Variability of Burkholderia pseudomallei Strain Sensitivities to Chlorine Disinfection

Burkholderia pseudomallei is a select agent and the causative agent of melioidosis. Variations in previously reported chlorine and monochloramine concentration time (Ct) values for disinfection of this organism make decisions regarding the appropriate levels of chlorine in water treatment systems difficult. This study identified the variation in Ct values for 2-, 3-, and 4-log₁₀ reductions of eight environmental and clinical isolates of B. pseudomallei in phosphate-buffered water. The greatest calculated Ct values for a 4-log₁₀ inactivation were 7.8 mg·min/liter for free available chlorine (FAC) at pH 8 and 5°C and 550 mg·min/liter for monochloramine at pH 8 and 5°C. Ionic strength of test solutions, culture hold times in water, and cell washing were ruled out as sources of the differences in prior observations. Tolerance to FAC was correlated with the relative amount of extracellular material produced by each isolate. Solid-phase cytometry analysis using an esterase-cleaved fluorochrome assay detected a 2-log₁₀-higher level of organisms based upon metabolic activity than did culture, which in some cases increased Ct values by fivefold. Despite strain-to-strain variations in Ct values of 17-fold for FAC and 2.5-fold for monochloramine, standard FAC disinfection practices utilized in the United States should disinfect planktonic populations of these B. pseudomallei strains by 4 orders of magnitude in less than 10 min at the tested temperatures and pH levels.Burkholderia pseudomallei, a gram-negative soil saprophyte, is endemic to Southeast Asia (30) and northern Australia and has been linked to waterborne illness in these areas (9, 15). B. pseudomallei has been identified by the Centers for Disease Control and Prevention (CDC) as a select agent. The organism can tolerate a wide variety of soil and temperature conditions (33), which enables it to exist in soil and water in subtropical zones (10, 19, 32, 35). Information on survival of B. pseudomallei in drinking water containing disinfectants is necessary to ensure that the end users of water distribution systems are sufficiently protected from exposure to this organism.Free available chlorine (FAC) and monochloramine are the most commonly used drinking water disinfectants (1, 2, 28). Factors such as ionic strength, pH, turbidity and chlorine demand, biofilm growth, and the physiological state of the cells can alter disinfection efficacy (6, 16, 18, 22, 31). The contact concentration and time values (Ct) (mg·min/liter) required to achieve disinfection have been observed to vary between strains of the same organism (23, 36). Additionally, stress-related phenotypic changes in Vibrio species have been linked to increased tolerance to chlorine (20, 24, 34).Several strains of B. pseudomallei endemic to northern Australia were found by Howard and Inglis (13) to be more chlorine and monochloramine tolerant than other strains via techniques such as plate culture, a most-probable-number (MPN) recovery method, and flow cytometry using a membrane integrity stain. Disinfection studies using cultural methods performed indicated that B. pseudomallei is very sensitive to FAC (26) and monochloramine (25). However, other published data indicate high levels of chlorine resistance for this organism (13). In order to determine if the differences between these published reports are due to differences in methodology or strain type, disinfection studies were performed with multiple strains of B. pseudomallei isolated from both clinical and environmental sources. These studies were designed to evaluate the range of B. pseudomallei sensitivities to FAC and monochloramine.Cells injured by chlorine disinfection may enter nonculturable states (21), making this still-viable portion of the population difficult to enumerate. An alternative assay which evaluates membrane integrity and metabolic activity was also employed in this study. This method allows direct counts of metabolically active organisms, which may be able to resume growth under more favorable conditions, and can be compared to plate culture recovery methods.     

Detection of Toxoplasma gondii Oocysts in Water Sample Concentrates by Real-Time PCR

PCR techniques in combination with conventional parasite concentration procedures have potential for the sensitive and specific detection of Toxoplasma gondii oocysts in water. Three real-time PCR assays based on the B1 gene and a 529-bp repetitive element were analyzed for the detection of T. gondii tachyzoites and oocysts. Lower sensitivity and specificity were obtained with the B1 gene-based PCR than with the 529-bp repeat-based PCR. New procedures for the real-time PCR detection of T. gondii oocysts in concentrates of surface water were developed and tested in conjunction with a method for the direct extraction of inhibitor-free DNA from water. This technique detected as few as one oocyst seeded to 0.5 ml of packed pellets from water samples concentrated by Envirocheck filters. Thus, this real-time PCR may provide a detection method alternative to the traditional mouse assay and microscopy.Toxoplasma gondii is a ubiquitous parasite found in all classes of warm-blooded vertebrates. Nearly one-third of humans have been exposed to this parasite (15). In immunocompetent adults, acute infection normally results in transient influenza-like symptoms, but in immunocompromised persons retinochoroiditis and encephalitis are more common. Infected individuals can retain the parasite as quiescent tissue cysts for long periods, but invasive infection can occur if the immune status of the infected person deteriorates (42). If women become infected during pregnancy, the parasite can cause abortion or seriously damage the fetus. The potential morbidity from the ingestion of oocysts of T. gondii and the organism''s low infectious dose are a great concern for public health. There are at least four reported waterborne outbreaks of toxoplasmosis (2, 3, 14, 44), and endemic toxoplasmosis in Brazil is associated with the consumption of water or ice contaminated with T. gondii oocysts (1, 23), demonstrating the potential for the waterborne transmission of this disease (15).There is no rapid detection method for T. gondii oocysts recovered from water or other environmental samples. Traditionally, the detection of protozoa in water required their concentration from large volumes of water by filtration or centrifugation, isolation from concentrated particulates by immunomagnetic separation (IMS) or other methods, and detection by immunofluorescence microscopy, the infection of cultured cells, biochemistry, animal infection tests, molecular techniques, or combinations of these (17, 58). For T. gondii oocysts there are no commercially available IMS techniques, no widely available immunofluorescent staining reagents, and no standardized cultivation protocols. The identification of oocysts from environmental samples has included differential floatation and mouse inoculation (27). Recently, IMS techniques have been developed for the isolation of T. gondii oocysts and sporocysts in water (16, 18). Both the oocyst and sporocyst IMS assays, however, had poor specificity, because antibodies cross-reacted with water debris and the sporocyst wall of Hammondia hammondi, Hammondia heydorni, and Neospora caninum (16).PCR is becoming a favored technique for the detection of T. gondii oocysts in water (32, 35, 36, 46, 49, 55) over the conventional mouse bioassay (27, 55), as it reduces the detection time from weeks to 1 to 2 days. Although they have been developed for the detection of T. gondii in clinical specimens (50), no real-time PCR assays have been adapted for the detection of oocysts in water samples, possibly because of expected high concentrations of PCR inhibitors and low numbers of T. gondii oocysts in environmental samples (55).There are several unresolved issues regarding the effectiveness of the PCR detection of T. gondii oocysts in water. The most readily available method for the isolation of T. gondii oocysts from water samples is flocculation or sucrose floatation prior to DNA extraction (35, 36, 49, 55). Because sucrose flotation and flocculation result in oocyst losses, the recovery rate of using these methods is poor. For DNA extraction, the phenol-chloroform method or QIAamp mini kit frequently is used (16, 35, 36, 46, 55). When oocysts are recovered from water either by the Environmental Protection Agency (EPA) information collection rule method (53) or EPA Method 1623 (54) without purification by IMS, neither the conventional phenol-chloroform DNA extraction nor the QIAamp mini kit is effective at removing PCR inhibitors (30, 55, 57).Recently, a method was used effectively in the analysis of Cryptosporidium oocysts in surface water, storm water, and wastewater samples (30). This method extracted DNA directly from water concentrates without pathogen IMS, differential flotation, or enrichment cultures, and it utilized a commercial DNA extraction kit, the FastDNA spin kit for soil, and a high concentration of nonacetylated bovine serum albumin in PCR. The FastDNA soil kit has a higher capacity for PCR inhibitor removal than several other commercial extraction kits designed for environmental samples. The use of nonacetylated bovine serum in the PCR neutralizes residual PCR inhibitors that are coextracted with the DNA (30).In the present study, the performance of two published LightCycler real-time PCR assays based on the multicopy B1 gene and 529-bp repetitive element (13, 45) and a newly developed LightCycler real-time PCR assay using a common primer set were analyzed for the detection of T. gondii, using pure DNA and DNA extracted by the aforementioned extraction method (30) from water sample concentrates seeded with known number of oocysts.     

