Flow-Based Microfluidic Device for Quantifying Bacterial Chemotaxis in Stable,Competing Gradients

Chemotaxis is the migration of cells in gradients of chemoeffector molecules. Although multiple, competing gradients must often coexist in nature, conventional approaches for investigating bacterial chemotaxis are suboptimal for quantifying migration in response to gradients of multiple signals. In this work, we developed a microfluidic device for generating precise and stable gradients of signaling molecules. We used the device to investigate the effects of individual and combined chemoeffector gradients on Escherichia coli chemotaxis. Laminar flow-based diffusive mixing was used to generate gradients, and the chemotactic responses of cells expressing green fluorescent protein were determined using fluorescence microscopy. Quantification of the migration profiles indicated that E. coli was attracted to the quorum-sensing molecule autoinducer-2 (AI-2) but was repelled from the stationary-phase signal indole. Cells also migrated toward higher concentrations of isatin (indole-2,3-dione), an oxidized derivative of indole. Attraction to AI-2 overcame repulsion by indole in equal, competing gradients. Our data suggest that concentration-dependent interactions between attractant and repellent signals may be important determinants of bacterial colonization of the gut.Bacteria sense chemoeffectors using cell surface receptors (13, 29). Cells constantly monitor the concentration of specific molecules, comparing the current concentration to the concentration detected a few seconds earlier. This comparison determines the net direction of movement (6, 22). Chemotaxis allows bacteria to approach sources of attractant chemicals or to avoid sources of repellent chemicals. Natural habitats for Escherichia coli, such as the gastrointestinal (GI) tract, are typically heterogeneous and contain multiple chemoeffectors with potentially opposing effects. The integrated chemotactic response in such environments is thus likely to be an important factor in bacterial colonization.Conventional approaches for investigating bacterial chemotaxis, such as the swim plate and capillary (1) assays, are not ideal for quantifying bacterial migration. Chemotactic-ring formation in semisolid agar requires metabolizable attractants and is subject to multiple factors, and both it and the traditional capillary assay are poorly designed to investigate repellent taxis. Mao et al. (23) were the first to investigate bacterial taxis in a microfluidic flow cell. In their device, a concentration gradient is formed by the diffusive mixing of two inlet streams. However, the exposure to a fully developed gradient in this device is limited because it takes time for the gradient to develop.Variations of this technique, such as three-channel microfluidic devices (7, 8) in which a linear gradient is generated in the absence of flow or a T-channel device that monitors chemotaxis perpendicular to the direction of fluid flow (18), were developed subsequently. The T-channel system has many of the limitations of the device developed by Mao et al. (23), and nonflow systems, like the capillary assay (1), suffer from a lack of temporal stability of the gradients.Here, we report a flow-based microfluidic chemotaxis device that is coupled to a gradient generator. Bacteria are exposed to precise and temporally stable concentration gradients of chemoeffectors over the length of the microfluidic channel. This device was used to quantify E. coli chemotaxis in response to the canonical chemoeffectors l-aspartate and Ni²⁺. The device was also used to investigate chemotaxis toward cell-cell communication signals such as autoinducer-2 (AI-2), indole, and isatin that are likely to be present in the in vivo microenvironment in which E. coli is present (e.g., the human GI tract). The data obtained reinforce the idea that concentration-dependent interactions between different chemical signals could be important determinants of bacterial colonization in natural environments.     

Chemotaxis to Pyrimidines and Identification of a Cytosine Chemoreceptor in Pseudomonas putida

