In Situ Phylogenetic Structure and Diversity of Wild Bradyrhizobium Communities

Bacteria often infect their hosts from environmental sources, but little is known about how environmental and host-infecting populations are related. Here, phylogenetic clustering and diversity were investigated in a natural community of rhizobial bacteria from the genus Bradyrhizobium. These bacteria live in the soil and also form beneficial root nodule symbioses with legumes, including those in the genus Lotus. Two hundred eighty pure cultures of Bradyrhizobium bacteria were isolated and genotyped from wild hosts, including Lotus angustissimus, Lotus heermannii, Lotus micranthus, and Lotus strigosus. Bacteria were cultured directly from symbiotic nodules and from two microenvironments on the soil-root interface: root tips and mature (old) root surfaces. Bayesian phylogenies of Bradyrhizobium isolates were reconstructed using the internal transcribed spacer (ITS), and the structure of phylogenetic relatedness among bacteria was examined by host species and microenvironment. Inoculation assays were performed to confirm the nodulation status of a subset of isolates. Most recovered rhizobial genotypes were unique and found only in root surface communities, where little bacterial population genetic structure was detected among hosts. Conversely, most nodule isolates could be classified into several related, hyper-abundant genotypes that were phylogenetically clustered within host species. This pattern suggests that host infection provides ample rewards to symbiotic bacteria but that host specificity can strongly structure only a small subset of the rhizobial community.Symbiotic bacteria often encounter hosts from environmental sources (32, 48, 60), which leads to multipartite life histories including host-inhabiting and environmental stages. Research on host-associated bacteria, including pathogens and beneficial symbionts, has focused primarily on infection and proliferation in hosts, and key questions about the ecology and evolution of the free-living stages have remained unanswered. For instance, is host association ubiquitous within a bacterial lineage, or if not, do host-infecting genotypes represent a phylogenetically nonrandom subset? Assuming that host infection and free-living existence exert different selective pressures, do bacterial lineages diverge into specialists for these different lifestyles? Another set of questions addresses the degree to which bacteria associate with specific host partners. Do bacterial genotypes invariably associate with specific host lineages, and is such specificity controlled by one or both partners? Alternatively, is specificity simply a by-product of ecological cooccurrence among bacteria and hosts?Rhizobial bacteria comprise several distantly related proteobacterial lineages, most notably the genera Azorhizobium, Bradyrhizobium, Mesorhizobium, Rhizobium, and Sinorhizobium (52), that have acquired the ability to form nodules on legumes and symbiotically fix nitrogen. Acquisition of nodulation and nitrogen fixation loci has likely occurred through repeated lateral transfer of symbiotic loci (13, 74). Thus, the term “rhizobia” identifies a suite of symbiotic traits in multiple genomic backgrounds rather than a taxonomic classification. When rhizobia infect legume hosts, they differentiate into specialized endosymbiotic cells called bacteroids, which reduce atmospheric nitrogen in exchange for photosynthates from the plant (35, 60). Rhizobial transmission among legume hosts is infectious. Rhizobia can spread among hosts through the soil (60), and maternal inheritance (through seeds) is unknown (11, 43, 55). Nodule formation on hosts is guided by reciprocal molecular signaling between bacteria and plant (5, 46, 58), and successful infection requires a compatible pairing of legume and rhizobial genotypes. While both host and symbiont genotypes can alter the outcome of rhizobial competition for adsorption (34) and nodulation (33, 39, 65) of legume roots, little is known about how this competition plays out in nature.Rhizobia can achieve reproductive success via multiple lifestyles (12), including living free in the soil (14, 44, 53, 62), on or near root surfaces (12, 18, 19, 51), or in legume nodules (60). Least is known about rhizobia in bulk soil (not penetrated by plant roots). While rhizobia can persist for years in soil without host legumes (12, 30, 61), it appears that growth is often negligible in bulk soil (4, 10, 14, 22, 25). Rhizobia can also proliferate in the rhizosphere (soil near the root zone) of legumes (4, 10, 18, 19, 22, 25, 51). Some rhizobia might specialize in rhizosphere growth and infect hosts only rarely (12, 14, 51), whereas other genotypes are clearly nonsymbiotic because they lack key genes (62) and must therefore persist in the soil. The best-understood rhizobial lifestyle is the root nodule symbiosis with legumes, which is thought to offer fitness rewards that are superior to life in the soil (12). After the initial infection, nodules grow and harbor increasing populations of bacteria until the nodules senesce and the rhizobia are released into the soil (11, 12, 38, 40, 55). However, rhizobial fitness in nodules is not guaranteed. Host species differ in the type of nodules they form, and this can determine the degree to which differentiated bacteroids can repopulate the soil (11, 12, 38, 59). Furthermore, some legumes can hinder the growth of nodules with ineffective rhizobia, thus punishing uncooperative symbionts (11, 27, 28, 56, 71).Here, we investigated the relationships between environmental and host-infecting populations of rhizobia. A main objective was to test the hypothesis that rhizobia exhibit specificity among host species as well as among host microenvironments, specifically symbiotic nodules, root surfaces, and root tips. We predicted that host infection and environmental existence exert different selective pressures on rhizobia, leading to divergent patterns of clustering, diversity, and abundance of rhizobial genotypes.     

