Occurrence and Molecular Characteristics of Methicillin-Resistant Staphylococcus aureus and Methicillin-Resistant Staphylococcus pseudintermedius in an Academic Veterinary Hospital

Recently, methicillin-resistant Staphylococcus aureus (MRSA) and methicillin-resistant Staphylococcus pseudintermedius (MRSP) have been increasingly isolated from veterinarians and companion animals. With a view to preventing the spread of MRSA and MRSP, we evaluated the occurrence and molecular characteristics of each in a veterinary college. MRSA and MRSP were isolated from nasal samples from veterinarians, staff members, and veterinary students affiliated with a veterinary hospital. Using stepwise logistic regression, we identified two factors associated with MRSA carriage: (i) contact with an identified animal MRSA case (odds ratio [OR], 6.9; 95% confidence interval [95% CI], 2.2 to 21.6) and (ii) being an employee (OR, 6.2; 95% CI, 2.0 to 19.4). The majority of MRSA isolates obtained from individuals affiliated with the veterinary hospital and dog patients harbored spa type t002 and a type II staphylococcal cassette chromosome mec (SCCmec), similar to the hospital-acquired MRSA isolates in Japan. MRSA isolates harboring spa type t008 and a type IV SCCmec were obtained from one veterinarian on three different sampling occasions and also from dog patients. MRSA carriers can also be a source of MRSA infection in animals. The majority of MRSP isolates (85.2%) carried hybrid SCCmec type II-III, and almost all the remaining MRSP isolates (11.1%) carried SCCmec type V. MRSA and MRSP were also isolated from environmental samples collected from the veterinary hospital (5.1% and 6.4%, respectively). The application of certain disinfection procedures is important for the prevention of nosocomial infection, and MRSA and MRSP infection control strategies should be adopted in veterinary medical practice.Methicillin-resistant Staphylococcus aureus (MRSA) is an important cause of nosocomial infections in human hospitals. The prevalence of hospital-acquired MRSA (HA-MRSA) infection among inpatients in intensive care units (ICUs) continues to increase steadily in Japan. Recently, cases of community-acquired MRSA (CA-MRSA) have been documented in persons without an established risk factor for HA-MRSA infection (14, 32, 36, 49).There has also been an increase in the number of reports of the isolation of MRSA from veterinarians and companion animals (5, 21, 23-26, 28, 31, 34, 38, 44, 50, 51, 53). Values reported for the prevalence of MRSA among veterinary staff include 17.9% in the United Kingdom (21), 10% in Japan (38), 3.9% in Scotland (13), and 3.0% in Denmark (28). Loeffler et al. reported that the prevalence of MRSA among dog patients and healthy dogs owned by veterinary staff members was 8.9% (21). In Japan, an MRSA isolate was detected in only one inpatient dog (3.8%) and could not be detected in any of 31 outpatient dogs (38). In the United States, MRSA isolates were detected in both dog (0.1%) and cat (0.1%) patients (31). The prevalence of MRSA among healthy dogs has been reported to be 0.7% (5). Hanselman et al. suggested that MRSA colonization may be an occupational risk for large-animal veterinarians (12). Recently, Burstiner et al. reported that the frequency of MRSA colonization among companion-animal veterinary personnel was equal to the frequency among large-animal veterinary personnel (6).In addition, other methicillin-resistant coagulase-positive staphylococci (MRCPS), such as methicillin-resistant Staphylococcus pseudintermedius (MRSP) and methicillin-resistant Staphylococcus schleiferi (MRSS), isolated from dogs, cats, and a veterinarian have been reported (11, 31, 38, 40, 52). MRSP isolates have also been detected among inpatient dogs (46.2%) and outpatient dogs (19.4%) in a Japanese veterinary teaching hospital (38). In Canada, however, MRSP and MRSS isolates were detected in only 2.1% and 0.5% of dog patients, respectively (11).Methicillin-resistant staphylococci produce penicillin-binding protein 2′, which reduces their affinity for β-lactam antibiotics. This protein is encoded by the mecA gene (48), which is carried on the staphylococcal cassette chromosome mec (SCCmec). SCCmec is a mobile genetic element characterized by the combination of the mec and ccr complexes (16), and it is classified into subtypes according to differences in the junkyard regions (43). SCCmec typing can be used as a molecular tool (22, 27, 30, 33, 36, 55) for examining the molecular epidemiology of methicillin-resistant staphylococci.In this study, we investigated the occurrence and characteristics of MRCPS isolates in a veterinary hospital in order to establish the transmission route of MRCPS in a veterinary hospital and with a view to preventing the spread of MRCPS infection. In addition, we evaluated the factors associated with MRCPS. Further, as Heller et al. have reported the distribution of MRSA within veterinary hospital environments and suggested the necessity to review cleaning protocols of hospital environments (13), we also attempted to isolate MRCPS from environmental samples collected in a veterinary hospital for an evaluation of MRSA transmission cycle though environmental surfaces in the veterinary hospital.     