We developed a high-throughput quantitative capillary assay and demonstrated that Pseudomonas putida strains F1 and PRS2000 were attracted to cytosine, but not thymine or uracil. In contrast, Pseudomonas aeruginosa PAO1 was not chemotactic to any pyrimidines. Chemotaxis assays with a mutant strain of F1 in which the putative methyl-accepting chemotaxis protein-encoding gene Pput_0623 was deleted revealed that this gene (designated mcpC) encodes a chemoreceptor for positive chemotaxis to cytosine. P. putida F1 also responded weakly to cytidine, uridine, and thymidine, but these responses were not mediated by mcpC. Complementation of the F1 ΔmcpC mutant XLF004 with the wild-type gene restored chemotaxis to cytosine. In addition, introduction of this gene into P. aeruginosa PAO1 conferred the ability to respond to cytosine. To our knowledge, this is the first report of a chemoreceptor for cytosine.Motile bacteria are capable of detecting chemical gradients in the environment and swim toward or away from them, a behavior known as chemotaxis. Historically, the enteric bacterium Escherichia coli has been the model organism for chemotaxis studies. E. coli has four transmembrane chemoreceptors called methyl-accepting chemotaxis proteins (MCPs), each of which binds a set of chemicals directly or in complex with specific periplasmic binding proteins. MCPs send signals to the flagellar motor via a complex signal transduction system that is composed of six soluble chemotaxis proteins, through which the bacterium modifies its swimming behavior based on the signal(s) received (for reviews, see references 5 and 15). The MCPs of E. coli sense a variety of stimuli, including amino acids, sugars, and dipeptides (30, 44). We recently reported that E. coli also responds to the pyrimidines thymine and uracil and demonstrated that Tap, the MCP known to mediate chemotaxis to dipeptides, is required for pyrimidine taxis (29).Pseudomonads are environmental bacteria that are widespread in nature, and all Pseudomonas species are motile. They have conserved chemotaxis proteins that are homologous to those present in E. coli, but their chemosensory systems appear to be more complex (6, 39, 55). Unlike E. coli, which has only one set of chemotaxis (che) genes in a single gene cluster, Pseudomonas species have multiple che gene homologs organized in several unlinked gene clusters (39). In addition, genome sequence analyses have revealed that Pseudomonas strains have numerous putative MCP genes. For example, the genome of Pseudomonas aeruginosa PAO1 (46) encodes 26 MCP-like proteins, Pseudomonas putida KT2440 (34) has 27, and Pseudomonas syringae DC3000 (9) has 49 (39).The best-studied chemotaxis system in Pseudomonas is that of the opportunistic pathogen P. aeruginosa. More than 75 different chemoattractants have been identified for P. aeruginosa (39), and 13 of its 26 MCP-like proteins have been functionally characterized. Eight MCPs have been shown to mediate positive responses to amino acids (PctABC), inorganic phosphate (CtpH and CtpL), malate (PA2652), ethylene (TlpQ), and chloroethylenes (McpA) (3, 25-27, 42, 47, 54). Two MCPs (McpA and McpB) were shown to be required for general optimal chemotaxis (16), and one MCP-like protein (Aer) was found to mediate energy taxis (22). The MCP-like proteins BdlA and PilJ were shown to be involved in biofilm formation and biosynthesis of type IV pili, respectively (10, 12, 32).P. putida is a common soil bacterium and, unlike P. aeruginosa, is not known to be pathogenic. Although P. putida and P. aeruginosa each have approximately the same number of MCP-like genes in their genomes, most of the protein products show relatively low amino acid sequence similarity. Based on our BLAST searches, three putative P. putida F1 MCPs have no obvious counterparts in P. aeruginosa PAO1. Most of the others share between 30 and 70% amino acid sequence identity, with the highest sequence conservation in the C-terminal signaling domains. The most highly conserved MCP-like proteins in the two species are Aer and PilJ (both are 77% identical to the corresponding homologs). These observations suggest that the two organisms respond to different subsets of attractants, which most likely reflects their different lifestyles and environmental niches. P. putida is known for its catabolic versatility (45), and we expect that members of the species are capable of responding to a correspondingly wide range of organic attractants. We are interested in defining the range of attractant and repellent responses and the functions of the MCPs present in P. putida compared to those of P. aeruginosa. In this study, we used P. putida strains F1 and PRS2000 and P. aeruginosa strain PAO1 to investigate the chemotactic responses to pyrimidines.     

Conjugative Plasmid Transfer and Adhesion Dynamics in an Escherichia coli Biofilm

A conjugative plasmid from the catheter-associated urinary tract infection strain Escherichia coli MS2027 was sequenced and annotated. This 42,644-bp plasmid, designated pMAS2027, contains 58 putative genes and is most closely related to plasmids belonging to incompatibility group X (IncX1). Plasmid pMAS2027 encodes two important virulence factors: type 3 fimbriae and a type IV secretion (T4S) system. Type 3 fimbriae, recently found to be functionally expressed in E. coli, played an important role in biofilm formation. Biofilm formation by E. coli MS2027 was specifically due to expression of type 3 fimbriae and not the T4S system. The T4S system, however, accounted for the conjugative ability of pMAS2027 and enabled a non-biofilm-forming strain to grow as part of a mixed biofilm following acquisition of this plasmid. Thus, the importance of conjugation as a mechanism to spread biofilm determinants was demonstrated. Conjugation may represent an important mechanism by which type 3 fimbria genes are transferred among the Enterobacteriaceae that cause device-related infections in nosocomial settings.Bacterial biofilms are complex communities of bacterial cells living in close association with a surface (17). Bacterial cells in these protected environments are often resistant to multiple factors, including antimicrobials, changes in the pH, oxygen radicals, and host immune defenses (19, 38). Biofilm formation is a property of many bacterial species, and a range of molecular mechanisms that facilitate this process have been described (2, 3, 11, 14, 16, 29, 33, 34). Often, the ability to form a biofilm is dependent on the production of adhesins on the bacterial cell surface. In Escherichia coli, biofilm formation is enhanced by the production of certain types of fimbriae (e.g., type 1 fimbriae, type 3 fimbriae, F1C, F9, curli, and conjugative pili) (14, 23, 25, 29, 33, 39, 46), cell surface adhesins (e.g., autotransporter proteins such as antigen 43, AidA, TibA, EhaA, and UpaG) (21, 34, 35, 40, 43), and flagella (22, 45).The close proximity of bacterial cells in biofilms creates an environment conducive for the exchange of genetic material. Indeed, plasmid-mediated conjugation in monospecific and mixed E. coli biofilms has been demonstrated (6, 18, 24, 31). The F plasmid represents the best-characterized conjugative system for biofilm formation by E. coli. The F pilus mediates adhesion to abiotic surfaces and stabilizes the biofilm structure through cell-cell interactions (16, 30). Many other conjugative plasmids also contribute directly to biofilm formation upon derepression of the conjugative function (16).One example of a conjugative system employed by gram-negative Enterobacteriaceae is the type 4 secretion (T4S) system. The T4S system is a multisubunit structure that spans the cell envelope and contains a secretion channel often linked to a pilus or other surface filament or protein (8). The Agrobacterium tumefaciens VirB-VirD4 system is the archetypical T4S system and is encoded by 11 genes in the virB operon and one gene (virD4) in the virD operon (7, 8). Genes with strong homology to genes in the virB operon have also been identified on other conjugative plasmids. For example, the pilX1 to pilX11 genes on the E. coli R6K IncX plasmid and the virB1 to virB11 genes are highly conserved at the nucleotide level (28).We recently described identification and characterization of the mrk genes encoding type 3 fimbriae in a uropathogenic strain of E. coli isolated from a patient with a nosocomial catheter-associated urinary tract infection (CAUTI) (29). The mrk genes were located on a conjugative plasmid (pMAS2027) and were strongly associated with biofilm formation. In this study we determined the entire sequence of plasmid pMAS2027 and revealed the presence of conjugative transfer genes homologous to the pilX1 to pilX11 genes of E. coli R6K (in addition to the mrk genes). We show here that biofilm formation is driven primarily by type 3 fimbriae and that the T4S apparatus is unable to mediate biofilm growth in the absence of the mrk genes. Finally, we demonstrate that conjugative transfer of pMAS2027 within a mixed biofilm confers biofilm formation properties on recipient cells due to acquisition of the type 3 fimbria-encoding mrk genes.     