Free-Living Protozoa in Two Unchlorinated Drinking Water Supplies,Identified by Phylogenic Analysis of 18S rRNA Gene Sequences

Free-living protozoan communities in water supplies may include hosts for Legionella pneumophila and other undesired bacteria, as well as pathogens. This study aimed at identifying free-living protozoa in two unchlorinated groundwater supplies, using cultivation-independent molecular approaches. For this purpose, samples (<20°C) of treated water, distributed water, and distribution system biofilms were collected from supply A, with a low concentration of natural organic matter (NOM) (<0.5 ppm of C), and from supply B, with a high NOM concentration (7.9 ppm of C). Eukaryotic communities were studied using terminal restriction fragment length polymorphism and clone library analyses of partial 18S rRNA gene fragments and a Hartmannella vermiformis-specific quantitative PCR (qPCR). In both supplies, highly diverse eukaryotic communities were observed, including free-living protozoa, fungi, and metazoa. Sequences of protozoa clustered with Amoebozoa (10 operational taxonomic units [OTUs]), Cercozoa (39 OTUs), Choanozoa (26 OTUs), Ciliophora (29 OTUs), Euglenozoa (13 OTUs), Myzozoa (5 OTUs), and Stramenopiles (5 OTUs). A large variety of protozoa were present in both supplies, but the estimated values for protozoan richness did not differ significantly. H. vermiformis was observed in both supplies but was not a predominant protozoan. One OTU with the highest similarity to Acanthamoeba polyphaga, an opportunistic human pathogen and a host for undesired bacteria, was observed in supply A. The high level of NOM in supply B corresponded with an elevated level of active biomass and with elevated concentrations of H. vermiformis in distributed water. Hence, the application of qPCR may be promising in elucidating the relationship between drinking water quality and the presence of specific protozoa.Free-living protozoa are ubiquitous in natural freshwater environments (7, 38, 51, 71) but also proliferate in engineered water systems, including water treatment systems (3, 47, 70), distribution systems (6, 75), and tap water installations inside buildings (54, 69). Concentrations of protozoa, determined using cultivation methods and microscopy, range from <1 to 10⁴ cells liter⁻¹ in treated water (3, 47, 70, 75) and from <1 to 7 × 10⁵ cells liter⁻¹ in distribution systems (6, 61, 64, 75). Genera of free-living protozoa commonly observed in these systems and in tap water installations include Acanthamoeba, Echinamoeba, Hartmannella, Platyamoeba, Vahlkampfia, and Vannella (47, 58, 69, 70). In warm water systems, certain free-living protozoa, e.g., Acanthamoeba spp. (57), Balamuthia mandrillaris (62), Echinamoeba exandans (16), Hartmannella spp. (39, 56), Naegleria spp. (49, 57), Tetrahymena spp. (18, 33), and Vahlkampfia jugosa (56), serve as hosts for Legionella pneumophila, the etiologic agent of Legionnaires'' disease. High concentrations of L. pneumophila are generally associated with the proliferation of host protozoa in biofilms (38, 53). In addition, other amoeba-resistant, potentially pathogenic bacteria, e.g., Burkholderia spp. (28) and Mycobacterium spp. (37), have been observed in man-made aquatic environments (24). Free-living protozoa may enhance the multiplication of bacteria, serve as a transmission vector, or serve as a shelter against unfavorable environmental conditions, such as the presence of disinfectants. Furthermore, certain free-living protozoa are human pathogens, e.g., Naegleria fowleri (81), Balamuthia mandrillaris (77), and Acanthamoeba spp. (12) can cause encephalitis. Acanthamoeba spp. have also been associated with keratitis in persons wearing contact lenses (31).Free-living protozoa feed on bacteria, algae, fungi, other protozoa, and organic detritus in biofilms or in the planktonic phase, thereby affecting the structure of microbial communities. In turn, the community of free-living protozoa depends on the diversity and abundance of bacteria in the biofilm and in the planktonic phase (26, 50, 51, 55, 63, 65). Water quality is a critical factor for biofilm formation in distribution systems and tap water installations and therefore will affect the abundance and diversity of free-living protozoa in these systems (72, 78). However, information about the presence and identity of free-living protozoa in water supplies in relation to the quality of treated water is scarce, which may be attributed to the limitations of microscopic techniques and cultivation methods for detection and identification of these organisms, e.g., low detection limits and selectivity for specific groups (19).In this study, we applied a variety of cultivation-independent techniques, viz., quantitative PCR, terminal restriction fragment length polymorphism (T-RFLP) analysis, and cloning and sequencing of eukaryotic 18S rRNA gene fragments, for the detection and identification of free-living protozoa predominating in two unchlorinated groundwater supplies. The concentrations of dissolved natural organic matter (NOM) in treated water at the plant were <0.5 mg C liter⁻¹ and 7.9 mg C liter⁻¹, covering the entire range of NOM concentrations in drinking water in The Netherlands. The objectives of the study were (i) to elucidate the identities of and diversity in the free-living protozoa predominating in these two different water supplies and (ii) to trace the presence of host protozoa for L. pneumophila and pathogenic free-living protozoa. The study revealed that treated water and biofilms in the distribution systems of both water supplies contained a large variety of free-living protozoa, including protozoan hosts for Legionella bacteria.     

