investigation examined the capability of biochars produced from three different feedstocks along with a compost blend to improve switchgrass growth conditions in a mine-impacted soil by examining influences on soil pH, grass metal contents, and soil-extractable metal concentrations. Cadmium (Cd)- and zinc (Zn)-contaminated mine soil was collected from a site near Webb City, Missouri, USA-a location within the Tri-State Mining District. In a full factorial design, soil was treated with a 0%, 2.5%, and 5% (w/w) compost mixture (wood chips + beef cattle manure), and 0%, 2.5% and 5% of each biochar pyrolyzed from beef cattle manure, poultry litter, and lodgepole pine feedstocks. Switchgrass (Panicum virgatum, 'Cave-In-Rock' variety) was grown in a greenhouse for 50 days and the mass of shoots (above-ground biomass) and roots was assessed, while soil pH, deionized H2O- and 0.01 M CaCl2-extractable Cd and Zn concentrations were measured. Poultry litter biochar and compost had the greatest ability to raise soil pH (from 4.40 to 6.61), beef cattle manure biochar and compost moderately raised pH (from 4.4 to 5.92), and lodgepole pine biochar and compost weakly raised pH (from 4.40 to 5.05). Soils treated with beef cattle manure biochar, poultry litter biochar significantly reduced deionized H2O- and 0.01 M CaCl2-extractable Cd and Zn concentrations, while lodgepole pine biochar-treated soils showed mixed results. Switchgrass shoot and root masses were greatest in soil treated with compost in combination with either beef cattle manure biochar or poultry litter biochar. Soils treated with 5% beef cattle manure biochar + 5% compost had greater reductions in total Cd and Zn concentrations measured in switchgrass shoots and roots compared to the other two treatments. The three biochars and compost mixtures applied to heavy metal, mine-impacted soil had considerable performance dissimilarities for improving switchgrass productivity. Switchgrass growth was noticeably improved after treatment with the compost in combination with biochar from beef cattle manure or poultry litter. This may be explained by the increased soil pH that promoted Zn and Cd precipitation and organic functional groups that reduced soil-available heavy metal concentrations. Our results imply that creating designer biochars is an important management component in developing successful mine-site phytostabilization programs.

Remediation of an acidic mine spoil: Miscanthus biochar and lime amendment affects metal availability, plant growth, and soil enzyme activity.

Biochar may be a tool for mine spoil remediation; however, its mechanisms for achieving this goal remain unclear. In this study, Miscanthus (Miscanthus giganteus) biochar was evaluated for its ability to reclaim acidic mine spoils (pH < 3) through reducing metal availability, improving soil microbial enzymatic activity, and initial growth of grass seedlings. Biochar was applied at 0, 1, 2.5 and 5% (w/w) along with lime/no lime and fertilizer additions. Blue Wildrye (Elymus glaucus cv. 'Elkton') was planted and later the shoots and roots were collected and metal concentrations determined. Afterwards, each pot was leached with deionized water, and the leachate analyzed for pH, electrical conductivity (EC), dissolved organic carbon (DOC) and soluble metal concentrations. After drying, the spoil was extracted with 0.01 M CaCl2 and Mehlich 3 (M3) to determine extractable Al, Cu, and Zn concentrations. Additionally, microbial activity was measured using a fluorescent ß-glucosidase and N-acetyl-ß-d-glucosaminidase assay. Spoil treated with lime and biochar had significantly greater pH and EC values. Significantly greater ß-glucosidase activity occurred only in the 5% biochar plus lime treatment, while N-acetyl-ß-d-glucosaminidase activities were not altered. Metal concentrations in rye shoot and roots were mixed. Lime additions significantly reduced extractable metal concentrations. Increasing biochar rates alone significantly reduced leachate DOC concentrations, and subsequently reduced leachable metal concentrations. Surprisingly, miscanthus biochar, by itself, was limited at mitigation, but when combined with lime, the combination was capable of further reducing extractable metal concentrations and improving ß-glucosidase enzyme activity.

Microbial community analysis of swine wastewater anaerobic lagoons by next-generation DNA sequencing.

