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.     

Complete Genome Sequence of the Representative γ-Hexachlorocyclohexane-Degrading Bacterium Sphingobium japonicum UT26

Sphingobium japonicum strain UT26 utilizes γ-hexachlorocyclohexane (γ-HCH), a man-made chlorinated pesticide that causes serious environmental problems due to its toxicity and long persistence, as a sole source of carbon and energy. Here, we report the complete genome sequence of UT26, which consists of two chromosomes and three plasmids. The 15 lin genes involved in γ-HCH degradation are dispersed on the two chromosomes and one of the three plasmids.γ-Hexachlorocylohexane (γ-HCH) is a man-made insecticide that has caused serious environmental problems worldwide (9). Although only several decades have passed since the initial release of γ-HCH into the environment, many γ-HCH-degrading bacterial strains have been isolated (9), suggesting that γ-HCH-degrading ability was acquired by such strains within a short period (14). Sphingobium japonicum UT26 was isolated from upland γ-HCH-contaminated soil in Japan and utilizes γ-HCH as its sole source of carbon and energy (8, 16). In UT26, 15 lin genes are involved in γ-HCH utilization (2, 11, 12). Our clarification of the complete genome sequence of this strain is expected to provide insights into the mechanisms by which bacteria adapt to xenobiotics.One S. japonicum UT26 colony was designated UT26S (NBRC 101211), and its complete genome sequence was determined by a whole-genome shotgun sequencing strategy using the Sanger method (10, 15, 17). The sequences of ca. 92,400 reads were assembled by the Phrap and CONSED assembly tools (4, 5, 7), and the gaps between contigs were closed by sequencing PCR products which were amplified from genomic DNA using the appropriate primers. The prediction and annotation of open reading frames (ORFs) were performed using Glimmer3 (1), BLASTP, the In Silico Molecular Cloning software suite (In Silico Biology, Inc.), and the GenomeMatcher software (13). Nontranslating genes were predicted using the Rfam, tRNAscan-SE, and ARAGORN programs.The genome of S. japonicum UT26S consists of two circular chromosomes (Chr), Chr 1 (3,514,822 bp, 64.8% G+C, 3,529 ORFs) and Chr 2 (681,892 bp, 65.9% G+C, 589 ORFs), and three circular plasmids, pCHQ1 (190,974 bp, 63.0% G+C, 224 ORFs), pUT1 (31,776 bp, 63.7% G+C, 44 ORFs), and pUT2 (5,398 bp, 61.0% G+C, 8 ORFs). Chr 1 and Chr 2 have one and two copies, respectively, of rRNA operons. Fifty-one and 4 tRNA genes were located on Chr 1 and Chr 2, respectively. One hundred ninety-six out of 206 bacterial essential genes proposed by Gil et al. (6) were all located on Chr 1, indicating that this is clearly a “main” chromosome. The 15 lin genes for γ-HCH degradation are dispersed on Chr 1, Chr 2, and pCHQ1. Comparison of the UT26S genome with those of five other sphingomonad (TM1) strains revealed that the lin genes (linA, linB, linC, linRED, and linF) specific for the conversion of γ-HCH to β-ketoadipate are located in the DNA regions unique to the UT26S genome. On the other hand, linGHIJ for the β-ketoadipate pathway (12) and linKLMN for the ABC transporter system (3) are located in conserved genomic regions of these sphingomonads. Based on these results, we propose a model in which UT26S was established by recruiting the specific lin genes into an ancestral strain having core functions of sphingomonads.     