Comparison and Utilization of Repetitive-Element PCR Techniques for Typing Lactobacillus Isolates from the Chicken Gastrointestinal Tract

Three repetitive-element PCR techniques were evaluated for the ability to type strains of Lactobacillus species commonly identified in the chicken gastrointestinal tract. Enterobacterial repetitive intergenic consensus PCR (ERIC-PCR) produced species- and strain-specific profiles for Lactobacillus crispatus, Lactobacillus gallinarum, Lactobacillus johnsonii, and Lactobacillus reuteri isolates. The technique typed strains within these species equally as well as pulsed-field gel electrophoresis. DNA concentration and quality did not affect the ERIC-PCR profiles, indicating that this method, unlike other high-resolution methods, can be adapted to high-throughput analysis of isolates. Subsequently, ERIC-PCR was used to type Lactobacillus species diversity of a large collection of isolates derived from chickens grown under commercial and necrotic enteritis disease induction conditions. This study has illustrated, for the first time, that there is great strain diversity within each Lactobacillus species present and has revealed that chickens raised under commercial conditions harbor greater species and strain diversity than chickens raised under necrotic enteritis disease induction conditions.Lactobacilli are normal inhabitants within the microflora of the chicken gastrointestinal tract (GIT) (27, 39). Species frequently identified within the chicken GIT include Lactobacillus crispatus, Lactobacillus gallinarum, Lactobacillus johnsonii, and Lactobacillus reuteri (1, 7, 27). The first three of these species are members of the Lactobacillus acidophilus complex (LAC) (22, 32), a closely related group of species which are difficult to differentiate using traditional techniques, such as physiological and biochemical tests (34). While molecular methods, such as DNA-DNA hybridization (22), ribotyping (71), sodium dodecyl sulfate-polyacrylamide gel electrophoresis (19), randomly amplified polymorphic DNA PCR (19), and 16S rRNA gene sequencing (41), have been used with some success, many of these techniques are not readily adaptable to high-throughput applications required for large-scale ecological studies.While numerous studies have reported Lactobacillus species distribution within the chicken GIT, the strain diversity within species has not been explored. Previously, Hagen et al. (28) investigated L. gallinarum isolates present within the crops of commercial chickens, revealing a high level of strain diversity among the isolates examined (17 strains represented among 38 isolates). These results indicate that there could be great diversity within and among the lactobacilli present within the chicken GIT. Examining and typing large numbers of lactobacilli from the chicken GIT to the strain level may facilitate a better understanding of microflora dynamics and niche competition. These studies may also result in the identification of strains which could be used in competitive exclusion applications and potentially in the development of probiotics or live vectors for the delivery of therapeutic recombinant proteins to specific sites within the chicken GIT.Lactobacilli have been proposed as possible competitive exclusion agents or probiotics (30, 36, 42) against Clostridium perfringens, the causative agent of necrotic enteritis (NE) in broiler chickens. The withdrawal of antimicrobial growth promoters in Europe has led to an increase in the incidence of NE (10, 64), prompting the investigation of alternative methods for controlling C. perfringens in the broiler chicken GIT. Various models have been developed to study NE under research conditions, as recently reviewed by Dahiya et al. (15). The conditions used in these NE models and the effects they have on GIT microflora may adversely impact the identification of antimicrobial alternatives, such as probiotic strains, for use within commercial broiler chickens. Application of a high-throughput typing method capable of distinguishing species and strains is needed to determine if differences exist between the Lactobacillus populations of chickens raised under NE and commercial conditions.While pulsed-field gel electrophoresis (PFGE) has been used widely for genotyping Lactobacillus strains (49, 50, 65), it is time-consuming, labor-intensive, expensive, and suitable only for low-throughput analysis of isolates (54, 65). In contrast, repetitive-element PCR (Rep-PCR) has been developed for genotypically fingerprinting bacteria and is a fast and reliable high-throughput genotyping system (66, 67). Rep-PCR has been used successfully to identify strains of a variety of genera (33, 44, 48, 55). Several Rep-PCR primers, including the repetitive extragenic palindromic (REP) primers (6, 9, 16, 65), the enterobacterial repetitive intergenic consensus (ERIC) primers (6, 65), and the (GTG)₅ primer (24, 40, 62), have been used in the typing of lactobacilli in studies which have generally focused on species important to the dairy industry and food fermentations. Very few studies have examined the application of Rep-PCR to species commonly identified in the chicken GIT (62, 65). To our knowledge, only a single study has actually applied Rep-PCR to type Lactobacillus isolates from chickens (62) for the identification of potential probiotics to control Salmonella enterica serovar Enteritidis in egg-laying hens.The aim of this study was to investigate whether Rep-PCR could be used to analyze Lactobacillus species and strains in the chicken GIT. Several Rep-PCR techniques [REP-, ERIC-, and (GTG)₅-PCR] were compared for the ability to type strains of several Lactobacillus species commonly isolated from the chicken GIT (L. crispatus, L. gallinarum, L. johnsonii, and L. reuteri). ERIC-PCR was able to simultaneously type isolates to the species and strain levels, and its strain differentiation ability was comparable with that of PFGE. ERIC-PCR was further applied to high-throughput analysis of a large number of isolates collected from chickens raised under NE and commercial conditions.     