Anaerobic lagoons are a standard practice for the treatment of swine wastewater. This practice relies heavily on microbiological processes to reduce concentrated organic material and nutrients. Despite this reliance on microbiological processes, research has only recently begun to identify and enumerate the myriad and complex interactions that occur in this microbial ecosystem. To further this line of study, we utilized a next-generation sequencing (NGS) technology to gain a deeper insight into the microbial communities along the water column of four anaerobic swine wastewater lagoons. Analysis of roughly one million 16S rDNA sequences revealed a predominance of operational taxonomic units (OTUs) classified as belonging to the phyla Firmicutes (54.1%) and Proteobacteria (15.8%). At the family level, 33 bacterial families were found in all 12 lagoon sites and accounted for between 30% and 50% of each lagoon's OTUs. Analysis by nonmetric multidimensional scaling (NMS) revealed that TKN, COD, ORP, TSS, and DO were the major environmental variables in affecting microbial community structure. Overall, 839 individual genera were classified, with 223 found in all four lagoons. An additional 321 genera were identified in sole lagoons. The top 25 genera accounted for approximately 20% of the OTUs identified in the study, and the low abundances of most of the genera suggests that most OTUs are present at low levels. Overall, these results demonstrate that anaerobic lagoons have distinct microbial communities which are strongly controlled by the environmental conditions present in each individual lagoon.

An analysis of the link between strokes and soils in the South Carolina coastal plains.

The Stroke Belt is a geographical region of the Southeastern United States where resident individuals suffer a disproportionately higher rate of strokes than the rest of the population. While the "buckle" of this Stroke Belt coincides with the Southeastern Coastal Plain region of North and South Carolina and Georgia, there is a paucity of information pinpointing specific causes for this phenomenon. A number of studies posit that an exposure event-potentially microbial in nature-early in life, could be a risk factor. The most likely vector for such an exposure event would be the soils of the Southeastern Coastal Plain region. These soils may have chemical and physical properties which are conducive to the growth and survival of microorganisms which may predispose individuals to stroke. To this aim, we correlated SC stroke mortality data to soil characteristics found in the NRCS SSURGO database. In statewide comparisons, depth to water table (50 to 100 cm, R = 0.62) and soil drainage class (poorly drained, R = 0.59; well drained, R = -0.54) both showed statistically significant relationships with stroke rate. In a 20 county comparison, depth to water table, drainage class, hydric rating (hydric soils, R = 0.56), and pH (very strongly acid, R = 0.66) all showed statistically significant relationships with stroke rate. These data should help direct future research and epidemiology efforts to pinpoint the exact exposure events which predispose individuals to an increased stroke rate.

Nitrification and denitrification gene abundances in swine wastewater anaerobic lagoons.

Although anaerobic lagoons are used globally for livestock waste treatment, their detailed microbial cycling ofN is only beginning to become understood. Within this cycling, nitrification can be performed by organisms that produce the enzyme ammonia monooxygenase. For denitrification, the reduction of nitrite to nitric oxide can be catalyzed by two forms of nitrite reductases, and N,O can be reduced by nitrous oxide reductase encoded by the gene nosZ The objectives of this investigation were to (i) quantify the abundance of the amoA, nirK, nirS, and nosZ genes; (ii) evaluate the influence of environmental conditions on their abundances; and (iii) evaluate their abundance relative to denitrification enzyme activity (DEA). Samples were analyzed via real-time quantitative polymerase chain reaction and collected from eight typical, commercial anaerobic, swine wastewater lagoons located in the Carolinas. The four genes assayed in this study were present in all eight lagoons. Their abundances relative to total bacterial populations were 0.04% (amoA), 1.33% (nirS), 5.29% (nirK), and 0.27% (nosZ). When compared with lagoon chemical characteristics, amoA and nirK correlated with several measured variables. Neither nirS nor nosZ correlated with any measured environmental variables. Although no gene measured in this study correlated with actual or potential DEA, nosZ copy numbers did correlate with the disparity between actual and potential DEA. Phylogenetic analysis ofnosZdid not reveal any correlations to DEA rates. As with other investigations, analyses of these genes provide useful insight while revealing the underlying greater complexity of N cycling within swine waste lagoons.

Characterization of a microbial community capable of nitrification at cold temperature.