Genome Sequence of Lactobacillus crispatus ST1

Lactobacillus crispatus is a common member of the beneficial microbiota present in the vertebrate gastrointestinal and human genitourinary tracts. Here, we report the genome sequence of L. crispatus ST1, a chicken isolate displaying strong adherence to vaginal epithelial cells.Lactobacillus crispatus can persist in the vertebrate gastrointestinal tract and is among the most prevalent species of the Lactobacillus-dominated human vaginal microbiota (2, 9, 13, 14). It belongs to the so-called acidophilus group (3), which has attracted interest because some of its species are important factors in the production of fermented foods (12) and some can, at least transiently, colonize the human host (2, 9, 13, 14). Moreover, some specific strains, mainly L. acidophilus NCFM and L. johnsonii NCC 533, have received prominence as intestinal-health-promoting microbes (4). Although the genomes of seven members of the acidophilus complex have been sequenced to date (12), the genome sequences of L. crispatus and other predominant lactobacillar species in the urogenital flora have mostly remained obscure. Vaginal lactobacilli can have an important role in controlling the health of the host (2, 14). They can, for example, positively influence and stabilize the host''s vaginal microbiota via the production of compounds that are acidic or exert a direct inhibiting action toward pathogenic bacteria (2, 14). In addition to the antimicrobial compounds, the competitive exclusion of pathogens is another mechanism by which the host''s microbiota can be balanced (2). L. crispatus ST1 was originally isolated from the crop of a chicken, and PCR profiling of L. crispatus isolates has verified it to be an abundant colonizer of the chicken crop (6, 8). It also displays a strong protein-dependent adhesion to the epithelial cells of the human vagina and has been shown to inhibit the adhesion of avian pathogenic Escherichia coli (6, 7).The genome was sequenced (18× coverage) using a 454 pyrosequencer with GS FLX chemistry (Roche). The contig order was confirmed and gaps were filled by sequencing PCR fragments from the genomic DNA template using ABI 3730 and Big Dye chemistry (Applied Biosystems). Genomic data were processed using the Staden Package (11) and gsAssembler (Roche). Coding sequences (CDSs) were predicted using Glimmer3 (5) followed by manual curation of the start sites. The remaining intergenic regions were reanalyzed for missed CDSs by using BlastX (1). Annotation transfer was performed based on a BlastP search, followed by Blannotator analysis using default settings (http://ekhidna.biocenter.helsinki.fi/poxo/blannotator) and manual verification. Orthologous groups between the different lactobacillar proteomes were identified using OrthoMCL (10).The genome of L. crispatus ST1 consists of a single circular chromosome 2.04 Mbp in size, with an overall G+C content of 37%, without any plasmids. There are 64 tRNA genes, 4 rRNA operons, and 2 CRISPR loci. Out of the 2,024 predicted CDSs, a putative function was assigned to 77%, whereas 10% of the CDSs were annotated as conserved and 13% as novel. Based on the orthologous grouping, 302 (15%) of the CDSs encoded by ST1 have no detectable homologs in any of the Lactobacillus proteomes published to date.     

Complete Genome Sequence of Lactobacillus salivarius CECT 5713, a Probiotic Strain Isolated from Human Milk and Infant Feces