Roles of Small,Acid-Soluble Spore Proteins and Core Water Content in Survival of Bacillus subtilis Spores Exposed to Environmental Solar UV Radiation

Spores of Bacillus subtilis contain a number of small, acid-soluble spore proteins (SASP) which comprise up to 20% of total spore core protein. The multiple α/β-type SASP have been shown to confer resistance to UV radiation, heat, peroxides, and other sporicidal treatments. In this study, SASP-defective mutants of B. subtilis and spores deficient in dacB, a mutation leading to an increased core water content, were used to study the relative contributions of SASP and increased core water content to spore resistance to germicidal 254-nm and simulated environmental UV exposure (280 to 400 nm, 290 to 400 nm, and 320 to 400 nm). Spores of strains carrying mutations in sspA, sspB, and both sspA and sspB (lacking the major SASP-α and/or SASP-β) were significantly more sensitive to 254-nm and all polychromatic UV exposures, whereas the UV resistance of spores of the sspE strain (lacking SASP-γ) was essentially identical to that of the wild type. Spores of the dacB-defective strain were as resistant to 254-nm UV-C radiation as wild-type spores. However, spores of the dacB strain were significantly more sensitive than wild-type spores to environmental UV treatments of >280 nm. Air-dried spores of the dacB mutant strain had a significantly higher water content than air-dried wild-type spores. Our results indicate that α/β-type SASP and decreased spore core water content play an essential role in spore resistance to environmentally relevant UV wavelengths whereas SASP-γ does not.Spores of Bacillus spp. are highly resistant to inactivation by different physical stresses, such as toxic chemicals and biocidal agents, desiccation, pressure and temperature extremes, and high fluences of UV or ionizing radiation (reviewed in references 33, 34, and 48). Under stressful environmental conditions, cells of Bacillus spp. produce endospores that can stay dormant for extended periods. The reason for the high resistance of bacterial spores to environmental extremes lies in the structure of the spore. Spores possess thick layers of highly cross-linked coat proteins, a modified peptidoglycan spore cortex, a low core water content, and abundant intracellular constituents, such as the calcium chelate of dipicolinic acid and α/β-type small, acid-soluble spore proteins (α/β-type SASP), the last two of which protect spore DNA (6, 42, 46, 48, 52). DNA damage accumulated during spore dormancy is also efficiently repaired during spore germination (33, 47, 48). UV-induced DNA photoproducts are repaired by spore photoproduct lyase and nucleotide excision repair, DNA double-strand breaks (DSB) by nonhomologous end joining, and oxidative stress-induced apurinic/apyrimidinic (AP) sites by AP endonucleases and base excision repair (15, 26-29, 34, 43, 53, 57).Monochromatic 254-nm UV radiation has been used as an efficient and cost-effective means of disinfecting surfaces, building air, and drinking water supplies (31). Commonly used test organisms for inactivation studies are bacterial spores, usually spores of Bacillus subtilis, due to their high degree of resistance to various sporicidal treatments, reproducible inactivation response, and safety (1, 8, 19, 31, 48). Depending on the Bacillus species analyzed, spores are 10 to 50 times more resistant than growing cells to 254-nm UV radiation. In addition, most of the laboratory studies of spore inactivation and radiation biology have been performed using monochromatic 254-nm UV radiation (33, 34). Although 254-nm UV-C radiation is a convenient germicidal treatment and relevant to disinfection procedures, results obtained by using 254-nm UV-C are not truly representative of results obtained using UV wavelengths that endospores encounter in their natural environments (34, 42, 50, 51, 59). However, sunlight reaching the Earth''s surface is not monochromatic 254-nm radiation but a mixture of UV, visible, and infrared radiation, with the UV portion spanning approximately 290 to 400 nm (33, 34, 36). Thus, our knowledge of spore UV resistance has been constructed largely using a wavelength of UV radiation not normally reaching the Earth''s surface, even though ample evidence exists that both DNA photochemistry and microbial responses to UV are strongly wavelength dependent (2, 30, 33, 36).Of recent interest in our laboratories has been the exploration of factors that confer on B. subtilis spores resistance to environmentally relevant extreme conditions, particularly solar UV radiation and extreme desiccation (23, 28, 30, 34 36, 48, 52). It has been reported that α/β-type SASP but not SASP-γ play a major role in spore resistance to 254-nm UV-C radiation (20, 21) and to wet heat, dry heat, and oxidizing agents (48). In contrast, increased spore water content was reported to affect B. subtilis spore resistance to moist heat and hydrogen peroxide but not to 254-nm UV-C (12, 40, 48). However, the possible roles of SASP-α, -β, and -γ and core water content in spore resistance to environmentally relevant solar UV wavelengths have not been explored. Therefore, in this study, we have used B. subtilis strains carrying mutations in the sspA, sspB, sspE, sspA and sspB, or dacB gene to investigate the contributions of SASP and increased core water content to the resistance of B. subtilis spores to 254-nm UV-C and environmentally relevant polychromatic UV radiation encountered on Earth''s surface.     

Heterologous Expression of Tulip Petal Plasma Membrane Aquaporins in Pichia pastoris for Water Channel Analysis