Characterization of Variation in Rumen Methanogenic Communities under Different Dietary and Host Feed Efficiency Conditions,as Determined by PCR-Denaturing Gradient Gel Electrophoresis Analysis

Understanding ruminal methanogens is essential for greenhouse gas mitigation, as well as for improving animal performance in the livestock industry. It has been speculated that ruminal methanogenic diversity affects host feed efficiency and results in differences in methane production. This study examined methanogenic profiles in the rumen using culture-independent PCR-denaturing gradient gel electrophoresis (PCR-DGGE) analysis for 56 beef cattle which differed in feed efficiency, as well as diet (the cattle were fed a low-energy diet or a high-energy diet). The methanogenic PCR-DGGE profiles detected were greatly affected by diet, and the major pattern changed from a community containing predominantly Methanobrevibacter ruminantium NT7 with the low-energy diet to a community containing predominantly Methanobrevibacter smithii, Methanobrevibacter sp. AbM4, and/or M. ruminantium NT7 with the high-energy diet. For each diet, the methanogenic PCR-DGGE pattern was strongly associated with the feed efficiency of the host. Diet-associated bands for Methanobrevibacter sp. AbM4 and M. smithii SM9 and a feed efficiency-related band for M. smithii PS were identified. The abundance of total methanogens was estimated by determining the numbers of copies of the 16S rRNA genes of methanogens. However, the size of the methanogen population did not correlate with differences in feed efficiency, diet, or metabolic measurements. Thus, the structure of the methanogenic community at the species or strain level may be more important for determining host feed efficiency under different dietary conditions.Ruminal methanogens use methanogenesis pathways to maintain low hydrogen partial pressure and to facilitate fiber digestion in the rumen by converting hydrogen into methane gas (24, 37). However, although it is necessary, this process also has adverse effects because the released methane represents a significant loss of dietary energy for the host animal (14) and it constitutes a large proportion of the agricultural greenhouse gas emitted (4, 10). Many studies to obtain a better understanding of rumen methanogens have been conducted in order to improve the efficiency of ruminal function and to mitigate methane release. Assessments by both cultivation-dependent and cultivation-independent methods have found that members of the genus Methanobrevibacter account for the majority of the methanogens in the rumens of sheep and cattle (11, 18, 21-23, 28, 31, 33, 34). In addition, Methanosphaera stadtmanae, Methanobacterium species, and Methanosarcina barkeri have also been found in some studies (13, 32). Although the phylogenetic positions of the methanogens in the rumen are diverse, these organisms utilize only three major pathways for methanogenesis: the CO₂ reduction pathway, the C₁ compound (e.g., methanol and methylamine) conversion pathway, and the acetate fermentation pathway. Each methanogen species has a substrate preference, and most methanogens can use only one or two substrates (37).Previous studies of rumen methanogens focused primarily on determining the methanogen species composition in different samples and developing strategies to reduce the methane yield from ruminants. Recently, there has been a strong desire to understand the impact of methanogens on host biology. Two primary studies found that feedlot beef cattle with higher feed efficiency (designated “efficient” animals) produced about 20% less methane gas than animals with lower feed efficiency (designated “inefficient” animals) (8, 19). The methanogenic communities of efficient and inefficient animals fed a low-energy diet have been compared, and divergence between the two communities has been reported (36). However, it is not clear how the methanogens in the rumen of cattle change when the animals are fed a different diet.The aims of this study were to describe the methanogenic communities in 56 steers with different feed efficiencies that were fed two distinct diets (a low-energy diet and a high-energy diet) and to understand how methanogenic communities change in response to diet modification using PCR-denaturing gradient gel electrophoresis (PCR-DGGE) and sequence analysis. Multivariate analysis was used to analyze the association of PCR-DGGE bands with the daily dry matter intake (DMI), average daily gain (ADG), feed conversion ratio (FCR), and residual feed intake (RFI). Methanogens that were associated with diet and with host feed efficiency were identified. In addition, the methanogen population of each rumen sample was examined by quantitative real-time PCR (qRT-PCR), and the results for different RFI groups and both diets were compared.     

Anaerobic Digestion of Renewable Biomass: Thermophilic Temperature Governs Methanogen Population Dynamics

Beet silage and beet juice were digested continuously as representative energy crops in a thermophilic biogas fermentor for more than 7 years. Fluorescence microscopy of 15 samples covering a period of 650 days revealed that a decrease in temperature from 60°C to 55°C converted a morphologically uniform archaeal population (rods) into a population of methanogens exhibiting different cellular morphologies (rods and coccoid cells). A subsequent temperature increase back to 60°C reestablished the uniform morphology of methanogens observed in the previous 60°C period. In order to verify these observations, representative samples were investigated by amplified rRNA gene restriction analysis (ARDRA) and fluorescence in situ hybridization (FISH). Both methods confirmed the temperature-dependent population shift observed by fluorescence microscopy. Moreover, all samples investigated demonstrated that hydrogenotrophic Methanobacteriales dominated in the fermentor, as 29 of 34 identified operational taxonomic units (OTUs) were assigned to this order. This apparent discrimination of acetoclastic methanogens contradicts common models for anaerobic digestion processes, such as anaerobic digestion model 1 (ADM1), which describes the acetotrophic Euryarchaeota as predominant organisms.The replacement of fossil fuels by renewable energy sources such as agricultural crops is gaining momentum internationally as a means to decrease emissions from conventional fuel sources impacting global warming (39). Thereby, biogasification using energy crops is the only fuel-producing process with a closed CO₂ and nutrient cycle (8). The production of biogas from plant waste or other organic materials is a feasible strategy in view of both ecology and economy (63). Fodder beet was chosen as the renewable biomass source for a thermophilic biogas fermentor because the European Union decreased the regulatory price for sugar beets in 2006, and therefore many farmers are looking for an alternative use. Furthermore, fodder beet was considered an attractive renewable energy crop due to its high methane yield per hectare (67), as well as the ideal ensiling conditions enabling the storage of beet silage for many years. Furthermore, the sugar beet was only recently identified as one of the most sustainable energy crops with regard to its water footprint when used for biofuel production (22).A long-term experiment was started on 4 July 2001 (see reference 48 for startup details), and the same biogas fermentors are still running stable due to the use of fuzzy logic control (16, 48). During the conversion of biomass to methane, four different microbial processes can be distinguished: hydrolysis, acidogenesis, acetogenesis, and methanogenesis (17, 69). Population changes might therefore impact the entire community by triggering an imbalance that is reflected in the bioreactor performance via accumulation of intermediates such as volatile fatty acids (mainly C₂ and C₃), via pH changes, or via reduced efficiency (52). This work focused on the methanogens which directly reduce CO₂ to CH₄ or use acetate or methylated C₁ compounds as the main substrate to yield methane (35). However, about 65 to 70% of methane produced by methanogens is assumed to originate from acetate (4, 5), and the so-called acetoclastic Euryarchaeota are also dominant in many biogas fermentors used for anaerobic wastewater treatment and sewage sludge digestion (17, 24, 30, 53).Our results seem to contradict these assumptions, as they clearly demonstrate that hydrogenotrophic methanogens can dominate during a thermophilic fermentation process with renewable biomass (16, 49-51). It appears that temperature has a decisive influence on the type of archaeal morphotypes present, as rod-like methanogens dominated at 60°C periods, whereas different morphotypes of methanogens appeared when 55°C conditions were enabled. However, studies elucidating the population dynamics of both acetotrophic and hydrogenotrophic methanogens during the anaerobic digestion of particulate solid biomass for biogas production are rather scarce. These population processes remain somewhat of a “black box” (12) due to the lack of data concerning the microbial consortia involved therein. Molecular biological techniques such as those targeting the 16S rRNA gene represent a valuable addition to culture-based techniques for studying the biodiversity and structure of complex microbial communities. By targeting methanogens, this study aimed to improve our insight into the poorly understood population dynamics of anaerobic digestion processes and how they are linked to operating conditions such as temperature.     