Lactobacillus salivarius is a homofermentative lactic acid bacterium and is frequently isolated from mucosal surfaces of healthy humans. L. salivarius CECT 5713, a strain isolated simultaneously from breast milk and infant feces of a healthy mother-infant pair, has immunomodulatory, anti-inflammatory, and anti-infectious properties, as revealed by several in vitro and in vivo assays. Here, we report its complete and annotated genome sequence.In the last years, culture-dependent and -independent analyses of the bacterial diversity of human milk and colostrum have revealed that these biological fluids are a source of live staphylococci, streptococci, lactic acid bacteria, and bifidobacteria in the infant gut (5, 6, 8, 9, 11, 13), where they play a key role in the initiation and development of the gut microbiota (12). In a previous study, we isolated L. salivarius CECT 5713 from human milk and infant feces of a mother-child pair (10). Subsequent studies revealed that this strain was a good probiotic candidate since it achieved high survival rates when exposed to the gastrointestinal tract conditions, showed a strong adherence to intestinal cells, stimulated the expression of mucin-encoding genes, produced antimicrobial compounds (lactate, acetate, and hydrogen peroxide), and displayed in vivo and in vitro immunomodulatory, anti-inflammatory, and antibacterial properties against pathogenic bacteria (2, 10, 15). Moreover, oral administration of L. salivarius CECT 5713 appears to be an efficient alternative for the treatment of infectious mastitis in lactating women (7). Similarly, studies with other L. salivarius strains in animal models and clinical trials have demonstrated their probiotic function and, particularly, their anti-inflammatory effects (3, 14, 16).In order to interrogate the genome sequence of L. salivarius CECT 5713 with regard to its probiotic properties, the complete genome sequence was determined by a whole-genome shotgun strategy using pyrosequencing technology (454 Life Sciences, Banford, CT). The initial draft assembly provided by 454 Life Sciences was based on 444,604 high-quality pyrosequencing reads, which assembled into 59 contigs. The genome sequence of L. salivarius UCC118 (1), a well-characterized probiotic strain, was used to order these contigs into large scaffolds.The genome of L. salivarius CECT 5713 consists of a circular chromosome of 1,828,169 bp, two plasmids (pHN1, 44,581 bp; pHN2, 20,426 bp), and a megaplasmid (pHN3, 242,962 bp). The overall GC content of the chromosome is 32.93%, similar to that of the megaplasmid but lower than those of the plasmids (>38%). The entire genome of CECT 5713 contains 1,558 protein-, 87 tRNA-, and 51 rRNA-encoding genes. A comparison between the genomes of L. salivarius CECT 5713 and UCC118 revealed the presence of 52 protein-encoding genes that are exclusive for CECT 5713, including genes encoding a 6-phospho-β-glucosidase and three collagen-binding proteins, which may explain the high potential for competitive exclusion of pathogens displayed by this strain. The genes responsible for the bacteriocin activity of L. salivarius CECT 5713 are located in pHN3. This megaplasmid contains six open reading frames (ORFs) closely related, but not identical, to the genes responsible for the biosynthesis of salivaricin ABP-118, a two-component class II bacteriocin (4), in L. salivarius UCC118. Globally, several features of the L. salivarius CECT 5713 genome suggest a strong probiotic potential in humans.     

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.     

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.     

The Genome of the Amoeba Symbiont “Candidatus Amoebophilus asiaticus” Reveals Common Mechanisms for Host Cell Interaction among Amoeba-Associated Bacteria