Water channels formed by aquaporins (AQPs) play an important role in the control of water homeostasis in individual cells and in multicellular organisms. Plasma membrane intrinsic proteins (PIPs) constitute a subclass of plant AQPs. TgPIP2;1 and TgPIP2;2 from tulip petals are members of the PIP family. In this study, we overexpressed TgPIP2;1 and TgPIP2;2 in Pichia pastoris and monitored their water channel activity (WCA) either by an in vivo spheroplast-bursting assay performed after hypo-osmotic shock or by growth assay. Osmolarity, pH, and inhibitors of AQPs, protein kinases (PKs), and protein phosphatases (PPs) affect the WCA of heterologous AQPs in this expression system. The WCA of TgPIP2;2-expressing spheroplasts was affected by inhibitors of PKs and PPs, which indicates that the water channel of this homologue is regulated by phosphorylation in P. pastoris. From the results reported herein, we suggest that P. pastoris can be employed as a heterologous expression system to assay the WCA of PIPs and to monitor the AQP-mediated channel gating mechanism, and it can be developed to screen inhibitors/effectors of PIPs.The movement of water across cell membranes has long been thought to occur by free diffusion through the lipid bilayer. However, the discovery of the membrane protein CHIP28 in red blood cells has suggested the involvement of protein channels (29), and it is now well established that transmembrane water permeability is facilitated by aquaporins (AQPs), water channel proteins that are found in bacteria, fungi, plants, and animals (1, 7, 13, 24). AQPs contain six transmembrane α-helices and five connecting loops, and both the N and C termini are located in the cytosol. The monomers assemble into tetrameric complexes, with each monomer forming an individual water channel (11, 14, 24, 33). Apart from the exceptions of AQP11 and AQP12 from mice, as described by K. Ishibashi (15), AQPs have two signature Asn-Pro-Ala motifs, which are located in the second intracellular and the fifth extracellular loops, B and E.While 13 different AQPs have been identified in mammals (16), more than 33 AQP homologues have been discovered in plants (6, 17, 30). Plant AQPs fall into four subclasses: (i) the plasma membrane (PM) intrinsic proteins (PIPs), which are localized in the PM; (ii) the tonoplast intrinsic proteins (TIPs), which are localized in the vacuolar membranes; (iii) the nodulin-26-like intrinsic proteins; and (iv) the small basic intrinsic proteins (24). In Arabidopsis and maize, there are 13 PIPs, which can be divided further into two subfamilies, PIP1 and PIP2 (6, 17).The functions and mechanisms of regulation of plant AQPs have been extensively investigated (7, 13, 18, 24). There have been several reports on the water channel activity (WCA) of specific AQPs and their regulation by protein phosphorylation (3, 4, 8, 12, 18, 25, 32, 33). It has been shown that the WCA of the PIP2 member SoPIP2;1 from spinach is regulated by phosphorylation at two Ser residues (19, 33).The physiologically interesting temperature-dependent opening and closing of tulip (Tulipa gesneriana) petals occur concomitantly with water transport and are regulated by reversible phosphorylation of an undefined PIP (4, 5). Recently, four PIP homologues were isolated from tulip petals, and their WCAs have been analyzed by heterologous expression in Xenopus laevis oocytes (3). It has been shown that the tulip PIP TgPIP2;2 (DDBJ/EMBL/GenBank accession no. {"type":"entrez-nucleotide","attrs":{"text":"AB305617","term_id":"196259707","term_text":"AB305617"}}AB305617) is ubiquitously expressed in all organs of the tulip and that TgPIP2;2 is the most likely of the TgPIP homologues to be modulated by the reversible phosphorylation that regulates transcellular water transport and mediates petal opening and closing (3, 4). However, while the members of the PIP2 subfamily are characterized as water channels (6), TgPIP2;1 (DDBJ/EMBL/GenBank accession no. {"type":"entrez-nucleotide","attrs":{"text":"AB305616","term_id":"196259705","term_text":"AB305616"}}AB305616) shows no significant WCA in the oocyte expression system (3). There is growing interest in research on AQPs due to their crucial roles in the physiology of plants and animals (1, 16, 21-24, 26-28, 36). The assay of AQP channel activity is usually performed using either a X. laevis oocyte expression system (29) or a stopped-flow light-scattering spectrophotometer (35), both of which are not widely available. Furthermore, the complexity of these methods and requirement of expertise limit their high-throughput applications. In contrast, a Pichia pastoris expression system is simple to use, inexpensive, and feasible and can be used in high-throughput applications. Although a P. pastoris expression system has been shown to assay the WCA of a TIP (9), extensive research is necessary with other AQPs such as PIPs or AQPs present in intragranular membranes to establish whether this assay system can be used to characterize a water channel and study its regulation mechanisms. With this in view, in the study reported herein, TgPIP2;1 and TgPIP2;2 have been heterologously expressed in P. pastoris, and their WCAs have been assayed. The effects of several factors, such as osmolarity, pH, and inhibitors of protein kinases (PKs) and protein phosphatases (PPs), on the WCA of the recombinant P. pastoris have been investigated. Based on the results, we demonstrate that the P. pastoris heterologous expression system can be used to rapidly characterize PIP channels, to monitor the effects of mutations, and to score the effects of inhibitors and abiotic factors.     

Failure To Detect Helicobacter pylori DNA in Drinking and Environmental Water in Dhaka,Bangladesh, Using Highly Sensitive Real-Time PCR Assays

The main transmission pathway of Helicobacter pylori has not been determined, but several reports have described detection of H. pylori DNA in drinking and environmental water, suggesting that H. pylori may be waterborne. To address this possibility, we developed, tested, and optimized two complementary H. pylori-specific real-time PCR assays for quantification of H. pylori DNA in water. The minimum detection level of the assays including collection procedures and DNA extraction was shown to be approximately 250 H. pylori genomes per water sample. Using our assays, we then analyzed samples of drinking and environmental water (n = 75) and natural water biofilms (n = 21) from a high-endemicity area in Bangladesh. We could not identify H. pylori DNA in any of the samples, even though other pathogenic bacteria have been found previously in the same water samples by using the same methodology. A series of control experiments were performed to ensure that the negative results were not falsely caused by PCR inhibition, nonspecific assays, degradation of template DNA, or low detection sensitivity. Our results suggest that it is unlikely that the predominant transmission route of H. pylori in this area is waterborne.Helicobacter pylori is the most common human bacterial pathogen in the world (15), and it has been estimated that 50% of the world''s population is infected. The prevalence of H. pylori infection varies greatly worldwide, with infection rates of more than 80% in some developing countries and below 20% in some developed countries (29). H. pylori causes peptic ulcers in 10 to 15% and stomach cancer in another 1 to 2% of those infected (29).H. pylori naturally resides in the human stomach, and except for some primate species, no other host has been identified. Outside its host, H. pylori is fastidious and can grow only under microaerophilic conditions at 34 to 40°C in nutrient-rich media (29). Under suboptimal conditions, H. pylori transforms into nonculturable spherical or coccoid forms. To date, it is not clear if this process is reversible or if the coccoid form is infectious or even viable, but it has been reported to retain some metabolic activity, its genome, and an intact membrane (1, 6, 12, 28, 38, 47).Transmission of H. pylori has been proposed to occur via gastric-oral, oral-oral, or fecal-oral routes, with studies suggesting transmission through saliva and dental plaque (14, 23), normal and diarrheal stools (18, 23, 41, 43), and vomitus (30, 41). Infected mothers or older siblings, low standards of living, and crowded households have been shown to be major risk factors for contracting H. pylori (25, 35, 50). Other studies have shown a relation between infection, water sanitation, and drinking water sources (24, 26, 39), further supported by reports of H. pylori DNA in drinking, river, lake, or seawater (3, 7, 16, 19-22, 25, 33, 34, 37, 40, 43, 49).Since none of the latter group of studies have shown a causative relation between traces of H. pylori in water and new infections, our original aim was to perform a 2-year prospective study tracing H. pylori in water in a high-endemicity area and relate the findings with new infections in children. For this purpose, we developed highly sensitive and specific quantitative real-time PCR assays for detecting H. pylori DNA in water or human samples while allowing analysis of clonal relatedness between samples of different origins by sequencing of recovered DNA. Using these assays, we conducted a study in a slum area in Dhaka, Bangladesh, where we have recently shown a very high rate of H. pylori infections, i.e., that 60% of the children were infected by the age of 2 years (4). Drinking, waste, and environmental water samples and natural drinking water biofilm samples were collected and analyzed, with rigorous controls for falsely positive or negative results.     