Multiple Interaction Domains in FtsL,a Protein Component of the Widely Conserved Bacterial FtsLBQ Cell Division Complex

A bioinformatic analysis of nearly 400 genomes indicates that the overwhelming majority of bacteria possess homologs of the Escherichia coli proteins FtsL, FtsB, and FtsQ, three proteins essential for cell division in that bacterium. These three bitopic membrane proteins form a subcomplex in vivo, independent of the other cell division proteins. Here we analyze the domains of E. coli FtsL that are involved in the interaction with other cell division proteins and important for the assembly of the divisome. We show that FtsL, as we have found previously with FtsB, packs an enormous amount of information in its sequence for interactions with proteins upstream and downstream in the assembly pathway. Given their size, it is likely that the sole function of the complex of these two proteins is to act as a scaffold for divisome assembly.The division of an Escherichia coli cell into two daughter cells requires a complex of proteins, the divisome, to coordinate the constriction of the three layers of the Gram-negative cell envelope. In E. coli, there are 10 proteins known to be essential for cell division; in the absence of any one of these proteins, cells continue to elongate and to replicate and segregate their chromosomes but fail to divide (29). Numerous additional nonessential proteins have been identified that localize to midcell and assist in cell division (7-9, 20, 25, 34, 56, 59).A localization dependency pathway has been determined for the 10 essential division proteins (FtsZ→FtsA/ZipA→FtsK→FtsQ→FtsL/FtsB→FtsW→FtsI→FtsN), suggesting that the divisome assembles in a hierarchical manner (29). Based on this pathway, a given protein depends on the presence of all upstream proteins (to the left) for its localization and that protein is then required for the localization of the downstream division proteins (to the right). While the localization dependency pathway of cell division proteins suggests that a sequence of interactions is necessary for divisome formation, recent work using a variety of techniques reveals that a more complex web of interactions among these proteins is necessary for a functionally stable complex (6, 10, 19, 23, 24, 30-32, 40). While numerous interactions have been identified between division proteins, further work is needed to define which domains are involved and which interactions are necessary for assembly of the divisome.One subcomplex of the divisome, composed of the bitopic membrane proteins FtsB, FtsL, and FtsQ, appears to be the bridge between the predominantly cytoplasmic cell division proteins and the predominantly periplasmic cell division proteins (10). FtsB, FtsL, and FtsQ share a similar topology: short amino-terminal cytoplasmic domains and larger carboxy-terminal periplasmic domains. This tripartite complex can be divided further into a subcomplex of FtsB and FtsL, which forms in the absence of FtsQ and interacts with the downstream division proteins FtsW and FtsI in the absence of FtsQ (30). The presence of an FtsB/FtsL/FtsQ subcomplex appears to be evolutionarily conserved, as there is evidence that the homologs of FtsB, FtsL, and FtsQ in the Gram-positive bacteria Bacillus subtilis and Streptococcus pneumoniae also assemble into complexes (18, 52, 55).The assembly of the FtsB/FtsL/FtsQ complex is important for the stabilization and localization of one or more of its component proteins in both E. coli and B. subtilis (11, 16, 18, 33). In E. coli, FtsB and FtsL are codependent for their stabilization and for localization to midcell, while FtsQ does not require either FtsB or FtsL for its stabilization or localization to midcell (11, 33). Both FtsL and FtsB require FtsQ for localization to midcell, and in the absence of FtsQ the levels of full-length FtsB are significantly reduced (11, 33). The observed reduction in full-length FtsB levels that occurs in the absence of FtsQ or FtsL results from the degradation of the FtsB C terminus (33). However, the C-terminally degraded FtsB generated upon depletion of FtsQ can still interact with and stabilize FtsL (33).While a portion of the FtsB C terminus is dispensable for interaction with FtsL and for the recruitment of the downstream division proteins FtsW and FtsI, it is required for interaction with FtsQ (33). Correspondingly, the FtsQ C terminus also appears to be important for interaction with FtsB and FtsL (32, 61). The interaction between FtsB and FtsL appears to be mediated by the predicted coiled-coil motifs within the periplasmic domains of the two proteins, although only the membrane-proximal half of the FtsB coiled coil is necessary for interaction with FtsL (10, 32, 33). Additionally, the transmembrane domains of FtsB and FtsL are important for their interaction with each other, while the cytoplasmic domain of FtsL is not necessary for interaction with FtsB, which has only a short 3-amino-acid cytoplasmic domain (10, 33).In this study, we focused on the interaction domains of FtsL. We find that, as with FtsB, the C terminus of FtsL is required for the interaction of FtsQ with the FtsB/FtsL subcomplex while the cytoplasmic domain of FtsL is involved in recruitment of the downstream division proteins. Finally, we provide a comprehensive analysis of the presence of FtsB, FtsL, and FtsQ homologs among bacteria and find that the proteins of this complex are likely more widely distributed among bacteria than was previously thought.     