Protozoa play host for many intracellular bacteria and are important for the adaptation of pathogenic bacteria to eukaryotic cells. We analyzed the genome sequence of “Candidatus Amoebophilus asiaticus,” an obligate intracellular amoeba symbiont belonging to the Bacteroidetes. The genome has a size of 1.89 Mbp, encodes 1,557 proteins, and shows massive proliferation of IS elements (24% of all genes), although the genome seems to be evolutionarily relatively stable. The genome does not encode pathways for de novo biosynthesis of cofactors, nucleotides, and almost all amino acids. “Ca. Amoebophilus asiaticus” encodes a variety of proteins with predicted importance for host cell interaction; in particular, an arsenal of proteins with eukaryotic domains, including ankyrin-, TPR/SEL1-, and leucine-rich repeats, which is hitherto unmatched among prokaryotes, is remarkable. Unexpectedly, 26 proteins that can interfere with the host ubiquitin system were identified in the genome. These proteins include F- and U-box domain proteins and two ubiquitin-specific proteases of the CA clan C19 family, representing the first prokaryotic members of this protein family. Consequently, interference with the host ubiquitin system is an important host cell interaction mechanism of “Ca. Amoebophilus asiaticus”. More generally, we show that the eukaryotic domains identified in “Ca. Amoebophilus asiaticus” are also significantly enriched in the genomes of other amoeba-associated bacteria (including chlamydiae, Legionella pneumophila, Rickettsia bellii, Francisella tularensis, and Mycobacterium avium). This indicates that phylogenetically and ecologically diverse bacteria which thrive inside amoebae exploit common mechanisms for interaction with their hosts, and it provides further evidence for the role of amoebae as training grounds for bacterial pathogens of humans.Free-living amoebae, such as Acanthamoeba spp., are ubiquitous protozoa which can be found in such diverse habitats as soil, marine water, and freshwater and in many engineered environments (62, 100). They are important predators of prokaryotic and eukaryotic microorganisms, thereby having great influence on microbial community composition, soil mineralization, plant growth, and nutrient cycles (14, 100). Interestingly, many well-known pathogens of humans are able to infect, survive, and multiply within amoebae (39, 51). These protozoa can thus serve as reservoirs and vectors for the transmission of pathogenic bacteria to humans, as demonstrated for L. pneumophila and Mycobacterium avium (2, 115). It is also increasingly being recognized that protozoa are important for the adaptation of (pathogenic) bacteria to higher eukaryotic cells as a niche for growth (2, 24, 42, 78, 89).In addition to the many recognized transient associations between amoeba and pathogens, stable and obligate relationships between bacteria and amoebae also were described for members of the Alphaproteobacteria (11, 34, 48), the Betaproteobacteria (49), the Bacteroidetes (50), and the Chlamydiae (4, 12, 35, 52). These obligate amoeba symbionts show a worldwide distribution, since phylogenetically highly similar strains were found in amoeba isolates from geographically distant sources (51, 107). The phylogenetic diversity and the different lifestyles of these obligate intracellular bacteria—some are located directly in the host cell cytoplasm (11, 34, 48-50, 52), while others are enclosed in host-derived vacuoles (4, 35, 44)—suggest fundamentally different mechanisms of host cell interaction. However, with the exception of chlamydia-related amoeba symbionts (37, 46, 47), our knowledge of the biology of obligate intracellular symbionts of amoebae is still scarce.Comparative genomics has been extremely helpful for the analysis of intracellular bacteria. Numerous genome sequences from the Alpha- and Gammaproteobacteria and the Chlamydiae are available and have contributed significantly to our understanding of genome evolution, the biology of intracellular bacteria, and the interactions with their host cells (24, 26, 46, 79, 82). In this study, we determined and analyzed the complete genome sequence of “Candidatus Amoebophilus asiaticus” strain 5a2 in order to gain novel insights into its biology. “Ca. Amoebophilus asiaticus” is a Gram-negative, obligate intracellular amoeba symbiont belonging to the Bacteroidetes which has been discovered within an amoeba isolated from lake sediment (107). “Ca. Amoebophilus asiaticus” shows highest 16S rRNA similarity to “Candidatus Cardinium hertigii,” an obligate intracellular parasite of arthropods able to manipulate the reproduction of its hosts (131). According to 16S rRNA trees, both organisms are members of a monophyletic group within the phylogenetically diverse phylum Bacteroidetes, consisting only of symbionts and sequences which were directly retrieved from corals (113). Among members of the Bacteroidetes, the genome sequences of only three symbionts, which are only distantly related (75 to 80% 16S rRNA sequence similarity) to “Ca. Amoebophilus asiaticus,” have been determined to date: two strains of “Candidatus Sulcia muelleri,” a symbiont of sharpshooters, and “Azobacteroides pseudotrichonymphae,” a symbiont of an anaerobic termite gut ciliate (45, 72, 74, 127).The genome of “Ca. Amoebophilus asiaticus” is only moderately reduced in size compared to those of many other obligate intracellular bacteria (75, 123), but nevertheless, its biosynthetic capabilities are extremely limited. A large fraction of the genome consists of IS elements and an unparalleled high number of proteins with eukaryotic domains, such as ankyrin repeats, TPR/SEL1 repeats, leucine-rich repeats, and domains from the eukaryotic ubiquitin system, all of them most likely important for host cell interaction. Feature enrichment analysis across a nonredundant data set of all bacterial genomes showed that these domains are enriched in the genomes of bacteria (including several pathogens of humans) known to be able to infect amoebae, providing further evidence for an important role of amoebae in the evolution of mechanisms for host cell interaction in intracellular bacteria.     