Thermotolerant Coliforms Are Not a Good Surrogate for Campylobacter spp. in Environmental Water

This study aimed to assess the importance of quantitatively detecting Campylobacter spp. in environmental surface water. The prevalence and the quantity of Campylobacter spp., thermotolerant coliforms, and Escherichia coli in 2,471 samples collected weekly, over a 2-year period, from 13 rivers and 12 streams in the Eastern Townships, Québec, Canada, were determined. Overall, 1,071 (43%), 1,481 (60%), and 1,463 (59%) samples were positive for Campylobacter spp., thermotolerant coliforms, and E. coli, respectively. There were weak correlations between the weekly distributions of Campylobacter spp. and thermotolerant coliforms (Spearman''s ρ coefficient = 0.27; P = 0.008) and between the quantitative levels of the two classes of organisms (Kendall tau-b correlation coefficient = 0.233; P < 0.0001). Well water samples from the Eastern Townships were also tested. Five (10%) of 53 samples from private surface wells were positive for Campylobacter jejuni, of which only 2 were positive for thermotolerant coliforms. These findings suggest that microbial monitoring of raw water by using only fecal indicator organisms is not sufficient for assessing the occurrence or the load of thermophilic Campylobacter spp. Insights into the role of environmental water as sources for sporadic Campylobacter infection will require genus-specific monitoring techniques.Campylobacter jejuni is the leading reported cause of bacterial gastroenteritis in developed countries (2). In 2004 in Canada, Campylobacter enteritis was the leading notifiable enteric food- and waterborne disease, with 9,345 reported cases (http://dsol-smed.phac-aspc.gc.ca). In Quebec province alone, nearly 3,000 cases of diarrheal illness are attributed annually to Campylobacter enteritis, more than the combined total caused by Salmonella and Shigella species, Escherichia coli O157:H7, and Yersinia enterocolitica (15). Thomas et al. recently concluded that even these numbers appear to represent a substantial underestimate of the public health burden of this enteric pathogen and that for every case of Campylobacter infection reported in Canada each year, there are an additional 23 to 49 unreported cases (47).Raw milk, untreated surface water, and poultry have all been well documented as sources of Campylobacter outbreaks (1, 8, 22, 23, 28, 32, 33, 37, 39, 42, 49). Nevertheless, most clinical cases appear as isolated, sporadic infections for which the source is rarely identified (6). Identifying the sources and routes of transmission of campylobacteriosis is essential for developing effective, targeted preventive measures.There is ample opportunity for Campylobacter spp. to contaminate environmental water, including streams, rivers, and lakes. The genus colonizes a wide variety of hosts, from domestic animals to wild birds, and thus an extensive burden of organisms is excreted via animal fecal material (2, 8). Other potential sources include discharges from wastewater treatment plants.Testing for indicator organisms (typically thermotolerant coliforms or E. coli) has generally been considered to reflect adequately the presence of enteric pathogens; consequently, campylobacters have not been explicitly monitored in water. Numerous studies (most of which were small and of short duration) have reported conflicting results regarding the value of detecting E. coli to predict Campylobacter sp. presence (4, 9, 11, 12, 16, 17, 21, 27, 29, 31, 38, 40, 43, 48). We report here a large study that analyzed 2,471 water samples from 32 different sites over 2 years to resolve this question.     

Quantification of Human Polyomaviruses JC Virus and BK Virus by TaqMan Quantitative PCR and Comparison to Other Water Quality Indicators in Water and Fecal Samples

In the United States, total maximum daily load standards for bodies of water that do not meet bacterial water quality standards are set by each state. The presence of human polyomaviruses (HPyVs) can be used as an indicator of human-associated sewage pollution in these waters. We have developed and optimized a TaqMan quantitative PCR (QPCR) assay based on the conserved T antigen to both quantify and simultaneously detect two HPyVs; JC virus and BK virus. The QPCR assay was able to consistently quantify ≥10 gene copies per reaction and is linear over 5 orders of magnitude. HPyVs were consistently detected in human waste samples (57 of 64) and environmental waters with known human fecal contamination (5 of 5) and were not amplified in DNA extracted from 127 animal waste samples from 14 species. HPyV concentrations in sewage decreased 81.2 and 84.2% over 28 days incubation at 25 and 35°C, respectively. HPyVs results were compared to Escherichia coli, fecal coliform, and enterococci concentrations and the presence of three other human-associated microbes: Bacteroidetes, Methanobrevibacter smithii, and adenovirus. HPyVs were the most frequently detected of these in human and contaminated environmental samples and were more human specific than the Bacteroidetes (HF183) or M. smithii. HPyVs and M. smithii more closely mimicked the persistence of adenovirus in sewage than the other microbes. The use of this rapid and quantitative assay in water quality research could help regulatory agencies to identify sources of water pollution for improved remediation of contaminated waters and ultimately protect humans from exposure to pathogens.Maintaining healthy coastal water systems is essential, since poor water quality can have detrimental effects on mangroves, seagrass beds, coral reefs, the fishing and shellfish harvesting industries, and the health of recreational water users (1, 5, 15, 17, 20, 44). Since 1972 in the United States, each state has been required to set total maximum daily loads (TMDLs) for pollutants in water bodies according to section 303(d) of the Clean Water Act (50). The probability that microbial pathogens are present is estimated by enumerating indicator bacteria, which are shed in the feces of humans and most animals. The U.S. Environmental Protection Agency recommends using Escherichia coli and enterococci to assess the quality of freshwater and saline water, respectively (47); however, Florida currently uses fecal coliforms and enterococci as indicators of fecal pollution (42).When bacterial indicators exceed regulatory levels, a plan of action (TMDL implementation) must be developed to reduce pathogens. TMDL plans for “pathogen” reduction are particularly problematic because they rely upon surrogate indicator bacteria, which yield little or no insight as to the source of pollution. High indicator bacteria concentrations can be attributed to many sources, including agricultural runoff, storm water runoff, wildlife, pets, faulty septic systems (onsite wastewater treatment and disposal systems), and a failing central sewer infrastructure (5, 12, 28).To address the issue of source identification, methods have been developed in which the biochemistry or genetics of certain microorganisms are used to indirectly identify probable source(s) of fecal pollution, which is termed microbial source tracking (MST) (48). MST methods based on detection of a source-associated gene (marker) by PCR have proliferated over the past 10 years due to the additional information they can provide to watershed managers on fecal contamination sources (43). Although marker detection by endpoint (binary) PCR can give important insights on the source(s) of fecal contamination, quantitative measurements can provide information about the relative magnitude of contamination from various sources. Moreover, epidemiological studies on the correlation between recreational water use, microbial contamination, and the risk of illness will greatly benefit from the ability to quantify MST markers, rather than simply assessing binary (+/−) detection.Although many bacterial targets have been proposed for MST of human sewage (8, 39, 46a), fewer viral targets have been investigated (19, 24, 33). Polyomavirus is the sole genus in the family Polyomaviridae (22). These viruses have a 5-kbp double-stranded DNA genome surrounded by a 40- to 50-nm icosahedral capsid (38). The JCV and BKV human polyomaviruses (HPyVs) have similarly structured genomes that show ∼75% identity (21). BK virus (BKV) and JC virus (JCV) gained much attention in the late 1970s as the etiological agents of kidney nephritis (i.e., BKV reactivation in the kidneys) and progressive multifocal leukoencephalopathy (i.e., JCV reactivation in brain tissue) in the immunocompromised (16, 34). Serological studies have shown that >70% of adults harbor antibodies to BKV or JCV (27, 30, 44). These viruses are known for producing lifelong, asymptomatic viruria in immunocompetent individuals (37). In 2000 it was first suggested that JCV would be a useful indicator of human sewage in water (11). The obligate host specificity and abundance of BKV and JCV in municipal sewage has led to the successful use of these viruses to indicate human fecal pollution in environmental water samples (12, 29).Due to the health implications of BKV and JCV, several methods have been developed to rapidly detect either BKV or JCV in clinical samples (6, 31, 35, 56). However, from an MST standpoint, it is advantageous to target both BKV and JCV. BKV has been found in feces (54), and both viruses are excreted in the urine (6, 11, 37, 55, 60) either simultaneously or individually. The focus of this research was the modification of the previously developed nested PCR protocol for HPyVs detection (29) to a TaqMan quantitative PCR (QPCR) assay to simultaneously detect and quantify both BKV and JCV. Furthermore, we compared measurements obtained with the newly developed QPCR assay to those of other water quality indicators and MST markers. These indicators included bacterial indicator concentrations (49) and PCR detection of human-associated markers currently used for MST. These included human-associated Bacteroidetes (8), Methanobrevibacter smithii (46a), and adenovirus (36). To assess the potential of HPyVs to mimic the fate of pathogens in water, the persistence of all of the water quality indicators was assessed, and relationships between bacterial indicator organisms and MST markers in both human waste samples as well as contaminated environmental samples were examined.     