While the oxidation of ammonia is an integral component of advanced aerobic livestock wastewater treatment, the rate of nitrification by ammonia-oxidizing bacteria is drastically reduced at colder temperatures. In this study we report an acclimated lagoon nitrifying sludge that is capable of high rates of nitrification at temperatures from 5 degrees C (11.2mg N/g MLVSS/h) to 20 degrees C (40.4 mg N/g MLVSS/h). The composition of the microbial community present in the nitrifying sludge was investigated by partial 16S rRNA gene sequencing. After DNA extraction and the creation of a plasmid library, 153 partial length 16S rRNA gene clones were sequenced and analyzed phylogenetically. Over 80% of these clones were affiliated with the Proteobacteria, and grouped with the beta- (114 clones), gamma- (7 clones), and alpha-classes (2 clones). The remaining clones were affiliated with the Acidobacteria (1 clone), Actinobacteria (8 clones), Bacteroidetes (16 clones), and Verrucomicrobia (5 clones). The majority of the clones belonged to the genus Nitrosomonas, while other clones affiliated with microorganisms previously identified as having floc forming or psychrotolerance characteristics.

Transcriptional and functional analysis of the Neisseria gonorrhoeae Fur regulon.

To ensure survival in the host, bacteria have evolved strategies to acquire the essential element iron. In Neisseria gonorrhoeae, the ferric uptake regulator Fur regulates metabolism through transcriptional control of iron-responsive genes by binding conserved Fur box (FB) sequences in promoters during iron-replete growth. Our previous studies showed that Fur also controls the transcription of secondary regulators that may, in turn, control pathways important to pathogenesis, indicating an indirect role for Fur in controlling these downstream genes. To better define the iron-regulated cascade of transcriptional control, we combined three global strategies--temporal transcriptome analysis, genomewide in silico FB prediction, and Fur titration assays (FURTA)--to detect genomic regions able to bind Fur in vivo. The majority of the 300 iron-repressed genes were predicted to be of unknown function, followed by genes involved in iron metabolism, cell communication, and intermediary metabolism. The 107 iron-induced genes encoded hypothetical proteins or energy metabolism functions. We found 28 predicted FBs in FURTA-positive clones in the promoters and within the open reading frames of iron-repressed genes. We found lower levels of conservation at critical thymidine residues involved in Fur binding in the FB sequence logos of FURTA-positive clones with intragenic FBs than in the sequence logos generated from FURTA-positive promoter regions. In electrophoretic mobility shift assay studies, intragenic FBs bound Fur with a lower affinity than intergenic FBs. Our findings further indicate that transcription under iron stress is indirectly controlled by Fur through 12 potential secondary regulators.

Transcript analysis of nrrF, a Fur repressed sRNA of Neisseria gonorrhoeae.

Like most microorganisms, Neisseria gonorrhoeae alters gene expression in response to iron availability. The ferric uptake regulator Fur has been shown to be involved in controlling this response, but the extent of this involvement remains unknown. It is known that in addition to working directly to repress gene expression, Fur may also work indirectly by controlling additional regulatory elements. Using in silico analysis, we identified a putative small RNA (sRNA) homolog of the meningococcal nrrF locus, and demonstrate that this sRNA is iron-repressible, suggesting that this is the gonococcal analog of the rhyB locus in Escherichia coli. Quantitative real-time RT-PCR analysis indicates that this transcript may also be temporally regulated. Transcript analysis identified the 5' start of the transcript, using a single reaction, fluorescent-based, primer extension assay. This protocol allows for the rapid identification of transcriptional start sites of RNA transcripts, and could be used for high-throughput transcript mapping.

Multilocus genotyping assays for single nucleotide polymorphism-based subtyping of Listeria monocytogenes isolates.