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).     

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

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

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

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

Cellular Localization of Predicted Transmembrane and Soluble Chemoreceptors in Sinorhizobium meliloti

Bacterial chemoreceptors primarily locate in clusters at the cell pole, where they form large sensory complexes which recruit cytoplasmic components of the signaling pathway. The genome of the soil bacterium Sinorhizobium meliloti encodes seven transmembrane and two soluble chemoreceptors. We have investigated the localization of all nine chemoreceptors in vivo using genome-encoded fusions to a variant of the enhanced green fluorescent protein and to monomeric red fluorescent protein. Six of the transmembrane (McpT to McpX and McpZ) and both soluble (McpY and IcpA) receptors localize to the cell pole. Only McpS, encoded from the symbiotic plasmid pSymA, is evenly distributed in the cell. While the synthesis of all polar localized receptors is confined to exponential growth correlating with the motility phase of cells, McpS is only weakly expressed throughout cell culture growth. Therefore, motile S. meliloti cells form one major chemotaxis cluster that harbors all chemoreceptors except for McpS. Colocalization and deletion analysis demonstrated that formation of polar foci by the majority of receptors is dependent on other chemoreceptors and that receptor clusters are stabilized by the presence of the chemotaxis proteins CheA and CheW. The transmembrane McpV and the soluble IcpA localize to the pole independently of CheA and CheW. However, in mutant strains McpV formed delocalized polar caps that spread throughout the cell membrane while IcpA exhibited increased bipolarity. Immunoblotting of fractionated cells revealed that IcpA, which lacks any hydrophobic domains, nevertheless is associated to the cell membrane.The chemosensory machinery of Escherichia coli and other bacteria is arranged in large protein clusters (22, 28, 43, 49). One individual signaling unit is formed by a ternary assembly of chemoreceptor dimers, the histidine kinase CheA, and the so-called adaptor protein CheW. E. coli cells contain 20,000 receptor molecules (22). Recent studies suggest that the stoichiometry of such chemosensory complexes is flexible (17, 32). Allosteric interactions among receptors in a chemosensory cluster facilitate amplification and integration of chemotactic stimuli (20, 21, 41, 42).In contrast to E. coli, which has a single set of che genes and only five receptors, some species from the alpha subgroup of the proteobacteria, such as Pseudomonas aeruginosa, Rhodobacter sphaeroides, and Sinorhizobium meliloti, encode multiple chemotaxis-like systems, reflecting their complex lifestyle. The opportunistic pathogen P. aeruginosa possesses four chemotaxis systems that together have 26 known receptor genes (47), while the nonsulfur bacterium R. sphaeroides has three separate che operons with 13 known receptor-like genes (27).The symbiotic soil bacterium S. meliloti possesses eight methyl-accepting chemotaxis proteins (MCPs), McpS to McpZ, and one transducer-like-protein, IcpA, which lacks the conserved Glu or Gln residues that serve as methyl-accepting sites (29). Seven of the MCP proteins are localized in the cytoplasmic membrane via two membrane-spanning regions, whereas McpY and IcpA lack such hydrophobic regions. The S. meliloti mcpS gene is the third gene of the che2 operon located on the symbiotic plasmid pSymA (4). The icpA gene is the first gene of the chromosomal che operon comprising a total of 10 genes (9). This operon is part of the flagellar gene cluster with 56 chemotaxis, motor, and flagellar genes residing on one contiguous 51.4-kb chromosomal region (7, 46). For bacteria with numerous chemoreceptor genes, it is not unusual to find most of them located outside chemotaxis operons. This is the case with six monocistronic S. meliloti mcp genes which are scattered throughout the genome. The remaining mcpW gene is cotranscribed with a putative cheW gene. In this study, we examined the localization of the nine receptor gene products in the S. meliloti cell by fluorescence microscopy in wild-type and various deletion strains. The cellular localization of the two soluble receptors, McpY and IcpA, was also analyzed in vitro using an immunoblot assay on fractionated cell components. Furthermore, timing of chemoreceptor gene expression during exponential and stationary phase was determined.     

Rapid Microarray-Based Genotyping of Enterohemorrhagic Escherichia coli Serotype O156:H25/H?/Hnt Isolates from Cattle and Clonal Relationship Analysis