Novel Clonal Complexes with an Unknown Animal Reservoir Dominate Campylobacter jejuni Isolates from River Water in New Zealand

Campylobacter jejuni is widely distributed in the environment, and river water has been shown to carry high levels of the organism. In this study, 244 C. jejuni isolates from three river catchment areas in New Zealand were characterized using multilocus sequence typing. Forty-nine of the 88 sequence types identified were new. The most common sequence types identified were ST-2381 (30 isolates), ST-45 (25 isolates), and ST-1225 (23 isolates). The majority of the sequence types identified in the river water could be attributed to wild bird fecal contamination. Two novel clonal complexes (CC) were identified, namely, CC ST-2381 (11 sequence types, 46 isolates) and CC ST-3640 (6 sequence types, 12 isolates), in which all of the sequence types were new. CC ST-2381 was the largest complex identified among the isolates and was present in two of the three rivers. None of the sequence types associated with the novel complexes has been identified among human isolates. The ST-2381 complex is not related to complexes associated with cattle, sheep, or poultry. The source of the novel complexes has yet to be identified.Contamination of the environment by bacterial pathogens is a significant health concern, as it provides a continuous source of organisms for the infection and reinfection of humans and animals. Enteric pathogens gain entry into the environment through the discharge of sewage into water and via contamination from animal feces (22). Fecal contamination is responsible for the continued presence and spread of a range of pathogenic organisms, including Campylobacter, norovirus, and Escherichia coli O157. Determining the roles of various environmental sources in human enteric disease requires an understanding of the distribution, survival, population structure, and pathogenic potential of the pathogens in the environment.Campylobacter is the most common cause of gastrointestinal illness in the industrialized world (17), imposing significant economic costs on health systems, and is associated with a number of neurological sequelae (32, 33). The majority of human campylobacter infections are caused by Campylobacter jejuni (90%), with Campylobacter coli mostly responsible for the remainder. Although Campylobacter has been isolated from a wide range of animals (41) and birds (47, 48), contaminated poultry and poultry products remain the most significant sources of human infections (10, 38, 50, 51). Campylobacter is a spiral gram-negative organism that grows best under low-oxygen conditions at 42°C. The organism is unable to grow outside an animal host, and survival in the environment is dependent on ambient temperature, oxygen levels, and sunlight.Studies worldwide examining rivers and waterways show that there is significant contamination by Campylobacter, with the sources being sewage outflow, direct fecal deposition, and pasture runoff (12, 22, 34, 37, 39). Similarly, coastal waters and estuaries can be contaminated by either sewage or bird fecal deposition (23, 35). The inability of Campylobacter to grow in the environment and its sensitivity to sunlight are thought to ensure that the organism is eventually purged from the system. However, the high levels of the organism identified in water systems have been highlighted as a risk for human infection.The characterization of campylobacter populations by multilocus sequence typing (MLST) has shown that the organism is weakly clonal and that certain clonal complexes are associated with particular animals (5, 9, 26). Isolates from human cases of infection show a wide variety of sequence types and many clonal complexes. Source attribution studies using MLST have identified poultry as causing approximately 60% of human infections (14, 38, 50). Cattle have been identified as a potential source of infection due to the high level of similarity between bovine and human strains (18, 19). There remains, however, a significant number of infections for which the source is not certain.New Zealand has one of the highest rates of campylobacteriosis in the developed world. This is due to the significant quantity of fresh chicken consumed coupled with high levels of contamination found in poultry products (1, 10, 51, 52). Campylobacter has been isolated from a range of environmental sources within New Zealand, including its rivers and streams (12, 37). Isolation rates for rivers in New Zealand range from 55 to 90%, comparable to results of studies overseas, and show the same seasonal variation as that seen elsewhere in the world (20). Pulsed-field gel electrophoresis (PFGE) analysis identified indistinguishable macrorestriction profiles for cattle, human, and river isolates, suggesting river water as a potential source of infection (8). In this study, C. jejuni isolates from three rivers in New Zealand, two on the South Island and one on the North Island, were characterized using MLST.     

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).     