Complete Genome Sequence of Methanothermobacter marburgensis,a Methanoarchaeon Model Organism

The circular genome sequence of the chemolithoautotrophic euryarchaeon Methanothermobacter marburgensis, with 1,639,135 bp, was determined and compared with that of Methanothermobacter thermautotrophicus. The genomes of the two model methanogens differ substantially in protein coding sequences, in insertion sequence (IS)-like elements, and in clustered regularly interspaced short palindromic repeats (CRISPR) loci.Methanothermobacter marburgensis (DSM 2133) (formerly Methanobacterium thermoautotrophicum strain Marburg), a member of the Methanobacteriales (2), was isolated in 1978 from anaerobic sewage sludge in Marburg, Germany (5). The hydrogenotrophic methanogen grows even faster (2 h versus 3 h doubling time) and to higher cell concentrations (3 g versus 1.5 g dry mass per liter) than Methanothermobacter thermautotrophicus (DSM 1053) (formerly Methanobacterium thermoautotrophicum strain ΔH) (20) (for other differences, see references 3 and 19). Both methanogens were used in the last 35 years for the elucidation of the enzymes and coenzymes involved in CO₂ reduction to methane with H₂ (4, 16-18). The genome sequence of M. thermautotrophicus was reported in 1997 (15); that of M. marburgensis is announced here.The genome size of M. marburgensis is 1,639,135 bp (that of M. thermautotrophicus is 1,751,377 bp), the genome G+C content is 48.64% (49.54% for M. thermautotrophicus), and the part coding is 90.94% (91.02% for M. thermautotrophicus). Comparison of the sequences (13) revealed that the two genomes have 1,607 protein coding sequences (CDS) in common and 411 CDS not in common (145 CDS are found only in M. marburgensis and 266 CDS only in M. thermautotrophicus) and show a high degree of synteny. The CDS not in common could be traced back to gene splitting (15%), gene deletion (30%), gene duplication (30%), and lateral gene transfer (24%) events (percentages given are for M. marburgensis). Of the 1,607 CDS in common, approximately 40% show BLAST search expectation values of >10⁻¹⁰⁰ at the protein level, reflecting large differences in sequence divergence. Almost 470 CDS encode conserved hypothetical proteins.The genome of M. marburgensis harbors 15 insertion sequence (IS)-like elements, whereas there is no evidence for a classically organized IS-like element in M. thermautotrophicus. Consistently, a CDS for a transposase is found only in M. marburgensis.In the genome of M. marburgensis there is only one clustered regularly interspaced short palindromic repeat (CRISPR) locus with 36 repeats and only one CRISPR-associated (cas) gene (csa3), indicating that the organism is not protected from invasion by phage and plasmid DNA (7, 8, 10, 12). By comparison, in the genome of M. thermautotrophicus there are three CRISPR loci with 124, 4, and 47 repeats and 18 cas genes that encode proteins involved in adaptation and interference (http://genoweb1.irisa.fr/Serveur-GPO/outils/repeatsAnalysis/CRISPR/). The spacer sequences from locus 2 match DNA sequences found in phage ΨM1 of M. marburgensis (6, 11) and ΨM100 of M. wolfei (9), which supports the observation that M. thermautotrophicus is not lysed by those two phages. Unfortunately, there is no DNA sequence available for phage ΦF1, which is able to lyse M. thermautotrophicus (14), to compare it with the spacer sequences of the CRISPR regions. In the plasmid pM2001 (= pMTBMA4) (4,439-bp circular multicopy plasmid found only in M. marburgensis) (1, 19), no sequence identities for CRISPR spacer sequences of M. thermautotrophicus were found (14).Approximately 200 CDS were identified that are required for the synthesis of the enzymes, coenzymes, and prosthetic groups involved in CO₂ reduction to methane and in the coupling of this process with energy conservation. Some of the genes have been found only recently; others, such as those for coenzyme F₄₃₀ biosynthesis, still remain to be discovered.     

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.     