Since enterohemorrhagic Escherichia coli (EHEC) isolates of serogroup O156 have been obtained from human diarrhea patients and asymptomatic carriers, we studied cattle as a potential reservoir for these bacteria. E. coli isolates serotyped by agglutination as O156:H25/H−/Hnt strains (n = 32) were isolated from three cattle farms during a period of 21 months and characterized by rapid microarray-based genotyping. The serotyping by agglutination of the O156 isolates was not confirmed in some cases by the results of DNA-based serotyping as only 25 of the 32 isolates were conclusively identified as O156:H25. In the multilocus sequence typing (MLST) analysis, all EHEC O156:H25 isolates were characterized as sequence type 300 (ST300) and ST688, which differ by a single-nucleotide exchange in the purA gene. Oligonucleotide microarrays allow simultaneous detection of a wider range of EHEC-associated and other E. coli virulence markers than other methods. All O156:H25 isolates showed a wide spectrum of virulence factors typical for EHEC. The stx₁ genes combined with the EHEC hlyA (hlyA_EHEC) gene, the eae gene of the ζ subtype, as well as numerous other virulence markers were present in all EHEC O156:H25 strains. The behavior of eight different cluster groups, including four that were EHEC O156:H25, was monitored in space and time. Variations in the O156 cluster groups were detected. The results of the cluster analysis suggest that some O156:H25 strains had the genetic potential for a long persistence in the host and on the farm, while other strains did not. As judged by their pattern of virulence markers, E. coli O156:H25 isolates of bovine origin may represent a considerable risk for human infection. Our results showed that the miniaturized E. coli oligonucleotide arrays are an excellent tool for the rapid detection of a large number of virulence markers.Shiga toxin-producing Escherichia coli (STEC) strains comprise a group of zoonotic enteric pathogens (45). In humans, infections with some STEC serotypes may result in hemorrhagic or nonhemorrhagic diarrhea, which can be complicated by the hemolytic uremic syndrome (HUS) (32). These STEC strains are also designated enterohemorrhagic Escherichia coli (EHEC). Consequently, EHEC strains represent a subgroup of STEC with high pathogenic potential for humans. Although E. coli O157:H7 is the most frequent EHEC serotype implicated in HUS, other serotypes can also cause this complication. Non-O157:H7 EHEC strains including serotypes O26:H11/H−, O103:H2/H−, O111:H8/H10/H−, and O145:H28/H25/H− and sorbitol-fermenting E. coli O157:H− isolates are present in about 50% of stool cultures from German HUS patients (10, 42). However, STEC strains that cause human infection belong to a large number of E. coli serotypes, although a small number of STEC isolates of serogroup O156 were associated with human disease (7). Strains of the serotypes O156:H1/H8/H21/H25 were found in human cases of diarrhea or asymptomatic infections (9, 22, 25, 26). The detection of STEC of serogroup O156 from healthy and diseased ruminants such as cattle, sheep, and goats was reported by several authors (1, 11-13, 21, 39, 46, 50, 52). Additional EHEC-associated virulence genes such as stx, eae, hlyA_EHEC, or nlaA were found preferentially in the serotypes O156:H25 and O156:H− (11-13, 21, 22, 50, 52).Numerous methods exist for the detection of pathogenic E. coli, including genotypic and phenotypic marker assays for the detection of virulence genes and their products (19, 47, 55, 57). All of these methods have the common drawback of screening a relatively small number of determinants simultaneously. A diagnostic DNA microarray based on the ArrayTube format of CLONDIAG GmbH was developed as a viable alternative due to its ability to screen multiple virulence markers simultaneously (2). Further microarray layouts working with the same principle but different gene targets were developed for the rapid identification of antimicrobial resistance genes in Gram-negative bacteria (5) and for the rapid DNA-based serotyping of E. coli (4). In addition, a protein microarray for E. coli O serotyping based on the ArrayTube format was described by Anjum et al. (3).The aim of our study was the molecular genotyping of bovine E. coli field isolates of serogroup O156 based on miniaturized E. coli oligonucleotide arrays in the ArrayStrip format and to combine the screening of E. coli virulence markers, antimicrobial resistance genes, and DNA serotyping targets, some of which were partially described previously for separate arrays (2, 4, 5). The epidemiological situation in the beef herds from which the isolates were obtained and the spatial and temporal behavior of the clonal distribution of E. coli serogroup O156 were analyzed during the observation period. The potential risk of the isolates inducing disease in humans was assessed.     

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.     

Enumeration and Characterization of Antimicrobial-Resistant Escherichia coli Bacteria in Effluent from Municipal,Hospital, and Secondary Treatment Facility Sources

We describe a modification of the most probable number (MPN) method for rapid enumeration of antimicrobial-resistant Escherichia coli bacteria in aqueous environmental samples. E. coli (total and antimicrobial-resistant) bacteria were enumerated in effluent samples from a hospital (n = 17) and municipal sewers upstream (n = 5) and downstream (n = 5) from the hospital, effluent samples from throughout the treatment process (n = 4), and treated effluent samples (n = 13). Effluent downstream from the hospital contained a higher proportion of antimicrobial-resistant E. coli than that upstream from the hospital. Wastewater treatment reduced the numbers of E. coli bacteria (total and antimicrobial resistant); however, antimicrobial-resistant E. coli was not eliminated, and E. coli resistant to cefotaxime (including extended-spectrum beta-lactamase [ESBL] producers), ciprofloxacin, and cefoxitin was present in treated effluent samples.The emergence and dissemination of antimicrobial resistance are well established as clinical problems that affect human and animal health. Escherichia coli is an important element of the flora of the human and animal intestine and a significant pathogen associated with gastrointestinal infection, urinary tract infections, and a variety of other extraintestinal infections (4). E. coli shed into the environment can survive for significant periods (7, 14, 23). Detection of E. coli in water and food is widely used as a microbiological indication of fecal contamination.Data on the significance of environmental contamination with antimicrobial-resistant E. coli for human health are limited. Previous reports have shown that antimicrobial-resistant strains of bacteria are present in various effluents, such as hospital effluent discharge (8, 10, 16, 21), inflow effluent to a wastewater treatment plant (WWTP) (15), and outflow-treated effluent from a wastewater treatment plant (2, 12, 13, 18, 27). A wastewater treatment plant treating effluent from hospitals may be associated with discharge of relatively high levels of antimicrobial-resistant E. coli compared with those of a plant treating municipal effluent that does not include hospital effluent discharge (22). There are few reports of quantitative data on antimicrobial-resistant E. coli bacteria in effluent, reflecting the lack of a convenient method for their enumeration (12, 15, 22). Previous methods available for the detection of antimicrobial-resistant E. coli in a water sample have generally involved the isolation of E. coli and the selection of some isolates for susceptibility testing. In such cases, the proportions of antimicrobial-resistant organisms are based only on those isolates selected and are therefore not representative of the entire population. By adding the antimicrobial agent of interest to the water sample before testing, we have adapted a commercial most probable number (MPN) method (the Colilert system) for enumerating the total number of E. coli isolates resistant to that agent in a sample.     