Viability PCR,a Culture-Independent Method for Rapid and Selective Quantification of Viable Legionella pneumophila Cells in Environmental Water Samples

PCR-based methods have been developed to rapidly screen for Legionella pneumophila in water as an alternative to time-consuming culture techniques. However, these methods fail to discriminate between live and dead bacteria. Here, we report a viability assay (viability PCR [v-PCR]) for L. pneumophila that combines ethidium monoazide bromide with quantitative real-time PCR (qPCR). The ability of v-PCR to differentiate viable from nonviable L. pneumophila cells was confirmed with permeabilizing agents, toluene, or isopropanol. v-PCR suppressed more than 99.9% of the L. pneumophila PCR signal in nonviable cultures and was able to discriminate viable cells in mixed samples. A wide range of physiological states, from culturable to dead cells, was observed with 64 domestic hot-water samples after simultaneous quantification of L. pneumophila cells by v-PCR, conventional qPCR, and culture methods. v-PCR counts were equal to or higher than those obtained by culture and lower than or equal to conventional qPCR counts. v-PCR was used to successfully monitor in vitro the disinfection efficacy of heating to 70°C and glutaraldehyde and chlorine curative treatments. The v-PCR method appears to be a promising and rapid technique for enumerating L. pneumophila bacteria in water and, in comparison with conventional qPCR techniques used to monitor Legionella, has the advantage of selectively amplifying only viable cells.Legionella organisms are ubiquitous bacteria found in many types of water sources in the environment. Their growth is especially favored in human-made warm water systems, including cooling towers, hot tubs, showerheads, and spas (3, 14, 15, 38). Legionella bacteria replicate as intracellular parasites of amoebae and persist in the environment as free-living microbes or in biofilms. In aerosol form, they enter the lungs and can cause an acute form of pneumonia known as Legionnaires'' disease or a milder form of pulmonary infection called Pontiac fever. The species Legionella pneumophila is responsible for the vast majority of the most severe form of this atypical pneumonia (52, 70). Legionellosis outbreaks are associated with high mortality rates (15 to 20%) (15, 16, 38, 46), which can reach up to 50% for people with weakened immune systems (immunocompromised patients) (69). Legionella surveillance programs include regular monitoring of environmental water samples (9, 13, 66). It is generally acknowledged that Legionella represents a health risk to humans when cell densities are greater than 10⁴ to 10⁵ CFU per liter of water, and epidemiological data show that outbreaks of legionellosis occur at these concentrations (36, 47).The evaluation of the risk associated with Legionella has traditionally been performed using culture-based methods (1, 24). Culture is essential for identifying and typing Legionella strains during epidemics. However, Legionella culture requires long incubation times (up to 10 days) before results can be scored. This problem makes culture unsuitable for preventive actions and rapid response in emergency situations. Moreover, under certain conditions (i.e., low-nutrient environments, oxidative or osmotic stress, etc.), Legionella cells can lose the ability to be cultured, although they are still viable (7, 17, 20, 22, 39, 45, 67). These viable but nonculturable (VBNC) Legionella cells may still represent a public health hazard because they can regain their ability to grow in new, more favorable conditions (12, 19, 23, 61).Molecular approaches, such as quantitative real-time PCR (qPCR), are faster and can mitigate the main drawbacks of culture-based methods. qPCR is an alternative tool that offers rapid, sensitive, and specific detection of Legionella bacteria in environmental water samples (4, 5, 12, 26, 65, 68). PCR results can be obtained in hours instead of days, and VBNC Legionella cells can also be detected (12, 26). However, the major disadvantage of qPCR lies in its inability to evaluate viability due to the persistence of DNA in cells after death (27, 34). The monitoring of Legionella contamination levels by conventional qPCR may thus result in an overestimation of the risk of infection because false-positive results can be scored. However, the real risk from Legionella is limited to the live fraction of the total Legionella population. Only live or viable Legionella cells are able to replicate in pulmonary macrophages and cause severe pneumonia (14, 15). The development of more rapid, culture-independent methods capable of discriminating between live and dead cells is of major interest for measuring Legionella infection risks and preventing legionellosis. The nucleic acid-binding dye ethidium monoazide bromide (EMA), used in combination with qPCR, is an attractive alternative for selectively detecting and enumerating viable bacteria. EMA is particularly useful because it selectively penetrates cells with damaged membranes and covalently binds to DNA after photoactivation (21, 53). DNA-bound EMA molecules prevent PCR amplification and thereby lead to a strong signal reduction during qPCR. DNA from viable cells with intact cell membranes prevents EMA molecules from entering the cell and therefore can be amplified and quantified (56). Nocker et al. (41, 42) suggested that the signal reduction was due to a selective loss of genomic DNA from dead cells (rendered insoluble after cross-linkage) during the DNA extraction procedure rather than to PCR inhibition. However, Soejima et al. (59, 60) recently reported that treatment with EMA followed by visible light irradiation directly cleaves the chromosomal DNA of dead bacteria.In this study we optimized the EMA-staining procedure in conjunction with qPCR with pure cultures of L. pneumophila. We analyzed the potential for the EMA-qPCR method to discriminate Legionella cells with compromised or intact cell membranes. We optimized this EMA-qPCR technique, viability PCR, hereafter named v-PCR, and used it to quantify viable Legionella cells in environmental water samples. We compared our results with those obtained by conventional qPCR and culture methods. In addition, we evaluated the ability of v-PCR to monitor the efficacy of different disinfection strategies.     

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.     