Development of a Markerless Genetic Exchange System for Desulfovibrio vulgaris Hildenborough and Its Use in Generating a Strain with Increased Transformation Efficiency

In recent years, the genetic manipulation of the sulfate-reducing bacterium Desulfovibrio vulgaris Hildenborough has seen enormous progress. In spite of this progress, the current marker exchange deletion method does not allow for easy selection of multiple sequential gene deletions in a single strain because of the limited number of selectable markers available in D. vulgaris. To broaden the repertoire of genetic tools for manipulation, an in-frame, markerless deletion system has been developed. The counterselectable marker that makes this deletion system possible is the pyrimidine salvage enzyme, uracil phosphoribosyltransferase, encoded by upp. In wild-type D. vulgaris, growth was shown to be inhibited by the toxic pyrimidine analog 5-fluorouracil (5-FU), whereas a mutant bearing a deletion of the upp gene was resistant to 5-FU. When a plasmid containing the wild-type upp gene expressed constitutively from the aph(3′)-II promoter (promoter for the kanamycin resistance gene in Tn5) was introduced into the upp deletion strain, sensitivity to 5-FU was restored. This observation allowed us to develop a two-step integration and excision strategy for the deletion of genes of interest. Since this in-frame deletion strategy does not retain an antibiotic cassette, multiple deletions can be generated in a single strain without the accumulation of genes conferring antibiotic resistances. We used this strategy to generate a deletion strain lacking the endonuclease (hsdR, DVU1703) of a type I restriction-modification system that we designated JW7035. The transformation efficiency of the JW7035 strain was found to be 100 to 1,000 times greater than that of the wild-type strain when stable plasmids were introduced via electroporation.The anaerobic sulfate-reducing bacteria (SRB) are found in a remarkable variety of habitats. These bacteria have received attention recently because they have a potential role in toxic metal bioremediation (23, 26). To fully understand the potential benefits and to maximize opportunities for successful manipulation of the SRB, it would be useful to create deletions in critically important genes. Several activities of particular interest are represented by multiple isozymes, suggesting that compensation may occur upon elimination of one or more of these genes. To fully elucidate alternative pathways, genetic approaches allowing the construction of multiple mutations are needed. The genetic manipulation of the SRB Desulfovibrio vulgaris Hildenborough has seen significant improvements in recent years (reviewed in reference 3). Chloramphenicol and kanamycin marker exchange mutagenesis methods have been developed (2, 10). Although gene deletions can be constructed, the necessary retention of antibiotic resistance limits sequential deletions, since each deletion would require an additional antibiotic cassette. To eliminate the necessity of marker retention, an in-frame markerless deletion system has been developed.A two-step method for marker exchange/deletion that used the counterselectable marker sacB (13) was used by Fu and Voordouw (10) to generate the first deletion by marker exchange in D. vulgaris. The sacB gene from Bacillus subtilis encodes levansucrase and confers sensitivity to sucrose in many gram-negative bacteria (7, 32, 35), including D. vulgaris (8, 10, 15, 18, 19, 21). In the first step of the process, a suicide plasmid carrying DNA regions from up- and downstream of a target gene flanking a chloramphenicol resistance (Cm^r) cassette was introduced into D. vulgaris by conjugation and single recombinants were selected as Cm^r colonies (10). After confirmation of the integration of this plasmid, the double-recombination event was selected on medium containing chloramphenicol and sucrose. This method, with some variation, has been used to make several mutants by the Voordouw group (8, 10, 15, 18, 19, 21). One unexpected complication was the observation that 50% of sucrose resistant colonies were due to events other than the removal of the sacB gene and plasmid through a second recombination as desired (11). Also, sensitivity to sucrose is apparently strongly affected by medium composition, initial culture density, and the time of exposure (11). This method involved a large time investment, but it ultimately resulted in a marker exchange mutant (Cm^r) and established the effectiveness of a two-step recombination process in D. vulgaris.Among alternative counterselectable markers are the purine and pyrimidine salvage enzymes, phosphoribosyl transferases (PRTases). These enzymes allow the recycling of free bases from internal or environmental sources, as well as the incorporation of base analogs into nucleoside monophosphates. Importantly, the incorporation of base analogs can be lethal and are the reason these nucleotide salvage pathways have been widely used as counterselectable markers for gene knockout systems in bacteria, archaea, and eukaryotes (4, 5, 8, 9, 12, 16, 22, 24, 27, 29, 34). Specifically, the incorporation of the pyrimidine analog 5-fluorouracil (5-FU) is lethal in a number of bacteria (8, 16, 22). Mutants whose genes encoding the pertinent PRTases have been deleted are resistant to the toxic base analogs (4, 5, 8, 9, 12, 16, 22, 24, 27, 29, 34). Reintroduction of these genes restores sensitivity. In order to utilize the genes for PRTases as counterselectable markers, a deletion of the endogenous PRTase gene must be created in the host strain. We have previously shown that wild-type D. vulgaris is extremely sensitive to low levels of 5-FU, as little as 0.1 μg/ml (3). In the present study, we deleted the upp gene (DVU1025) encoding the putative uracil phosphoribosyl transferase in D. vulgaris creating strain JW710 and showed that it was resistant to 5-FU. When the upp gene was reintroduced into JW710 (Δupp), it restored sensitivity to wild-type levels of 5-FU. These phenotypic observations indicate that the loss of the upp provides a selectable marker for a two-step integration and excision strategy for the deletion of target genes without a residual marker exchange. A second advantage of using this markerless method is the facile ability to generate in-frame deletions, eliminating potential polarity.To test the effectiveness of using the upp as a counterselectable marker in D. vulgaris, we deleted the gene encoding the endonuclease of a type I restriction-modification system, hsdR (DVU1703), and the downstream conserved hypothetical gene (CHP; DVU1702), creating strain JW7035 [Δupp Δ(hsdR-CHP)]. The type I restriction-modification system was targeted for deletion in hopes of increasing the transformation efficiency of D. vulgaris and facilitating the construction of future deletions. As anticipated, electroporation experiments with stable plasmids revealed an improvement in transformation efficiency for stable plasmids compared to wild-type D. vulgaris. Finally, Gateway Technology (Invitrogen) was applied to generate a destination vector (pMO727) containing the constitutively expressed wild-type upp gene. This vector will expedite the process of creating the required suicide deletion vectors for future markerless deletions.     