Assessment of the Microbial Ecology of Ruminal Methanogens in Cattle with Different Feed Efficiencies

Cattle with high feed efficiencies (designated “efficient”) produce less methane gas than those with low feed efficiencies (designated “inefficient”); however, the role of the methane producers in such difference is unknown. This study investigated whether the structures and populations of methanogens in the rumen were associated with differences in cattle feed efficiencies by using culture-independent methods. Two 16S rRNA libraries were constructed using ∼800-bp amplicons generated from pooled total DNA isolated from efficient (n = 29) and inefficient (n = 29) animals. Sequence analysis of up to 490 randomly selected clones from each library showed that the methanogenic composition was variable: less species variation (22 operational taxonomic units [OTUs]) was detected in the rumens of efficient animals, compared to 27 OTUs in inefficient animals. The methanogenic communities in inefficient animals were more diverse than those in efficient ones, as revealed by the diversity indices of 0.84 and 0.42, respectively. Differences at the strain and genotype levels were also observed and found to be associated with feed efficiency in the host. No difference was detected in the total population of methanogens, but the prevalences of Methanosphaera stadtmanae and Methanobrevibacter sp. strain AbM4 were 1.92 (P < 0.05) and 2.26 (P < 0.05) times higher in inefficient animals, while Methanobrevibacter sp. strain AbM4 was reported for the first time to occur in the bovine rumen. Our data indicate that the methanogenic ecology at the species, strain, and/or genotype level in the rumen may play important roles in contributing to the difference in methane gas production between cattle with different feed efficiencies.Microbial fermentation and ruminal nutrient absorption are key steps in the energy metabolism of cattle. The microbiota in the rumen is highly associated with the diet, age, antibiotic use, and health of host animals (32). Different types of symbiotic anaerobic microorganisms, including bacteria, archaea, ciliated protozoa, and fungi, inhabit the rumen (15), interact with each other, and play important roles in affecting the host''s performance. The microbial-host relationships are highly complex and varied, ranging from mutually beneficial cooperation to competition (10). Among ruminal microbes, bacteria decompose the feed into short-chain (C₁ to C₅) fatty acids, amino acids, H₂, and CO₂, etc. (20). To maintain the low hydrogen level in this habitat, hydrogen-utilizing microbes, such as methanogens, utilize H₂ and carbon substrates, mainly CO₂, acetate, or methanol, to generate methane gas and hence to reduce hydrogen pressure in the rumen (8). However, this process causes a significant (6%) loss of dietary energy in the form of methane emission (14), which contributes to 13 to 19% of global greenhouse gas (16), and is one of the significant agricultural “causative sectors” contributing to global warming (13). Therefore, the energy loss and the consequent methane emission arouse both nutritional and environmental concerns in the livestock industry.Archaeal methanogens are obligate anaerobes (38), and species of the order Methanobacteriales are the most common methanogens found in the rumen (11). Recent studies using culture-independent methods investigating the methanogenic communities in the rumens of sheep and cattle have identified 21 different strains belonging to 13 species in sheep (40, 41, 43, 44) and 13 different strains related to 8 species in cattle (23, 37, 42). In addition, the identification of novel uncultured methanogens in the rumen (23, 33, 40) suggests that the understanding of the methanogenic ecology is limited. Cattle with higher feed efficiencies are reported to produce 20 to 30% less methane (9, 24). However, the linkage between rumen methanogenic composition and the host''s feed efficiency and methane production has not been studied and reported.As one of the indicators of feed efficiency in cattle, residual feed intake (RFI) measures the difference between an animal''s actual feed intake and the expected feed requirements for growth (1, 2). Cattle with low RFI (L-RFI) are designated “efficient,” while animals with high RFI (H-RFI) are designated “inefficient.” A recent study reporting a correlation between bacterial profiles and cattle RFI has suggested the probable linkage between rumen microbial ecology and feed efficiency in cattle (7). Therefore, we hypothesized that the structures and populations of methanogens may be also associated with RFI and methane gas production by the host. In this study, the compositions of methanogens in the rumens of cattle with different RFIs were compared by sequence analysis of the partial 16S rRNA genes (∼800 bp) generated from two constructed libraries, using pooled DNA from efficient (L-RFI) and inefficient (H-RFI) animals. The population of selected species in each steer was evaluated using quantitative real-time PCR (qRT-PCR) analysis, and the correlation between methanogenic structure/population and cattle RFI was investigated.     