Complete Genome Sequence of Bacillus thuringiensis Mutant Strain BMB171

Bacillus thuringiensis has been widely used as a biopesticide for a long time. Here we report the finished and annotated genome sequence of B. thuringiensis mutant strain BMB171, an acrystalliferous mutant strain with a high transformation frequency obtained and stocked in our laboratory.Bacillus thuringiensis is an insect pathogen which is widely used as a biopesticide due to its various endogenous crystal proteins and spores (12). To improve the virulence and practical effectiveness of B. thuringiensis, genetic transformation of different genes with beneficial traits is a fundamental procedure. Simultaneously, genetic transformation can facilitate functional genomic research. However, wild-type strains are not suitable to be used as recipient strains because of low transformation efficiency. This obstacle is mainly caused by the thick cell wall layer of B. thuringiensis together with multiple plasmids inside the cell, which harbor genes encoding insecticidal crystal proteins. We used the method of elevating the growth temperature and adding 0.05% sodium dodecyl sulfate to treat several parental strains and finally obtained mutant strain BMB171, with no resident plasmid, from wild-type crystalliferous strain YBT-1463 (9). The electrotransformation frequency of mutant BMB171 could reach up to 10⁷ transformants/μg DNA after optimization of the electrotransformation parameters (7), which was 4.8 × 10⁴-fold higher than that of the parental strain (8). Moreover, mutant strain BMB171 exhibited the same characteristics as YBT-1463, such as metabolic abilities and growth properties, as well as sensitivity to 10 antibiotics (8). Of course, BMB171 could produce parasporal crystals with characteristic geometric shapes through the expression of relevant cry genes carried by plasmids (7). Thus, B. thuringiensis mutant strain BMB171 has become a major recipient strain and is widely used for insecticidal crystal protein-encoding gene expression (14, 15), cell surface display (10, 13), gene function and regulation researches (2, 5), etc.The B. thuringiensis mutant strain BMB171 genome was sequenced by using a massive parallel pyrosequencing technology (454 GS-FLX). A total of 448,963 high-quality reads with an average read length of 391 bp were produced, providing about 32-fold coverage of the genome. Assembly was performed using the Newbler software of the 454 suite package (454 Life Sciences), which resulted in 193 large (defined as >500 bp) contigs. The relationship of contigs was determined by multiplex PCR, and gaps were filled through sequencing of PCR products by primer walking or shotgun sequencing with an ABI 3730 sequencer. The Phred/Phrap/Consed software package (3) was used for final sequence assembly and quality assessment. Protein-coding genes were predicted by combining the results of Glimmer 3.02 (1) and ZCURVE (4), followed by manual inspection. Both tRNA and rRNA genes were identified by tRNAscan-SE (11) and RNAmmer (6), respectively. Functional annotation was performed by searching against a protein database of the microbial genome developed in house.The 5.64-Mb genome of B. thuringiensis mutant strain BMB171 contains two replicons: a circular chromosome (5.33 Mb) encoding 5,088 open reading frames (ORFs) and a circular plasmid (0.31 Mb), which is named pBMB171, encoding 276 predicted ORFs. The G+C content of the chromosome is 35.3%, while that of the plasmid is 33.3%. The mutant strain BMB171 genome encodes 104 tRNAs and 14 rRNA operons. A previous study indicated that BMB171 is a plasmid-free mutant (9); however, our sequencing results demonstrated that a large plasmid still remains. The reason why the plasmid was not detected previously might be its large size and low copy number. We did not find any crystal protein genes in either chromosome or plasmid sequences, which was consistent with previous observations (9).In summary, the complete B. thuringiensis mutant strain BMB171 genome provides a better-defined genetic background for gene expression and regulation studies, especially crystal protein production and metabolic network construction.     

Optical Mapping Reveals a Large Genetic Inversion between Two Methicillin-Resistant Staphylococcus aureus Strains