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

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

Genetic Characterization of Vibrio vulnificus Strains from Tilapia Aquaculture in Bangladesh

Outbreaks of Vibrio vulnificus wound infections in Israel were previously attributed to tilapia aquaculture. In this study, V. vulnificus was frequently isolated from coastal but not freshwater aquaculture in Bangladesh. Phylogenetic analyses showed that strains from Bangladesh differed remarkably from isolates commonly recovered elsewhere from fish or oysters and were more closely related to strains of clinical origin.Vibrio vulnificus causes severe wound infections and life-threatening septicemia (mortality, >50%), primarily in patients with underlying chronic diseases (10, 19, 23) and primarily from raw oyster consumption (21). This Gram-negative halophile is readily recovered from oysters (27, 35, 43) and fish (14) and was initially classified into two biotypes (BTs) based on growth characteristics and serology (5, 18, 39). Most human isolates are BT1, while BT2 is usually associated with diseased eels (1, 39). An outbreak of wound infections from aquacultured tilapia in Israel (6) revealed a new biotype (BT3). Phenotypic assays do not consistently distinguish biotypes (33), but genetic analyses have helped resolve relationships (20). A 10-locus multilocus sequence typing (MLST) scheme (8, 9) and a similar analysis of 6 loci (13) segregated V. vulnificus strains into two clusters. BT1 strains were in both clusters, while BT2 segregated into a single cluster and BT3 was a genetic mosaic of the two lineages. Significant associations were observed between MLST clusters and strain origin: most clinical strains (BT1) were in one cluster, and the other cluster was comprised mostly of environmental strains (some BT1 and all BT2). Clinical isolates were also associated with a unique genomic island (13).The relationship between genetic lineages and virulence has not been determined, and confirmed virulence genes are universally present in V. vulnificus strains from both clinical and environmental origins (19, 23). However, segregation of several polymorphic alleles agreed with the MLST analysis and correlated genotype with either clinical or environmental strain origin. Alleles include 16S rRNA loci (15, 26, 42), a virulence-correlated gene (vcg) locus (31, 41, 42), and repetitive sequence in the CPS operon (12). DiversiLab repetitive extrageneic palindromic (rep-PCR) analysis also confirmed these genetic distinctions and showed greater diversity among clinical strains (12).Wound infections associated with tilapia in Israel implicated aquaculture as a potential source of V. vulnificus in human disease (6, 40). Tilapia aquaculture is increasing rapidly, as shown by a 2.8-fold increase in tons produced from 1998 to 2007 (Food and Agriculture Organization; http://www.fao.org/fishery/statistics/en). Therefore, presence of V. vulnificus in tilapia aquaculture was examined in Bangladesh, a region that supports both coastal and freshwater sources of industrial-scale aquaculture. V. vulnificus strains were recovered from market fish, netted fish, and water samples, and the phylogenetic relationship among strains was examined relative to clinical and environmental reference strains collected elsewhere.     