mcrA-Targeted Real-Time Quantitative PCR Method To Examine Methanogen Communities

Methanogens are of great importance in carbon cycling and alternative energy production, but quantitation with culture-based methods is time-consuming and biased against methanogen groups that are difficult to cultivate in a laboratory. For these reasons, methanogens are typically studied through culture-independent molecular techniques. We developed a SYBR green I quantitative PCR (qPCR) assay to quantify total numbers of methyl coenzyme M reductase α-subunit (mcrA) genes. TaqMan probes were also designed to target nine different phylogenetic groups of methanogens in qPCR assays. Total mcrA and mcrA levels of different methanogen phylogenetic groups were determined from six samples: four samples from anaerobic digesters used to treat either primarily cow or pig manure and two aliquots from an acidic peat sample stored at 4°C or 20°C. Only members of the Methanosaetaceae, Methanosarcina, Methanobacteriaceae, and Methanocorpusculaceae and Fen cluster were detected in the environmental samples. The three samples obtained from cow manure digesters were dominated by members of the genus Methanosarcina, whereas the sample from the pig manure digester contained detectable levels of only members of the Methanobacteriaceae. The acidic peat samples were dominated by both Methanosarcina spp. and members of the Fen cluster. In two of the manure digester samples only one methanogen group was detected, but in both of the acidic peat samples and two of the manure digester samples, multiple methanogen groups were detected. The TaqMan qPCR assays were successfully able to determine the environmental abundance of different phylogenetic groups of methanogens, including several groups with few or no cultivated members.Methanogens are integral to carbon cycling, catalyzing the production of methane and carbon dioxide, both potent greenhouse gases, during organic matter degradation in anaerobic soils and sediment (8). Methanogens are widespread in anaerobic environments, including tundra (36), freshwater lake and wetland sediments (9, 12), estuarine and marine sediments (2), acidic peatlands (4, 14), rice field soil (10, 16), animal guts (41), landfills (30), and anaerobic digesters treating animal manure (1), food processing wastewater (27), and municipal wastewater and solid waste (37, 57). Methane produced in anaerobic digesters may be captured and used for energy production, thus offsetting some or all of the cost of operation and reducing the global warming potential of methane release to the atmosphere.Methanogens are difficult to study through culture-based methods, and therefore many researchers have instead used culture-independent techniques to study methanogen populations. The 16S rRNA gene is the most widely used target for gene surveys, and a number of primers and probes have been developed to target methanogen groups (9, 11, 31, 36, 38, 40, 46, 48, 57). To eliminate potential problems with nonspecific amplification, some researchers have developed primers for the gene sequence of the α-subunit of the methyl coenzyme M reductase (mcrA) (17, 30, 49). The Mcr is exclusive to the methanogens with the exception of the methane-oxidizing Archaea (18) and shows mostly congruent phylogeny to the 16S rRNA gene, allowing mcrA analysis to be used in conjunction with, or independently of, that of the 16S rRNA gene (3, 30, 49). A number of researchers have examined methanogen communities with mcrA and have found uncultured clades quite different in sequence from cultured methanogen representatives (9, 10, 12, 14, 17, 22, 28, 47).Previous studies described methanogen communities by quantitation of different clades through the use of rRNA-targeted or rRNA gene-targeted probes with techniques such as dot blot hybridization (1, 27, 37, 38, 48) and fluorescent in situ hybridization (11, 40, 44, 57). Real-time quantitative PCR (qPCR) is an alternate technique capable of determining the copy number of a particular gene present in the DNA extracted from an environmental sample. Only a few studies have used qPCR to quantitatively examine different clades within methanogen communities, and most of these studies have exclusively targeted the 16S rRNA gene (19, 41, 42, 54-56). Far fewer researchers have used qPCR to quantify methanogen clades by targeting the mcrA (21, 34, 45), and these studies were limited to only a few phylogenetic groups.In this paper we present a methodology for determining methanogen gene copy numbers through the use of qPCR targeting the mcrA. Methanogens were quantified in total using methanogen-specific primers in SYBR green assays and also as members of nine different phylogenetic groups using TaqMan probes targeting specific subsets of methanogens.     

Engineering of Clostridium phytofermentans Endoglucanase Cel5A for Improved Thermostability

A family 5 glycoside hydrolase from Clostridium phytofermentans was cloned and engineered through a cellulase cell surface display system in Escherichia coli. The presence of cell surface anchoring, a cellulose binding module, or a His tag greatly influenced the activities of wild-type and mutant enzymes on soluble and solid cellulosic substrates, suggesting the high complexity of cellulase engineering. The best mutant had 92%, 36%, and 46% longer half-lives at 60°C on carboxymethyl cellulose, regenerated amorphous cellulose, and Avicel, respectively.The production of biofuels from nonfood cellulosic biomass would benefit the economy, the environment, and national energy security (17, 32). The largest technological and economical obstacle is the release of soluble fermentable sugars at prices competitive with those from sugarcane or corn kernels (17, 31). One of the approaches is discovering new cellulases from cellulolytic microorganisms, followed by cellulase engineering for enhanced performance on pretreated solid substrates. However, cellulase engineering remains challenging because enzymatic cellulose hydrolysis is complicated, involving heterogeneous substrates (33, 37), different action mode cellulase components (18), synergy and/or competition among cellulase components (36, 37), and declining substrate reactivity over the course of conversion (11, 26). Directed enzyme evolution, independent of knowledge of the protein structure and the enzyme-substrate interactions (6, 34), has been conducted to generate endoglucanase mutants, such as enhanced activities on soluble substrates (14, 16, 22), prolonged thermostability (20), changed optimum pH (24, 28), or improved expression levels (21). Here, we cloned and characterized a family 5 glycoside hydrolase (Cel5A) from a cellulolytic bacterium, Clostridium phytofermentans ISDg (ATCC 700394) (29, 30), and engineered it for enhanced thermostability.     