Rapid Affinity Immunochromatography Column-Based Tests for Sensitive Detection of Clostridium botulinum Neurotoxins and Escherichia coli O157

Existing methods for detection of food-borne pathogens and their toxins are frequently time-consuming, require specialized equipment, and involve lengthy culture procedures and/or animal testing and are thus unsuitable for a rapid response to an emergency public health situation. A series of simple and rapid affinity immunochromatography column (AICC) assays were developed to detect Clostridium botulinum neurotoxin types A, B, E, and F and Escherichia coli O157 in food matrices. Specifically, for milk, grape juice with peach juice, and bottled water, the detection limit for the botulinum neurotoxin type A complex was 0.5 ng. Use of this method with a 10-ml sample would therefore result in a detection limit of 50 pg ml^−l. Thus, this assay is approximately 2 orders of magnitude more sensitive than a comparable lateral-flow assay. For botulinum neurotoxin complex types B, E, and F, the minimum detection limit was 5 ng to 50 ng. Sensitive detection of E. coli O157 was achieved, and the detection limit was 500 cells. The AICC test was also shown to be specific, rapid, and user friendly. This test takes only 15 to 30 min to complete without any specialized equipment and thus is suitable for use in the field. It has the potential to replace existing methods for presumptive detection of botulinum neurotoxin types A, B, E, and F and E. coli O157 in contaminated matrices without a requirement for preenrichment.The majority of conventional methods used for detection and identification of pathogenic microorganisms, viruses, and/or their toxins lack the speed and sensitivity necessary for use in the field (they typically are not completed in a single day) and also require specialized equipment (20). Rapid methods, including antibody-based and nucleic acid-based assays, have revolutionized the methodology for detection of microbial pathogens and their toxins in foods (16). However, while most antibody-based and nucleic acid-based assays are rapid, specialized equipment is often required, and specific enrichment is needed to achieve the necessary sensitivity. This means that the analysis time can still be several days (16). Lateral-flow assays (LFAs) and column flow assays are tests that have considerable merit in terms of rapidity and ease of use in the field without specialized equipment (4, 5, 8, 19, 34).Two contrasting agents were used as detection targets in this study: (i) a potent microbial toxin (Clostridium botulinum neurotoxin), including type A, B, E, and F neurotoxins; and (ii) an infectious pathogen, Escherichia coli O157. These two targets present different problems for detection; the first target is a protein toxin, and the second target is intact bacterial cells. The botulinum neurotoxin is the most potent toxin known, and as little as 30 to 100 ng has the potential to be fatal to humans (28). It is responsible for botulism, a severe neuroparalytic disease that affects humans and also animals and birds (28). There are seven antigenically distinct botulinum neurotoxins (types A to G), and a number of subtypes have also been described (9, 11, 15, 28, 36). Botulism in humans is associated principally with neurotoxin types A, B, E, and F (27, 29). Since the botulinum neurotoxins are the toxic agents and they can be produced by six physiologically distinct clostridia (28), considerable emphasis has been placed on detection of the neurotoxins rather than the bacteria. The “gold standard” method for detecting botulinum neurotoxins is the mouse bioassay due to its high levels of sensitivity and specificity. However, this technique is also problematic (33). It typically requires 24 to 48 h to yield results, is expensive, and is becoming less favored because of its use of animals (4). The alternative tests include enzyme-linked immunosorbent assays (ELISAs), lateral-flow assays (LFAs), a chemiluminescent slot blot immunoassay, surface plasmon resonance (SPR), the assay with a large immunosorbent surface area (ALISSA) test, and quantum dot immunoassays (4, 5, 7, 22, 43, 46). Lateral-flow assays are available and are convenient for toxin testing as they are easy to perform and rapid (<30 min) and no additional equipment is required. However, their poor sensitivity has limited their use (23).E. coli O157 produces a cytotoxin (verotoxin), and an E. coli O157 infection can lead to severe bloody diarrhea, kidney failure, brain damage, and death. Enumeration, identification, and control of this pathogen are challenging due to the low infectious dose necessary to cause disease, which is between 2 and 2,000 ingested cells (41). Sources of E. coli O157 infection include ground beef and unpasteurized milk and apple juice (1), raw milk (6), and spinach and lettuce (42). Isolation of E. coli O157:H7 from water, food, and environmental samples is laborious. Culture is difficult due to the large competing microflora that either overgrows or mimics the non-sorbitol-fermenting organism E. coli O157:H7 (12). According to Tokarskyy and Marshall (41), the largest group of rapid test kits commercially available for testing for the presence of E. coli O157 in food includes immunological methods, such as latex agglutination, reverse passive latex agglutination, immunodiffusion, ELISA, immunomagnetic separation (IMS), and immunoprecipitation. The other methods that have been developed include a dipstick test device (2), a lateral-flow immunoassay (8), real-time PCR (39), and an enzyme-linked immunomagnetic chemiluminescent assay (17). However, in many cases these tests require preenrichment or have limited sensitivity.The objective of the work described here was to develop a rapid sensitive diagnostic test for detection of botulinum neurotoxins A, B, E, and F and E. coli O157 that can be used without preenrichment.     