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).     

Multiple-Locus Variable-Number Tandem-Repeat Analysis for Clonal Identification of Vibrio parahaemolyticus Isolates by Using Capillary Electrophoresis

Epidemics of Vibrio parahaemolyticus in Chile have occurred since 1998. Direct genome restriction enzyme analysis (DGREA) using conventional gel electrophoresis permitted discrimination of different V. parahaemolyticus isolates obtained from these outbreaks and showed that this species consists of a highly diverse population. A multiple-locus variable-number tandem-repeat (VNTR) analysis (MLVA) approach was developed and applied to 22 clinical and 91 environmental V. parahaemolyticus isolates from Chile to understand their clonal structures. To this end, an advanced molecular technique was developed by applying multiplex PCR, fluorescent primers, and capillary electrophoresis, resulting in a high-resolution and high-throughput (HRHT) genotyping method. The genomic basis of this HRHT method was eight VNTR loci described previously by Kimura et al. (J. Microbiol. Methods 72:313-320, 2008) and two new loci which were identified by a detailed molecular study of 24 potential VNTR loci on both chromosomes. The isolates of V. parahaemolyticus belonging to the same DGREA pattern were distinguishable by the size variations in the indicative 10 VNTRs. This assay showed that these 10 VNTR loci were useful for distinguishing isolates of V. parahaemolyticus that had different DGREA patterns and also isolates that belong to the same group. Isolates that differed in their DGREA patterns showed polymorphism in their VNTR profiles. A total of 81 isolates was associated with 59 MLVA groups, providing fine-scale differentiation, even among very closely related isolates. The developed approach enables rapid and high-resolution analysis of V. parahaemolyticus with pandemic potential and provides a new surveillance tool for food-borne pathogens.Food-borne infections by Vibrio parahaemolyticus cause gastroenteritis, which is the most common clinical manifestation (38). An increasing number of V. parahaemolyticus infections and outbreaks caused by strains belonging to a pandemic clonal complex have been observed throughout the world since 1996 (2, 6, 9, 12, 13, 31, 32, 36, 40). Epidemics of Vibrio parahaemolyticus in Chile have occurred since the summer of 1998 and were caused by the pandemic clone O3:K6 that had emerged in Southeast Asia in 1996 (12, 13, 15). However, this strain was only a minor component of a highly diverse V. parahaemolyticus population in shellfish, as demonstrated by an improved method for restriction enzyme analysis, using total bacterial DNA, named direct genome restriction enzyme analysis (DGREA), in combination with conventional gel electrophoresis (12). This method has a discrimination index similar to that of restriction fragment length polymorphism-pulsed-field gel electrophoresis (PFGE) (12, 13, 19).A variety of molecular typing methods have been applied to V. parahaemolyticus, such as ribotyping (3, 10, 14), PFGE (3, 30), group-specific PCR (32), arbitrarily primed PCR (18, 32, 36), and multilocus sequence typing (7, 16). The use of DGREA permitted discrimination of different V. parahaemolyticus Chilean isolates and showed that these bacteria consist of a highly diverse population comprising at least 23 different genotypic groups among the environmental isolates obtained from shellfish and 5 different groups of clinical isolates (19).Epidemiological analyses of infections caused by pathogenic bacteria depend on the accurate identification of strains, preferably at the clonal level. Variable-number tandem repeats (VNTRs) comprising short sequence repeats constitute a rich source of genetic polymorphism and have been used extensively as markers for discrimination between strains of many different bacterial genera (27, 46). VNTRs have been used to discriminate among individual strains within several food- or waterborne pathogens with little genetic variation, including Escherichia coli O157:H7 (25, 35), Pseudomonas aeruginosa (37), Staphylococcus aureus (41), and Salmonella enterica subsp. enterica serovar Typhimurium (26), and to characterize other important human pathogens, such as Neisseria meningitidis (42), Listeria monocytogenes (28), Legionella pneumophila (34, 39), Leptospira interrogans (43), and Mycobacterium tuberculosis (45). VNTR loci have even been found in genetically highly homogenous pathogens, such as Bacillus anthracis (1, 21, 29). Multiple-locus VNTR analysis (MLVA) is defined as the analysis of a set of loci spread throughout the bacterial genome (23). Individual strains within a bacterial species often maintain the same sequence elements but with different copy numbers due to variations introduced by slipped-strand mispairing during DNA replication (33).Recently, a study of the polymorphism of tandem repeats in V. parahaemolyticus showed the utility of the MLVA approach for characterizing recently emerged and highly homogeneous pandemic strains of serotype O3:K6 (22). These authors reported a scheme of eight genomic VNTR loci, comparing PFGE results for clinical strains of V. parahaemolyticus serotype O3:K6. The study by Kimura et al. (22) comprised only strains of serogroup O3:K6 and used conventional gel electrophoresis to evaluate VNTRs. In epidemiological studies, a more rapid technique is needed for mass application of MLVA that also provides improved resolution and has been validated for nonserogroup O3:K6 isolates. Capillary electrophoresis has become the preferred technology to improve resolution and accuracy in bacterial VNTR analysis due to the availability of multiple fluorescent labels and better accuracy and reproducibility (27).In our study we describe the use of an improved MLVA for discriminating genotypically a diverse collection of clinical and environmental V. parahaemolyticus isolates from Chile. These very closely related isolates have been analyzed and grouped by DGREA previously (12). To this end, we developed and applied multiplex PCR of 10 VNTR loci, tagged with multiple fluorescent dyes, and analyzed the amplicons by capillary electrophoresis. The results demonstrated that MLVA typing is able to distinguish between V. parahaemolyticus isolates that have different DGREA patterns and isolates that belong to the same group, allowing accurate sizing of amplicons by assignment of the fragment size. Validation of this typing method with 113 Chilean isolates demonstrated the utility of this technique also for nonserogroup O3:K6 clinical isolates, thereby providing a new tool for the study of the molecular epidemiology of V. parahaemolyticus.     