Abundance of Novel and Diverse tfdA-Like Genes,Encoding Putative Phenoxyalkanoic Acid Herbicide-Degrading Dioxygenases,in Soil

Phenoxyalkanoic acid (PAA) herbicides are widely used in agriculture. Biotic degradation of such herbicides occurs in soils and is initiated by α-ketoglutarate- and Fe²⁺-dependent dioxygenases encoded by tfdA-like genes (i.e., tfdA and tfdAα). Novel primers and quantitative kinetic PCR (qPCR) assays were developed to analyze the diversity and abundance of tfdA-like genes in soil. Five primer sets targeting tfdA-like genes were designed and evaluated. Primer sets 3 to 5 specifically amplified tfdA-like genes from soil, and a total of 437 sequences were retrieved. Coverages of gene libraries were 62 to 100%, up to 122 genotypes were detected, and up to 389 genotypes were predicted to occur in the gene libraries as indicated by the richness estimator Chao1. Phylogenetic analysis of in silico-translated tfdA-like genes indicated that soil tfdA-like genes were related to those of group 2 and 3 Bradyrhizobium spp., Sphingomonas spp., and uncultured soil bacteria. Soil-derived tfdA-like genes were assigned to 11 clusters, 4 of which were composed of novel sequences from this study, indicating that soil harbors novel and diverse tfdA-like genes. Correlation analysis of 16S rRNA and tfdA-like gene similarity indicated that any two bacteria with D > 20% of group 2 tfdA-like gene-derived protein sequences belong to different species. Thus, data indicate that the soil analyzed harbors at least 48 novel bacterial species containing group 2 tfdA-like genes. Novel qPCR assays were established to quantify such new tfdA-like genes. Copy numbers of tfdA-like genes were 1.0 × 10⁶ to 65 × 10⁶ per gram (dry weight) soil in four different soils, indicating that hitherto-unknown, diverse tfdA-like genes are abundant in soils.Phenoxyalkanoic acid (PAA) herbicides such as MCPA (4-chloro-2-methyl-phenoxyacetic acid) and 2,4-D (2,4-dichlorophenoxyacetic acid) are widely used to control broad-leaf weeds in agricultural as well as nonagricultural areas (19, 77). Degradation occurs primarily under oxic conditions in soil, and microorganisms play a key role in the degradation of such herbicides in soil (62, 64). Although relatively rapidly degraded in soil (32, 45), both MCPA and 2,4-D are potential groundwater contaminants (10, 56, 70), accentuating the importance of bacterial PAA herbicide-degrading bacteria in soils (e.g., references 3, 5, 6, 20, 41, 59, and 78).Degradation can occur cometabolically or be associated with energy conservation (15, 54). The first step in the degradation of 2,4-D and MCPA is initiated by the product of cadAB or tfdA-like genes (29, 30, 35, 67), which constitutes an α-ketoglutarate (α-KG)- and Fe²⁺-dependent dioxygenase. TfdA removes the acetate side chain of 2,4-D and MCPA to produce 2,4-dichlorophenol and 4-chloro-2-methylphenol, respectively, and glyoxylate while oxidizing α-ketoglutarate to CO₂ and succinate (16, 17).Organisms capable of PAA herbicide degradation are phylogenetically diverse and belong to the Alpha-, Beta-, and Gammproteobacteria and the Bacteroidetes/Chlorobi group (e.g., references 2, 14, 29-34, 39, 60, 68, and 71). These bacteria harbor tfdA-like genes (i.e., tfdA or tfdAα) and are categorized into three groups on an evolutionary and physiological basis (34). The first group consists of beta- and gammaproteobacteria and can be further divided into three distinct classes based on their tfdA genes (30, 46). Class I tfdA genes are closely related to those of Cupriavidus necator JMP134 (formerly Ralstonia eutropha). Class II tfdA genes consist of those of Burkholderia sp. strain RASC and a few strains that are 76% identical to class I tfdA genes. Class III tfdA genes are 77% identical to class I and 80% identical to class II tfdA genes and linked to MCPA degradation in soil (3). The second group consists of alphaproteobacteria, which are closely related to Bradyrhizobium spp. with tfdAα genes having 60% identity to tfdA of group 1 (18, 29, 34). The third group also harbors the tfdAα genes and consists of Sphingomonas spp. within the alphaproteobacteria (30).Diverse PAA herbicide degraders of all three groups were identified in soil by cultivation-dependent studies (32, 34, 41, 78). Besides CadAB, TfdA and certain TfdAα proteins catalyze the conversion of PAA herbicides (29, 30, 35). All groups of tfdA-like genes are potentially linked to the degradation of PAA herbicides, although alternative primary functions of group 2 and 3 TfdAs have been proposed (30, 35). However, recent cultivation-independent studies focused on 16S rRNA genes or solely on group 1 tfdA sequences in soil (e.g., references 3-5, 13, and 41). Whether group 2 and 3 tfdA-like genes are also quantitatively linked to the degradation of PAA herbicides in soils is unknown. Thus, tools to target a broad range of tfdA-like genes are needed to resolve such an issue. Primers used to assess the diversity of tfdA-like sequences used in previous studies were based on the alignment of approximately 50% or less of available sequences to date (3, 20, 29, 32, 39, 47, 58, 73). Primers specifically targeting all major groups of tfdA-like genes to assess and quantify a broad diversity of potential PAA degraders in soil are unavailable. Thus, the objectives of this study were (i) to develop primers specific for all three groups of tfdA-like genes, (ii) to establish quantitative kinetic PCR (qPCR) assays based on such primers for different soil samples, and (iii) to assess the diversity and abundance of tfdA-like genes in soil.     