Absence of Escherichia coli Phylogenetic Group B2 Strains in Humans and Domesticated Animals from Jeonnam Province,Republic of Korea

Multiplex PCR analyses of DNAs from genotypically unique Escherichia coli strains isolated from the feces of 138 humans and 376 domesticated animals from Jeonnam Province, South Korea, performed using primers specific for the chuA and yjaA genes and an unknown DNA fragment, TSPE4.C2, indicated that none of the strains belonged to E. coli phylogenetic group B2. In contrast, phylogenetic group B2 strains were detected in about 17% (8 of 48) of isolates from feces of 24 wild geese and in 3% (3 of 96) of isolates obtained from the Yeongsan River in Jeonnam Province, South Korea. The distribution of E. coli strains in phylogenetic groups A, B1, and D varied depending on the host examined, and there was no apparent seasonal variation in the distribution of strains in phylogenetic groups among the Yeongsan River isolates. The distribution of four virulence genes (eaeA, hlyA, stx₁, and stx₂) in isolates was also examined by using multiplex PCR. Virulence genes were detected in about 5% (38 of 707) of the total group of unique strains examined, with 24, 13, 13, and 9 strains containing hlyA, eaeA, stx₂, and stx₁, respectively. The virulence genes were most frequently present in phylogenetic group B1 strains isolated from beef cattle. Taken together, results of these studies indicate that E. coli strains in phylogenetic group B2 were rarely found in humans and domesticated animals in Jeonnam Province, South Korea, and that the majority of strains containing virulence genes belonged to phylogenetic group B1 and were isolated from beef cattle. Results of this study also suggest that the relationship between the presence and types of virulence genes and phylogenetic groupings may differ among geographically distinct E. coli populations.Escherichia coli is a normal inhabitant of the lower intestinal tract of warm-blooded animals and humans. While the majority of E. coli strains are commensals, some are known to be pathogenic, causing intestinal and extraintestinal diseases, such as diarrhea and urinary tract infections (42). Phylogenetic studies done using multilocus enzyme electrophoresis and 72 E. coli strains in the E. coli reference collection showed that E. coli strains can be divided into four phylogenetic groups (A, B1, B2, and D) (20, 41, 48). Recently, a potential fifth group (E) has also been proposed (11). Since multiplex PCR was developed for analysis of phylogenetic groups (6), a number of studies have analyzed a variety of E. coli strains for their phylogenetic group association (10, 12, 17, 18, 23, 54). Duriez et al. (10) reported the possible influence of geographic conditions, dietary factors, use of antibiotics, and/or host genetic factors on the distribution of phylogenetic groups among 168 commensal E. coli strains isolated from human stools from three geographically distinct populations in France, Croatia, and Mali. Random-amplified polymorphic DNA analysis of the intraspecies distribution of E. coli in pregnant women and neonates indicated that there was a correlation between the distribution of phylogenetic groups, random-amplified polymorphic DNA groups, and virulence factors (54). Moreover, based on comparisons of the distribution of E. coli phylogenetic groups among humans of different sexes and ages, it has been suggested that E. coli genotypes are likely influenced by morphological, physiological, and dietary differences (18). In addition, climate has also been proposed to influence the distribution of strains within E. coli phylogenetic groups (12). There are now several reports indicating that there is a potential relationship between E. coli phylogenetic groups, age, and disease. For example, E. coli isolates belonging to phylogenetic group B2 have been shown to predominate in infants with neonatal bacterial meningitis (27) and among urinary tract and rectal isolates (55). Also, Nowrouzian et al. (39) and Moreno et al. (37) reported that strains belonging to phylogenetic group B2 persisted among the intestinal microflora of infants and were more likely to cause clinical symptoms.Boyd and Hartl (2) reported that among the E. coli strains in the E. coli reference and the diarrheagenic E. coli collections, strains in phylogenetic group B2 carry the greatest number of virulence factors, followed by those in group D. Virulence factors carried by group B2 strains are thought to contribute to their strong colonizing capacity; a greater number of virulence genes have been detected in resident strains than in transient ones (38). Moreover, a mouse model of extraintestinal virulence showed that phylogenetic group B2 strains killed mice at greater frequency and possessed more virulence determinants than strains in other phylogenetic groups, suggesting a link between phylogeny and virulence genes in E. coli extraintestinal infection (45). In contrast, Johnson and Kuskowski (25) suggested that a group B2 ancestral strain might have simply acquired virulence genes by chance and that these genes were vertically inherited by group members during clonal expansion. However, numerous studies published to date suggest that there is a relationship between the genomic background of phylogenetic group B2 and its association with virulence factors (12, 28, 35, 39, 45).Both enteropathogenic and enterohemorrhagic E. coli (EPEC and EHEC, respectively) strains are among the most important food-borne pathogens worldwide, often causing severe gastrointestinal disease and fatal infections (13). While EPEC strains cause diarrhea and generally do not produce enterotoxin, they possess an adherence factor which is controlled by the chromosomal gene eaeA, encoding intimin (8). Unlike the EPEC strains, however, the EHEC strains typically contain the hlyA, stx₁, and stx₂ virulence genes, encoding hemolysins and Shiga-like type 1 and 2 toxins, respectively, and eaeA. The ability to detect EHEC has been greatly facilitated by the use of multiplex PCR (13, 44, 53). Several studies have shown that strains producing Shiga-like toxin 2 are more frequently found in cases of hemolytic-uremic syndrome than are those containing Shiga-like toxin 1 (30, 43, 46, 49).In the study reported here, we examined the distribution of phylogenetic groups and the prevalence of virulence genes in 659 genotypically unique E. coli strains isolated from humans and domestic animals in South Korea. In addition, we also tested 48 and 96 nonunique E. coli isolates from wild geese and the Yeongsan River, respectively, for phylogenetic distribution and virulence gene profiles. Here, we report that contrary to what has been previously reported in other parts of the world, no E. coli strains belonging to phylogenetic group B2 were found in domesticated animals and in humans from Jeonnam Province, South Korea. We also report that among the strains we examined, virulence genes were mainly found in phylogenetic group B1 strains isolated from beef cattle. Results of these studies may prove to be useful for the development of risk management strategies to maintain public health.     