High Heterogeneity within Methicillin-Resistant Staphylococcus aureus ST398 Isolates,Defined by Cfr9I Macrorestriction-Pulsed-Field Gel Electrophoresis Profiles and spa and SCCmec Types

During recent years, the animal-associated methicillin-resistant Staphylococcus aureus clone ST398 has extensively been studied. The DNA of these isolates turned out to be refractory to SmaI restriction, and consequently, SmaI is unsuitable for subtyping this clone by standard pulsed-field gel electrophoresis (PFGE). Very recently, ST398 DNA was shown to be digested by Cfr9I, a neoschizomer of SmaI. In the present study, we employed Cfr9I PFGE on 100 German and 5 Dutch ST398 isolates and compared their PFGE profiles, protein A gene variable repeat regions (spa types), and types of the staphylococcal cassette chromosome mec (SCCmec). The isolates (from healthy carrier pigs, clinical samples from pigs, dust from farms, milk, and meat) were assigned to 35 profiles, which were correlated to the SCCmec type. A dendrogram with the Cfr9I patterns assigned all profiles to two clusters. Cluster A grouped nearly all isolates with SCCmec type V, and cluster B comprised all SCCmec type IVa and V* (a type V variant first identified as III) carriers plus one isolate with SCCmec type V. Both clusters also grouped methicillin-susceptible S. aureus isolates. The association of the majority of isolates with SCCmec type V in one large cluster indicated the presence of a successful subclone within the clonal complex CC398 from pigs, which has diversified. In general, the combination of Cfr9I PFGE with spa and SCCmec typing demonstrated the heterogeneity of the series analyzed and can be further used for outbreak investigations and traceability studies of the MRSA ST398 emerging clone.Methicillin-resistant Staphylococcus aureus (MRSA) strains are an important cause of hospital-acquired infections worldwide (8). However, MRSA strains are not confined to health care settings, and during the last 10 years community-acquired MRSA has increasingly been reported (8). In 2003, a clone of MRSA associated with pig farming and not related to the traditional hospital- and community-acquired MRSA emerged in the Netherlands (37), where it now amounts to >30% of human MRSA cases (16). This clone has also been detected in healthy and sick animals, in food of animal origin, and in humans from other European countries, Canada, the United States, the Dominican Republic, and China (5, 7, 31, 38, 39). This emerging MRSA clone belongs to the multilocus sequence type ST398, which includes different spa types (mainly t011, t034, and t108). The majority of the ST398 isolates reported are MRSA, although methicillin-susceptible (MSSA) strains have been described as well (15, 34). Resistance to methicillin and other β-lactam antibiotics is caused by the mecA gene, which is located on a mobile genetic element, the staphylococcal cassette chromosome mec (SCCmec). The SCCmec cassette consists of the mec gene complex, the ccr gene complex, and the junkyard regions. Based on the variability and combinations of these genetic elements, several types of SCCmec and several variants of the types have been described (9). Three SCCmec types (III, IVa, and V) were identified in ST398 isolates (25). However, recent investigations have shown that some ST398 isolates typed as SCCmec type III using the method of Zhang et al. (40) proved to be type V after further sequencing (21, 35).For typing S. aureus, pulsed-field gel electrophoresis (PFGE) of the whole genome by macrorestriction with the SmaI endonuclease is still considered as the “gold standard” (26). However, the isolates of the ST398 clone are nontypeable (NT) by PFGE using SmaI (3, 4). Consequently, comparison between these isolates and the typeable ones from humans and animals is not possible. The nontypeability is due to the action of a novel C5-cytosine methyltransferase which modifies the consensus sequence C^mCNGG at the second cytosine (3, 4). Other enzymes with a different recognition sequence from SmaI have been used for PFGE typing of the ST398 clone, including EagI and ApaI (22, 28, 31, 38), but the patterns obtained cannot be compared to S. aureus patterns generated with SmaI. XmaI, a neoschizomer of SmaI that recognizes the same sequence cutting at a different position, only generates partial digestions (3, 4). Recently, the use of Cfr9I, another neoschizomer of SmaI whose activity is not reduced on ST398 methylated DNA, has been recommended. This enzyme had been successfully used for typing SmaI NT macrolide-resistant Streptococcus pyogenes isolates (6, 30), and now it is being applied for typing ST398 isolates, i.e., from human origin (5, 11, 36) and, to a lesser extent, from animals (3, 36). The aim of this study was to characterize a large collection of recent ST398 isolates by Cfr9I PFGE as well as other methods (spa typing, multilocus sequence typing [MLST], and SCCmec typing). Most of them were recovered in Germany from different sources, including animals and foods.     

Polymorphic Mutation Frequencies of Clinical and Environmental Stenotrophomonas maltophilia Populations