Replacement of the Replication Factors of Porcine Circovirus (PCV) Type 2 with Those of PCV Type 1 Greatly Enhances Viral Replication In Vitro

Porcine circovirus type 1 (PCV1), originally isolated as a contaminant of PK-15 cells, is nonpathogenic, whereas porcine circovirus type 2 (PCV2) causes an economically important disease in pigs. To determine the factors affecting virus replication, we constructed chimeric viruses by swapping open reading frame 1 (ORF1) (rep) or the origin of replication (Ori) between PCV1 and PCV2 and compared the replication efficiencies of the chimeric viruses in PK-15 cells. The results showed that the replication factors of PCV1 and PCV2 are fully exchangeable and, most importantly, that both the Ori and rep of PCV1 enhance the virus replication efficiencies of the chimeric viruses with the PCV2 backbone.Porcine circovirus (PCV) is a single-stranded DNA virus in the family Circoviridae (34). Type 1 PCV (PCV1) was discovered in 1974 as a contaminant of porcine kidney cell line PK-15 and is nonpathogenic in pigs (31-33). Type 2 PCV (PCV2) was discovered in piglets with postweaning multisystemic wasting syndrome (PMWS) in the mid-1990s and causes porcine circovirus-associated disease (PCVAD) (1, 9, 10, 25). PCV1 and PCV2 have similar genomic organizations, with two major ambisense open reading frames (ORFs) (16). ORF1 (rep) encodes two viral replication-associated proteins, Rep and Rep′, by differential splicing (4, 6, 21, 22). The Rep and Rep′ proteins bind to specific sequences within the origin of replication (Ori) located in the intergenic region, and both are responsible for viral replication (5, 7, 8, 21, 23, 28, 29). ORF2 (cap) encodes the immunogenic capsid protein (Cap) (26). PCV1 and PCV2 share approximately 80%, 82%, and 62% nucleotide sequence identity in the Ori, rep, and cap, respectively (19).In vitro studies using a reporter gene-based assay system showed that the replication factors of PCV1 and PCV2 are functionally interchangeable (2-6, 22), although this finding has not yet been validated in a live infectious-virus system. We have previously shown that chimeras of PCV in which cap has been exchanged between PCV1 and PCV2 are infectious both in vitro and in vivo (15), and an inactivated vaccine based on the PCV1-PCV2 cap (PCV1-cap2) chimera is used in the vaccination program against PCVAD (13, 15, 18, 27).PCV1 replicates more efficiently than PCV2 in PK-15 cells (14, 15); thus, we hypothesized that the Ori or rep is directly responsible for the differences in replication efficiencies. The objectives of this study were to demonstrate that the Ori and rep are interchangeable between PCV1 and PCV2 in a live-virus system and to determine the effects of swapped heterologous replication factors on virus replication efficiency in vitro.     

Diversity of Formyltetrahydrofolate Synthetases in the Guts of the Wood-Feeding Cockroach Cryptocercus punctulatus and the Omnivorous Cockroach Periplaneta americana

We examined the diversity of a marker gene for homoacetogens in two cockroach gut microbial communities. Formyltetrahydrofolate synthetase (FTHFS or fhs) libraries prepared from a wood-feeding cockroach, Cryptocercus punctulatus, were dominated by sequences that affiliated with termite gut treponemes. No spirochete-like sequences were recovered from the omnivorous roach Periplaneta americana, which was dominated by Firmicutes-like sequences.The guts of wood-feeding termites and Cryptocercus punctulatus cockroaches share an unusual pattern of electron flow, as high rates of CO₂-reductive acetogenesis typically supplant methanogenesis as the terminal electron sink (2, 3). Past studies have shown that from 10 to 30% of gut acetate produced in environments of termites and wood-feeding cockroaches is microbially generated from CO₂ (3, 28), ultimately powering 18 to 26% of the host insect''s own respiratory energy metabolism (25). Nevertheless, most termites emit methane (2), and termite emissions constitute approximately 4% of the global methane budget (27). Cockroaches have been proposed to represent an additional source of note (9). Interestingly, methanogenic termites and cockroaches exhibit increased acetogenesis following addition of exogenous H₂ (3, 29). This suggests that these insects are host to a robust population of bacteria that are capable of homoacetogenesis but may be primarily using alternative electron donors (and other substrates and pathways) in vivo.Acetogenic bacteria belonging to two bacterial phyla, Firmicutes and Spirochaetes, have been isolated from the guts of termites (1, 4, 11, 12, 14). Several surveys have targeted and used the gene for formyltetrahydrofolate synthetase (FTHFS), a key gene in the Wood-Ljungdahl pathway of acetogenesis (16), as a potential marker for the pathway (15, 18). For the wood-feeding termites that have been examined, the studies have revealed an abundance of FTHFS sequences that form a coherent phylogenetic cluster, together with genes from homoacetogenic termite gut spirochetes belonging to the genus Treponema (24, 26, 30). This suggests that treponemes may be among the more abundant of the homoacetogens active in these environments.Little is known about the population structure and biology of CO₂-reducing, acetogenic bacteria in the guts of either omnivorous or wood-feeding cockroaches. The wood-feeding cockroach Cryptocercus hosts an abundance of flagellate protozoa closely related to those believed to dominate polysaccharide fermentation in the guts of termites (5, 6, 22), suggesting that at least one key environmental niche is filled by similar microbes in both termites and Cryptocercidae. Additionally, Cryptocercidae cockroaches, like termites, house diverse spirochetes and are the site of intense CO₂ reduction into acetate (3, 7). Possibly, spirochetes capable of CO₂ reduction into acetate are present in the hindguts of cockroaches. However, no evidence has yet been presented for the existence of homoacetogenic treponemes in environments other than the guts of termites, and FTHFS surveys of human (21) or horse (15) fecal matter and bovine rumen samples (20) revealed only Firmicutes-like and other FTHFS alleles that are distinct from those comprising the termite treponeme cluster.Here, by examining FTHFS gene diversity in Cryptocercus punctulatus and Periplaneta americana guts, we endeavored to learn more about the distribution and origins of homoacetogenic treponemes (and their genes) that are found in wood-feeding termites. In particular, we wished to ascertain whether FTHFS genes present in either of the two cockroaches are termite treponeme-like and, if so, whether analysis reveals any obvious signal indicating recent or ancient lateral community transfer events between insect lineages.     