YjhS (NanS) Is Required for Escherichia coli To Grow on 9-O-Acetylated N-Acetylneuraminic Acid

The nanATEK-yhcH, yjhATS, and yjhBC operons in Escherichia coli are coregulated by environmental N-acetylneuraminic acid, the most prevalent sialic acid in nature. Here we show that YjhS (NanS) is a probable 9-O-acetyl N-acetylneuraminic acid esterase required for E. coli to grow on this alternative sialic acid, which is commonly found in mammalian host mucosal sites.The coregulated nanATEK-yhcH, yjhATS, and yjhBC operons involved in sialic acid catabolism in Escherichia coli are thought to be induced by the most common sialic acid, N-acetylneuraminic acid (Neu5Ac), through reversible inactivation of the NanR repressor encoded by nanR mapping immediately upstream of nanA (15, 27, 28; http://vetmed.illinois.edu/path/sialobiology/). Sialic acids are a family of over 40 naturally occurring 9-carbon keto sugar acids found mainly in metazoans of the deuterostome (starfish to human) developmental lineage and in some, mostly pathogenic, bacteria, where sialic acids expressed at the microbial cell surface inhibit host innate immunity (27). By contrast, most bacterial commensals and pathogens catabolize sialic acids as sole carbon and nitrogen sources, indicating exploitation of the sialic acid-rich host mucosal environment by a wide range of species (2, 27, 28). Interestingly, in vivo experimental evidence further indicates that sialic acid catabolism functions directly (nutrition) or indirectly (surface decoration and cell signaling) in host-microbe commensal and pathogenic interactions in organisms such as E. coli, Haemophilus influenzae, Pasteurella multocida, Salmonella enterica serovar Typhi, Streptococcus pneumoniae, Vibrio vulnificus, and Vibrio cholerae (1, 3, 5, 6, 10, 14, 23, 24, 26, 29). The animal species used for these studies include rodent models and natural hosts such as cattle and turkeys. The structural diversity of sialic acids at the terminal positions on glycoconjugates (glycoproteins and glycolipids) of mucosal surfaces of these hosts requires sialidases, acetyl esterases, and probably other enzymes that convert alternative or at least minor sialic acids to the more digestible Neu5Ac form (8, 9). We have previously demonstrated that E. coli has an epicurean propensity for metabolizing alternative sialic acids (30, 31). In the current communication, we show that YjhS is required for growth of E. coli on 9-O-acetyl-N-acetylneuraminic acid (Neu5,9Ac₂).Because most sialic acids are bound to other sugars, including other sialic acids, as part of the oligosaccharide chains on glycoconjugates, either microbial or endogenous (host) sialidases (NanH, or N-acylneuraminate hydrolases) are needed to release free sugar, which is then transported by NanT in E. coli (15, 16, 26, 31). Once internalized, sialic acid is cleaved by an nanA-encoded aldolase or lyase to yield the 6-carbon hexosamine, N-acetylmannosamine (ManNAc), and pyruvate, with the latter entering the tricarboxylic acid cycle or gluconeogenesis. ManNAc is converted to its 6-phosphate derivative by a specific kinase encoded by nanK and epimerized by NanE to yield N-acetylglucosamine 6-phosphate, which is converted to fructose 6-phosphate by products of the nag operon (15, 17, 31, 32). The functions of the coregulated yjhS, yjhB, yjhC, and yhcH gene products are unknown but are not required for growth on Neu5Ac (15). However, YjhA (NanC) is an outer membrane porin required for diffusion of Neu5Ac in the absence of the major porins (7), while YjhT (NanM) is a mutarotase that catalyzes the conversion of the alpha sialic acid isomer to the more thermodynamically stable beta form (21). Neither nanC nor nanM is required for growth on Neu5Ac (15), suggesting that yjhS, yjhBC, and yhcH are involved in reactions that convert alternative sialic acids to Neu5Ac (22, 23). YhcH was crystallized and has been suggested to be an isomerase or epimerase involved in processing N-glycolylneuraminic acid (Neu5Gc) (25), but deletion of yhcH did not affect growth on this sialic acid as a sole carbon source (16).Computer-assisted analysis indicated that YjhB is a permease similar to NanT (16) whereas YjhC is a likely oxidoreductase or dehydrogenase. Orthologs of yhcH, nanC, nanM, and yjhBC are found in most bacterial species with intact Neu5Ac utilization systems, while yjhS is confined to E. coli and shigellae, either as part of the chromosomes in these strains or integrated with phages or phage remnants. However, a significant match (E value = 0.0007) was found between YjhS and AxeA in Rhodopirellula baltica, where AxeA is an acetyl xylan esterase (11), suggesting YjhS might be a sialate esterase. We propose that YjhS should be designated NanS to indicate its direct participation in utilization of an alternative sialic acid.