Mutation frequencies were studied in 174 Stenotrophomonas maltophilia isolates from clinical and nonclinical environments by detecting spontaneous rifampin-resistant mutants in otherwise-susceptible populations. The distribution of mutation frequencies followed a pattern similar to that found for other bacterial species, with a modal value of 1 × 10⁻⁸. Nevertheless, the proportion of isolates showing mutation frequencies below the modal value (hypomutators) was significantly higher for S. maltophilia than those so far reported in other organisms. Low mutation frequencies were particularly frequent among environmental S. maltophilia strains (58.3%), whereas strong mutators were found only among isolates with a clinical origin. These results indicate that clinical environments might select bacterial populations with high mutation frequencies, likely by second-order selection processes. In several of the strong-mutator isolates, functional-complementation assays with a wild-type allele of the mutS gene demonstrated that the mutator phenotype was due to the impairment of MutS activity. In silico analysis of the amino acid changes present in the MutS proteins of these hypermutator strains in comparison with the normomutator isolates suggests that the cause of the defect in MutS might be a H683P amino acid change.Stenotrophomonas maltophilia is a Gram-negative, nonfermenting environmental bacterial species often isolated from the rhizosphere and from water sources (11, 12, 63). Some S. maltophilia strains have been used for bioremediation (13, 24, 73) or bioaugmentation (37). However, besides its environmental origin and potential relevance for biotechnological purposes, S. maltophilia is also a relevant human opportunistic pathogen (44) associated with a broad spectrum of clinical syndromes, such as bacteremia (79, 81), endocarditis (18), infection in cancer patients (1), and respiratory tract infections, including those suffered by cystic fibrosis (CF) patients (72, 77). One of the most problematic characteristics of S. maltophilia is its intrinsic high resistance to several antibiotics (4). This intrinsic antibiotic resistance is at least partly due to the presence in the genome of S. maltophilia (17) of genes encoding antibiotic-inactivating enzymes (6, 9, 30, 39, 42, 58) and multidrug resistance (MDR) efflux pumps (2, 3, 43, 78). More recently, a chromosomally encoded Qnr protein that contributes to the intrinsic resistance to quinolones of S. maltophilia has been described (67, 68).A clear difference between infective (clinical) and environmental (nonclinical) S. maltophilia strains has not been reported (12, 63). However, although the available data fit the concept that opportunistic pathogens have not specifically evolved to infect humans (48), this does not mean that they do not evolve during the infective process. For most acute infections, we can presume that the time of in-host evolution is probably too short to detect relevant adaptive changes. Nevertheless, the situation might be different in chronic infections, such as those involving the bronchial compartment in CF patients. In this case, the same bacterial clone can be maintained and grow inside the host for years (62). This produces strong diversification over time and in different compartments of the lung (25, 71, 80), a process in which the acquisition of a mutator phenotype is important (52). Thus, isolates derived from an initial clone but presenting different morphotypes (47), different phenotypes of susceptibility to antibiotics (26) or in the expression of virulence determinants (14, 15, 36), or with different mutation frequencies (49, 60) are recovered from each individual patient suffering chronic infections. More recently, intraclonal diversification has also been described for Pseudomonas aeruginosa causing acute infections in intubated patients (38). Taken together, this indicates that bacteria can evolve during infection.For different bacterial species, strains isolated from CF patients with chronic lung infections show high mutation