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).     

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.     

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.     

Rearranged JC Virus Noncoding Control Regions Found in Progressive Multifocal Leukoencephalopathy Patient Samples Increase Virus Early Gene Expression and Replication Rate

Polyomavirus JC (JCV) infects ∼60% of the general population, followed by asymptomatic urinary shedding in ∼20%. In patients with pronounced immunodeficiency, including HIV/AIDS, JCV can cause progressive multifocal leukoencephalopathy (PML), a devastating brain disease of high mortality. While JCV in the urine of healthy people has a linear noncoding control region called the archetype NCCR (at-NCCR), JCV in brain and cerebrospinal fluid (CSF) of PML patients bear rearranged NCCRs (rr-NCCRs). Although JCV NCCR rearrangements are deemed pathognomonic for PML, their role as a viral determinant is unclear. We sequenced JCV NCCRs found in CSF of eight HIV/AIDS patients newly diagnosed with PML and analyzed their effect on early and late gene expression using a bidirectional reporter vector recapitulating the circular polyomavirus early and late gene organization. The rr-NCCR sequences were highly diverse, but all increased viral early reporter gene expression in progenitor-derived astrocytes, glia-derived cells, and human kidney compared to the expression levels with the at-NCCR. The expression of simian virus 40 (SV40) large T antigen or HIV Tat expression in trans was associated with a strong increase of at-NCCR-controlled early gene expression, while rr-NCCRs were less responsive. The insertion of rr-NCCRs into the JCV genome backbone revealed higher viral replication rates for rr-NCCR compared to those of the at-NCCR JCV in human progenitor-derived astrocytes or glia cells, which was abrogated in SV40 large T-expressing COS-7 cells. We conclude that naturally occurring JCV rr-NCCR variants from PML patients confer increased early gene expression and higher replication rates compared to those of at-NCCR JCV and thereby increase cytopathology.Polyomavirus JC (JCV) infects approximately 60% of the general population, followed by asymptomatic urinary shedding in 20% of healthy individuals (20). Although JCV-associated nephropathy may occur in kidney transplant (14, 33) and HIV/AIDS patients (6, 27), the most prominent JCV disease is progressive multifocal leukoencephalopathy (PML) (44, 60). The pathology of PML was first described in 1958 as a rare complication of patients with chronic lymphocytic leukemia or Hodgkin''s lymphoma (3). Today, PML is recognized as a rare, virus-mediated demyelinating disease of the white brain matter in highly immunocompromised patients, including HIV/AIDS, transplantation, and chemotherapy patients and those exposed to immunomodulatory or depleting biologicals for the treatment of autoimmune diseases (29, 40). During the human immunodeficiency virus type 1 (HIV-1) pandemic, the incidence of PML rose significantly to rates of 1 to 8% prior to the use of highly active antiretroviral therapy (2, 5, 34). The definitive diagnosis requires brain tissue, but the detection of JCV by PCR in cerebrospinal fluid (CSF) is generally accepted for a laboratory-confirmed diagnosis in immunocompromised patients with (multi-)focal neurological deficits and corresponding radiological findings (8, 26). Due to the lack of effective antiviral therapy (13), the treatment of PML is based on improving overall immune functions. While this is difficult to achieve in cancer, chemotherapy, and transplantation, prompt antiretroviral therapy in HIV/AIDS patients has significantly improved PML survival, with increasing JCV-specific immune responses and declining intracerebral JCV replication (7, 15, 23, 35, 37). In patients diagnosed with PML after treatment with natalizumab for multiple sclerosis or inflammatory bowel disease, the removal of the monoclonal antibody by plasmapheresis has been tried to restore lymphocyte homing to, and the immune surveillance of, JCV replication sites in the central nervous system (38, 40, 52). However, the success of immune reconstitution in HIV/AIDS- and natalizumab-associated PML cases is limited by the fact that PML is typically diagnosed clinically by neurological deficits resulting from significant brain damage, where mounting antiviral immunity often may be too slow to modify the outcome. On the other hand, rapid recovery may cause immune reconstitution inflammatory syndrome with paradoxical clinical worsening and fatal outcomes (9, 16, 25, 38, 46). Although the etiologic role of JCV in PML is well documented, the pathogenesis and, in particular, the role of viral determinants is less clear. Virtually all JCV strains isolated from the brain or CSF of PML patients are characterized by highly variable genomic rearrangements of the noncoding control region (NCCR), which governs viral early and late genes in opposite directions of the circular polyomavirus DNA genome (1, 4, 31, 39, 41, 43, 49, 54, 59). In contrast, JCV detected in the urine of immunocompetent individuals show a consistent linear architecture called the archetype NCCR (at-NCCR). Thus, detecting rearranged NCCRs (rr-NCCRs) JCV in the central nervous system has been viewed as being derived from the archetype and closely linked to PML (4), but the functional consequences of rearrangements are unclear. To address the consequences of the rr-NCCR for JCV gene expression and replication, we characterized the sequences of JCV rr-NCCR from patients with PML and analyzed their effect on viral gene expression and replication with JCV at-NCCR in a bidirectional reporter assay and in recombinant JCV.     