Staphylococcus aureus is a highly versatile and evolving bacterium of great clinical importance. S. aureus can evolve by acquiring single nucleotide polymorphisms and mobile genetic elements and by recombination events. Identification and location of novel genomic elements in a bacterial genome are not straightforward, unless the whole genome is sequenced. Optical mapping is a new tool that creates a high-resolution, in situ ordered restriction map of a bacterial genome. These maps can be used to determine genomic organization and perform comparative genomics to identify genomic rearrangements, such as insertions, deletions, duplications, and inversions, compared to an in silico (virtual) restriction map of a known genome sequence. Using this technology, we report here the identification, approximate location, and characterization of a genetic inversion of ∼500 kb of a DNA element between the NRS387 (USA800) and FPR3757 (USA300) strains. The presence of the inversion and location of its junction sites were confirmed by site-specific PCR and sequencing. At both the left and right junction sites in NRS387, an IS1181 element and a 73-bp sequence were identified as inverted repeats, which could explain the possible mechanism of the inversion event.Staphylococcus aureus is a gram-positive bacterium of immense clinical importance. This opportunistic pathogen is capable of causing a wide range of diseases from skin and soft-tissue infections to life-threatening bacteremia, endocarditis, and osteomyelitis (14). Approximately 75% of the S. aureus genome is composed of a core genome that is common in all strains, and 25% of the genome is composed of variable regions which can differ between different strains (4, 16, 24-26). S. aureus evolves primarily by introducing single nucleotide polymorphisms in its core genome and by acquiring mobile genetic elements (MGEs) through horizontal gene transfer. These MGEs include pathogenicity/genomic islands, plasmids, transposons, and bacteriophages that become integrated in the chromosome (4, 11, 16, 31, 32). Despite being a heterogeneous organism, genetic recombination in S. aureus is proposed to be rather rare (20, 24, 29, 35). Its clones are more likely to evolve by point mutations than by recombination events (12). The MGEs contribute to the phenotypic and genotypic diversity seen with the S. aureus population. Acquisition of the staphylococcal cassette chromosome (SCCmec) elements through site-specific recombinases has led to the epidemic of methicillin-resistant S. aureus (MRSA) strains in hospitals and communities all over the world (6, 10, 15). In recent years, the integration of arginine catabolite mobile element in the USA300 lineage of MRSA has been proposed to give the pathogen its epidemiological advantage, including traits for surviving in low-pH conditions and oxygen tension environments (11). In addition, chromosomal replacements have been observed within lineages of sequence type 34 (ST34) and ST42 (34) and ST8 and ST30 (13).Genomic rearrangements, such as inversions, have been observed with genomes of enteric bacteria, such as Salmonella enterica, Shigella flexneri, Yersinia pestis KIM, Escherichia coli (K12 and O157:H7), and group A Streptococcus pyogenes (8, 9, 18, 27, 28, 30, 37). No genomic inversions in S. aureus have been reported to date. With the use of optical mapping, large genomic rearrangements, such as inversions, that would otherwise be missed with other comparative genotyping approaches, including microarray analysis, can be identified. Optical mapping uses high-resolution restriction maps (optical maps) of a bacterial genome to determine its genomic organization (5, 21, 23, 33, 36). These optical maps can be compared to an in silico (virtual) restriction map of a known genome sequence and can be used to identify gene rearrangements and their locations. Using optical mapping in conjunction with subsequent site-specific PCR and sequencing, we report the identification, approximate location, and partial characterization of an ∼500-kb DNA element in NRS387, a USA800 strain that was found to be inverted relative to USA300FPR3757. Identification of IS1181 elements and a novel 73-bp element at both ends of the ∼500-kb element in NRS387 could suggest the possibility of an inversion event in an ancestral strain of NRS387.     

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.     

Spatiotemporal Homogeneity of Campylobacter Subtypes from Cattle and Sheep across Northeastern and Southwestern Scotland