Listeria monocytogenes is responsible for serious invasive illness associated with consumption of contaminated food and places a significant burden on public health and the agricultural economy. We recently developed a multilocus genotyping (MLGT) assay for high-throughput subtype determination of L. monocytogenes lineage I isolates based on interrogation of single nucleotide polymorphisms (SNPs) via multiplexed primer extension reactions. Here we report the development and validation of two additional MLGT assays that address the need for comprehensive DNA sequence-based subtyping of L. monocytogenes. The first of these novel MLGT assays targeted variation segregating within lineage II, while the second assay combined probes for lineage III strains with probes for strains representing a recently characterized fourth evolutionary lineage (IV) of L. monocytogenes. These assays were based on nucleotide variation identified in >3.8 Mb of comparative DNA sequence and consisted of 115 total probes that differentiated 93% of the 100 haplotypes defined by the multilocus sequence data. MLGT reproducibly typed the 173 isolates used in SNP discovery, and the 10,448 genotypes derived from MLGT analysis of these isolates were consistent with DNA sequence data. Application of the MLGT assays to assess subtype prevalence among isolates from ready-to-eat foods and food-processing facilities indicated a low frequency (6.3%) of epidemic clone subtypes and a substantial population of isolates (>30%) harboring mutations in inlA associated with attenuated virulence in cell culture and animal models. These mutations were restricted to serogroup 1/2 isolates, which may explain the overrepresentation of serotype 4b isolates in human listeriosis cases.

A single-nucleotide-polymorphism-based multilocus genotyping assay for subtyping lineage I isolates of Listeria monocytogenes.

Listeria monocytogenes is a facultative intracellular pathogen responsible for food-borne disease with high mortality rates in humans and is the leading microbiological cause of food recalls. Lineage I isolates of L. monocytogenes are a particular public health concern because they are responsible for most sporadic cases of listeriosis and the vast majority of epidemic outbreaks. Rapid, reproducible, and sensitive methods for differentiating pathogens below the species level are required for effective pathogen control programs, and the CDC PulseNet Task Force has called for the development and validation of DNA sequence-based methods for subtyping food-borne pathogens. Therefore, we developed a multilocus genotyping (MLGT) assay for L. monocytogenes lineage I isolates based on nucleotide variation identified by sequencing 23,251 bp of DNA from 22 genes distributed across seven genomic regions in 65 L. monocytogenes isolates. This single-well assay of 60 allele-specific probes captured 100% of the haplotype information contained in approximately 1.5 Mb of comparative DNA sequence and was used to reproducibly type a total of 241 lineage I isolates. The MLGT assay provided high discriminatory power (Simpson's index value, 0.91), uniquely identified isolates from the eight listeriosis outbreaks examined, and differentiated serotypes 1/2b and 4b as well as epidemic clone I (ECI), ECIa, and ECII. In addition, the assay included probes for a previously characterized truncation mutation in inlA, providing for the identification of a specific virulence-attenuated subtype. These results demonstrate that MLGT represents a significant new tool for use in pathogen surveillance, outbreak detection, risk assessment, population analyses, and epidemiological investigations. DNA sequences were deposited in the GenBank database under accession numbers DQ 812146 to DQ 812517, DQ 843664 to DQ 844598, and AY 512391 to AY 512502.

Identification of the iron-responsive genes of Neisseria gonorrhoeae by microarray analysis in defined medium.

To ensure survival, most bacteria must acquire iron, a resource that is sequestered by mammalian hosts. Pathogenic bacteria have therefore evolved intricate systems to sense iron limitation and regulate gene expression appropriately. We used a pan-Neisseria microarray to examine genes regulated in Neisseria gonorrhoeae in response to iron availability in defined medium. Overall, 203 genes varied in expression, 109 up-regulated and 94 down-regulated by iron deprivation. In iron-replete medium, genes essential to rapid bacterial growth were preferentially expressed, while iron transport functions, and predominantly genes of unknown function, were expressed in low-iron medium. Of those TonB-dependent proteins encoded in the FA1090 genome with unknown ligand specificity, expression of three was not controlled by iron availability, suggesting that these receptors may not be high-affinity transporters for iron-containing ligands. Approximately 30% of the operons regulated by iron appeared to be directly under control of Fur. Our data suggest a regulatory cascade where Fur indirectly controls gene expression by affecting the transcription of three secondary regulators. Our data also suggest that a second MerR-like regulator may be directly responding to iron availability and controlling transcription independent of the Fur protein. Comparison of our data with those recently published for Neisseria meningitidis revealed that only a small portion of genes were found to be similarly regulated in these closely related pathogens, while a large number of genes derepressed during iron starvation were unique to each organism.