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.     

Genome-Derived Criteria for Assigning Environmental narG and nosZ Sequences to Operational Taxonomic Units of Nitrate Reducers

Ninety percent of cultured bacterial nitrate reducers with a 16S rRNA gene similarity of ≥97% had a narG or nosZ similarity of ≥67% or ≥80%, respectively, suggesting that 67% and 80% could be used as standardized, conservative threshold similarity values for narG and nosZ, respectively (i.e., any two sequences that are less similar than the threshold similarity value have a very high probability of belonging to different species), for estimating species-level operational taxonomic units. Genus-level tree topologies of narG and nosZ were generally similar to those of the corresponding 16S rRNA genes. Although some genomes contained multiple copies of narG, recent horizontal gene transfer of narG was not apparent.Nitrate reducers (i.e., both dissimilatory nitrate reducers and denitrifiers) reduce nitrate to nitrite, which can then be reduced to ammonium by dissimilatory nitrate reducers or sequentially reduced to nitric oxide, nitrous oxide, and dinitrogen by denitrifiers (29). narG codes for the alpha subunit of the dissimilatory nitrate reductase, which reduces nitrate to nitrite and is thus common to both dissimilatory nitrate reducers and denitrifiers (29). nosZ codes for nitrous oxide reductase, which reduces nitrous oxide to dinitrogen and is common to denitrifiers but not dissimilatory nitrate reducers (29). Both narG and nosZ are commonly used as gene markers for community level analysis of nitrate reducers (2, 8, 9, 16, 18, 19, 20, 25). However, standardized criteria for assigning environmental narG and nosZ sequences to operational taxonomic units (OTUs) are required so that diverse data sets on nitrate-reducing communities can be normalized. The widespread ability of bacteria and archaea to denitrify (29) complicates the development of such criteria for genes involved in denitrification. Some closely related narG and closely related nosZ genes occur in distantly related taxa, and narG or nosZ phylogenies do not always reflect 16S rRNA phylogenies (17). However, nosZ-based phylogenies in general have a high degree of congruency with 16S rRNA gene-based phylogenies (3, 10, 30), and recent horizontal gene transfer of nosZ seems unlikely (10), indicating that denitrifier structural genes might be used for estimating the species-level novelty, as well as species-level diversity, of denitrifiers in environmental samples. The limited amount of data on horizontal gene transfer of narG (4, 24) identifies a need to extend such an approach to this gene. The limited number of studies that have compared 16S rRNA with narG or nosZ phylogenies accentuates the need for a more thorough analysis of the phylogenetic relatedness of these three genes (3, 4, 7). Thus, the main objectives of this study were to (i) resolve criteria for standardizing OTU assignment of environmental narG and nosZ sequences, (ii) determine whether those criteria can be used as indicators of novel species, and (iii) investigate the impact of horizontal gene transfer on narG.     

Development and Application of a Novel Peptide Nucleic Acid Probe for the Specific Detection of Cronobacter Genomospecies (Enterobacter sakazakii) in Powdered Infant Formula

Here, we report a fluorescence in situ hybridization (FISH) method for rapid detection of Cronobacter strains in powdered infant formula (PIF) using a novel peptide nucleic acid (PNA) probe. Laboratory tests with several Enterobacteriaceae species showed that the specificity and sensitivity of the method were 100%. FISH using PNA could detect as few as 1 CFU per 10 g of Cronobacter in PIF after an 8-h enrichment step, even in a mixed population containing bacterial contaminants.Cronobacter strains were originally described as Enterobacter sakazakii (12), but they are now known to comprise a novel genus consisting of six separate genomospecies (20, 21). These opportunistic pathogens are ubiquitous in the environment and various types of food and are occasionally found in the normal human flora (11, 12, 16, 32, 47). Based on case reports, Cronobacter infections in adults are generally less severe than Cronobacter infections in newborn infants, with which a high fatality rate is associated (24).The ability to detect Cronobacter and trace possible sources of infection is essential as a means of limiting the impact of these organisms on neonatal health and maintaining consumer confidence in powdered infant formula (PIF). Conventional methods, involving isolation of individual colonies followed by biochemical identification, are more time-consuming than molecular methods, and the reliability of some currently proposed culture-based methods has been questioned (28). Recently, several PCR-based techniques have been described (23, 26, 28-31, 38). These techniques are reported to be efficient even when low levels of Cronobacter cells are found in a sample (0.36 to 66 CFU/100 g). However, PCR requires DNA extraction and does not allow direct, in situ visualization of the bacterium in a sample.Fluorescence in situ hybridization (FISH) is a method that is commonly used for bacterial identification and localization in samples. This method is based on specific binding of nucleic acid probes to particular DNA or RNA target regions (1, 2). rRNA has been regarded as the most suitable target for bacterial FISH, allowing differentiation of potentially viable cells. Traditionally, FISH methods are based on the use of conventional DNA oligonucleotide probes, and a commercial system, VIT-E sakazakii (Vermicon A.G., Munich, Germany), has been developed based on this technology (25). However, a recently developed synthetic DNA analogue, peptide nucleic acid (PNA), has been shown to provide improved hybridization performance compared to DNA probes, making FISH procedures easier and more efficient (41). Taking advantage of the PNA properties, FISH using PNA has been successfully used for detection of several clinically relevant microorganisms (5, 15, 17, 27, 34-36).