Source attribution using molecular subtypes has implicated cattle and sheep as sources of human Campylobacter infection. Whether the Campylobacter subtypes associated with cattle and sheep vary spatiotemporally remains poorly known, especially at national levels. Here we describe spatiotemporal patterns of prevalence, bacterial enumeration, and subtype composition in Campylobacter isolates from cattle and sheep feces from northeastern (63 farms, 414 samples) and southwestern (71 farms, 449 samples) Scotland during 2005 to 2006. Isolates (201) were categorized as sequence type (ST), as clonal complex (CC), and as Campylobacter jejuni or Campylobacter coli using multilocus sequence typing (MLST). No significant difference in average prevalence (cattle, 22%; sheep, 25%) or average enumeration (cattle, 2.7 × 10⁴ CFU/g; sheep, 2.0 × 10⁵ CFU/g) was found between hosts or regions. The four most common STs (C. jejuni ST-19, ST-42, and ST-61 and C. coli ST-827) occurred in both hosts, whereas STs of the C. coli ST-828 clonal complex were more common in sheep. Neither host yielded evidence for regional differences in ST, CC, or MLST allele composition. Isolates from the two hosts combined, categorized as ST or CC, were more similar within than between farms but showed no further spatiotemporal trends up to 330 km and 50 weeks between farm samples. In contrast, both regions yielded evidence for significant differences in ST, CC, and allele composition between hosts, such that 65% of isolates could be attributed to a known host. These results suggest that cattle and sheep within the spatiotemporal scales analyzed are each capable of contributing homogeneous Campylobacter strains to human infections.Campylobacter species are the largest cause of bacterial intestinal infection in the developed and developing world (3). Almost all reported human Campylobacter infections in the United Kingdom are caused by Campylobacter jejuni, which accounts for approximately 92% of cases, and Campylobacter coli, which accounts for most of the rest (8). Campylobacter species are carried asymptomatically in a wide range of host animals and excreted into the environment in feces (23). Humans can be infected by several routes including consumption of contaminated water (14) or food (23); indeed, case control studies indicate that consumption of poultry meat is a risk factor (11, 12, 28), but other foods including unpasteurized milk (33) and meat from cattle and sheep contaminated at the abattoir might be important (30).Cattle and sheep on farms are major hosts of Campylobacter, with up to 89% of cattle herds (31) and up to 55% of sheep flocks (26) being infected. The prevalence of C. jejuni and C. coli combined, estimated at the level of individual animals from fecal specimens, is 23 to 54% in cattle (22, 25) and up to 20% in sheep (37). Campylobacter enumeration in feces shed from individual animals ranges from <10² to 10⁷ CFU/g in both hosts (31), and the concentration shed varies with time. Meat products of cattle and sheep, by contrast, have generally lower levels of Campylobacter contamination. Prevalence values are 0.5 to 4.9% in surveys of retail beef (11a, 17, 36) and 6.9 to 12.6% in surveys of retail lamb and mutton (17, 35).Clinical Campylobacter strains can be attributed to infection sources in animals by comparing subtype frequencies in clinical cases with those in different candidate sources, including cattle, sheep, pigs, and the physical environment. Campylobacter subtype data sets are most transferable when subtypes are defined as sequence type (ST) using multilocus sequence typing (MLST). Three recent MLST-based studies based in northwestern England (34), mainland Scotland (29), northeastern Scotland (32), and New Zealand (24) have used source attribution models to infer the source of human clinical infection. The results suggest that retail chicken is the source with the highest (55 to 80%) attribution while cattle and sheep combined are ranked second (20 to 40%). These attribution models require further empirical validation but appear to be showing broadly similar results.Attribution of human Campylobacter infections to cattle and sheep raises the question of whether Campylobacter subtypes infecting farm cattle and sheep are generally homogeneous or tend to have spatiotemporal structure. Regarding spatial differences, isolates of C. jejuni from a 100-km² study of farmland area with dairy cattle and sheep in northwestern England displayed increased genetic similarity up to 1 km apart but no further trend over distances of 1 to 14 km apart (7), and isolates from three dairy cattle farms 2 or 5 km apart in the same area demonstrated differences in the frequencies of strains of clonal complexes (CCs) ST-42 and ST-61 (15). Regarding temporal differences, isolates of C. jejuni from five dairy cattle farms in the same area demonstrated differences in the frequency of strains of CC ST-61 between the spring and summer of 2003 (15). Lastly, regarding host-associated strains, STs of CCs ST-21, ST-42, and ST-61 are associated with cattle, and the more limited data for STs from sheep also show the presence of ST-21 and ST-61 (7, 15).The larger-scale spatiotemporal structure of Campylobacter strains from cattle and sheep is poorly known. The main aims of the present study were (i) to characterize C. jejuni and C. coli from cattle and sheep from two distinct geographical Scottish regions in terms of Campylobacter prevalence and enumeration and C. jejuni and C. coli ST composition and (ii) to estimate the extent of host association of C. jejuni and C. coli STs from cattle versus sheep.     

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.