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

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

Eukaryotic Viruses in Wastewater Samples from the United States

Human fecal matter contains a large number of viruses, and current bacterial indicators used for monitoring water quality do not correlate with the presence of pathogenic viruses. Adenoviruses and enteroviruses have often been used to identify fecal pollution in the environment; however, other viruses shed in fecal matter may more accurately detect fecal pollution. The purpose of this study was to develop a baseline understanding of the types of viruses found in raw sewage. PCR was used to detect adenoviruses, enteroviruses, hepatitis B viruses, herpesviruses, morbilliviruses, noroviruses, papillomaviruses, picobirnaviruses, reoviruses, and rotaviruses in raw sewage collected throughout the United States. Adenoviruses and picobirnaviruses were detected in 100% of raw sewage samples and 25% and 33% of final effluent samples, respectively. Enteroviruses and noroviruses were detected in 75% and 58% of raw sewage samples, respectively, and both viral groups were found in 8% of final effluent samples. This study showed that adenoviruses, enteroviruses, noroviruses, and picobirnaviruses are widespread in raw sewage. Since adenoviruses and picobirnaviruses were detected in 100% of raw sewage samples, they are potential markers of fecal contamination. Additionally, this research uncovered previously unknown sequence diversity in human picobirnaviruses. This baseline understanding of viruses in raw sewage will enable educated decisions to be made regarding the use of different viruses in water quality assessments.Millions of viruses and bacteria are excreted in human fecal matter (5, 17, 82), and current methods of sewage treatment do not always effectively remove these organisms (74, 76-78). The majority of treated wastewater, as well as untreated sewage, drains into the marine environment (1) and has the potential to threaten environmental (e.g., nutrients and chemicals) (45) and public (e.g., pathogen exposure via swimming and seafood consumption) (1, 24, 28, 29, 33, 44, 57, 63) health. Currently, the U.S. Environmental Protection Agency (EPA) mandates the use of bacterial indicators such as fecal coliforms and enterococci to assess water quality (75). Although monitoring of these bacteria is simple and inexpensive, it has been shown that fecal-associated bacteria are not ideal indicators of fecal pollution.Since fecal-associated bacteria are able to live in sediments in the absence of fecal pollution (18, 32, 55), their resuspension into the water column can result in false-positive results and mask correlations between their concentrations and the extent of recent fecal pollution. Another unfavorable characteristic of current bacterial indicators is their inability to predict or correlate with the presence of pathogenic viruses (25, 40, 41, 64, 80). Human-pathogenic viruses associated with feces are generally more robust than enteric bacteria and are not as easily eliminated by current methods of wastewater treatment (43, 80). For example, adenoviruses are more resilient to tertiary wastewater treatment and UV disinfection than are bacterial indicators of fecal pollution (74). Since bacterial indicators cannot accurately depict the risks to human health from fecal pollution, several studies have proposed the use of a viral indicator of wastewater contamination (35, 41, 61).While it is impractical to monitor the presence of all viral pathogens related to wastewater pollution, the development of an accurate viral indicator of sewage contamination is needed for enhanced water quality monitoring. Enteric viruses (including viruses belonging to the families Adenoviridae, Caliciviridae, Picornaviridae, and Reoviridae) are transmitted via the fecal-oral route and are known to be abundant in raw sewage. These viruses have been used to identify fecal pollution in coastal environments throughout the world (27, 35, 39, 40, 48, 50, 56, 57, 63, 64, 67-69, 71, 80). To determine which viruses are effective indicators of fecal pollution, it is first necessary to establish a broad, baseline understanding of the many diverse groups of eukaryotic viruses in raw sewage. Several studies have identified adenoviruses, noroviruses, reoviruses, rotaviruses, and other enteroviruses (e.g., polioviruses, coxsackie viruses, and echoviruses) in raw sewage in Australia, Europe, and South Africa (30, 47, 58, 76-78). However, no broad baseline data on the presence of eukaryotic viruses in raw sewage in the United States currently exist.This study determined the presence of 10 viral groups (adenoviruses, enteroviruses, hepatitis B viruses, herpesviruses, morbilliviruses, noroviruses, papillomaviruses, picobirnaviruses, reoviruses, and rotaviruses) in raw sewage samples collected throughout the United States. All viral groups that were detected in raw sewage were then examined further to determine if they were also present in final treated wastewater effluent. These 10 viral groups were chosen because of their potential to be transmitted via the fecal-oral route, suggesting that they might be found in raw sewage. Many of these viruses (excluding adenoviruses, enteroviruses, noroviruses, reoviruses, and rotaviruses) have not been studied in sewage despite their likely presence. Picobirnaviruses have been detected in individual fecal samples (12, 70, 79, 82); however, their presence has never been analyzed in collective waste, nor have they been proposed to be potential markers of fecal pollution. This study identified potential viral indicators of fecal pollution and will have important applications to water quality monitoring programs throughout the country.     

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

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

Detection of Protozoan Hosts for Legionella pneumophila in Engineered Water Systems by Using a Biofilm Batch Test

Legionella pneumophila proliferates in aquatic habitats within free-living protozoa, 17 species of which have been identified as hosts by using in vitro experiments. The present study aimed at identifying protozoan hosts for L. pneumophila by using a biofilm batch test (BBT). Samples (600 ml) collected from 21 engineered freshwater systems, with added polyethylene cylinders to promote biofilm formation, were inoculated with L. pneumophila and subsequently incubated at 37°C for 20 days. Growth of L. pneumophila was observed in 16 of 18 water types when the host protozoan Hartmannella vermiformis was added. Twelve of the tested water types supported growth of L. pneumophila or indigenous Legionella anisa without added H. vermiformis. In 12 of 19 BBT flasks H. vermiformis was indicated as a host, based on the ratio between maximum concentrations of L. pneumophila and H. vermiformis, determined with quantitative PCR (Q-PCR), and the composition of clone libraries of partial 18S rRNA gene fragments. Analyses of 609 eukaryotic clones from the BBTs revealed that 68 operational taxonomic units (OTUs) showed the highest similarity to free-living protozoa. Forty percent of the sequences clustering with protozoa showed ≥99.5% similarity to H. vermiformis. None of the other protozoa serving as hosts in in vitro studies were detected in the BBTs. In several tests with growth of L. pneumophila, the protozoa Diphylleia rotans, Echinamoeba thermarum, and Neoparamoeba sp. were identified as candidate hosts. In vitro studies are needed to confirm their role as hosts for L. pneumophila. Unidentified protozoa were implicated as hosts for uncultured Legionella spp. grown in BBT flasks at 15°C.Legionella pneumophila, the causative agent of Legionnaires'' disease, is a common inhabitant of natural freshwater environments and human-made water systems, including cooling towers, whirlpools, air-conditioning systems, and installations for warm tap water (14). In the aquatic environment L. pneumophila proliferates within certain free-living protozoa, which serve as its hosts (15, 30, 59). Environmental factors favoring the growth and survival of L. pneumophila in freshwater systems include a water temperature between 20°C and 45°C (41, 60) and the presence of biofilms and sediments on which the protozoan hosts can graze (30, 41, 56).Rowbotham (44) was the first to report the growth of L. pneumophila within free-living amoebae, which belonged to the genera Acanthamoeba and Naegleria. In vitro studies with cocultures have revealed that 14 species of amoebae, viz., Acanthamoeba spp. (1, 35, 44, 53), Balamuthia mandrillaris (47), Echinamoeba exundans (15), Hartmannella spp. (43), Naegleria spp. (38, 44, 53), and Vahlkampfia jugosa (43); the slime mold Dictyostelium discoideum (20, 48); and two species of the ciliate genus Tetrahymena (15, 26) can serve as hosts for L. pneumophila. Recently, it has been reported that L. pneumophila can also replicate within the intestinal tract of the microbiovorous nematode Caenorhabditis elegans (3).A number of the free-living protozoa mentioned above and others, e.g., Vannella spp. and Saccamoeba spp., have been observed in aquatic environments from which L. pneumophila was cultivated or in which it was detected with PCR (4, 42, 51, 52). However, it remains unknown which of these protozoa actually serve as hosts for L. pneumophila in the aquatic environment, including human-made water systems. Moreover, it cannot be excluded that free-living protozoa other than those tested in vitro can serve as hosts for L. pneumophila as well. Information is also lacking about protozoan hosts for Legionella anisa (13, 49), which is frequently present in water installations in temperate regions (11, 62). Furthermore, it is unknown which free-living protozoa serve as hosts for uncultured Legionella bacteria that can grow at temperatures of about 15°C (61; B. A. Wullings, G. Bakker, and D. van der Kooij, submitted for publication).L. pneumophila can proliferate in samples of surface water, effluent of wastewater treatment plants, potable water, and water from cooling towers incubated at 25°C, 35°C, or 37°C (28, 45, 56). Consequently, incubation of freshwater samples can be used to amplify protozoan hosts for L. pneumophila and other Legionella spp. In this study, different human-made water types were investigated using a biofilm batch test (BBT) system to (i) amplify and subsequently identify predominating, known, and yet-undescribed hosts for L. pneumophila and (ii) identify potential protozoan hosts for Legionella bacteria that can grow at 15°C.     

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

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