frequencies (hypermutable strains) (19, 60, 61, 66), whereas hypermutators have rarely been found in isolates from acute infections (33). An explanation for this difference could be that hypermutable strains tend to be selected for in the highly compartmentalized environment of the infected lung by intensive antibiotic therapy, as well as by the stressful conditions of the habitat. This is a second-order selection process (75, 76), in which mutations are selected because they confer an advantage in clinical environments in such a way that mutator strains are selected because they can produce more mutants (both advantageous and deleterious) for selection. In cases of chronic infections that are treated, strong and maintained selective local processes might occur, either by antibiotic treatment or by the actions of the anti-infective systems of the host. Natural out-of-host open environments obviously might have local stresses. However, the intensity of selection is expected to be lower in these habitats, and a constant replacement of potentially lost organisms by migration of neighbor populations probably mitigates the local selection of mutators and favors the enrichment of bacteria presenting low mutation frequencies. In the case of chronic infections, the replacement of mutators by neighbor normomutators is unlikely, because those infections are produced by a single clone that remains for several years in the host (62). Furthermore, although the infection process presents strong evolutionary bottlenecks for bacterial populations, the human host also provides a constant temperature, reliable nutrient supplies, and a habitat largely free from predators and competitors. Thus, while hypermutation might increase the capability of bacteria to adapt to some specific challenges in the clinical environment, the cost of hypermutation in terms of deleterious mutations might also be diminished, and these effects might be mutually reinforcing.The hypothesis explored in this paper is that S. maltophilia is adapted to deal with out-of-host fluctuating environmental variations but that once the organism enters a patient as an opportunistic pathogen, its adaptive needs significantly increase due to the actions of stressful local environmental conditions, such as the immune response and, when present, antibiotics. This enhanced stress under infective conditions might result in the selection of variants with increased mutation frequencies in a second-order selection process (75, 76). To test this hypothesis, the mutation frequencies of S. maltophilia clinical isolates (obtained from CF and non-CF patients) and from the environment (nonclinical origin) were compared. Most works that have been published on the different mutation frequencies in bacterial populations have focused on the detection of strains showing a high mutation frequency (mutators). In our work, we describe for the first time the presence of mutators in clinical isolates of S. maltophilia and demonstrate that hypermutation in several of those isolates is due to defects in MutS.Nevertheless, our main goal has been the analysis of the global distribution of mutation frequencies in an ample number of samples from clinical and nonclinical environments. Our results indicate not only that mutators are more frequent in clinical S. maltophilia isolates, but also that the overall distribution of mutation frequencies is different in S. maltophilia populations with environmental or clinical origins, with a tendency toward mutation frequencies lower than the modal mutation value (hypomutators) in the environmental isolates.     

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

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

Virus Entry via the Alternative Coreceptors CCR3 and FPRL1 Differs by Human Immunodeficiency Virus Type 1 Subtype

Human immunodeficiency virus type 1 (HIV-1) infects target cells by binding to CD4 and a chemokine receptor, most commonly CCR5. CXCR4 is a frequent alternative coreceptor (CoR) in subtype B and D HIV-1 infection, but the importance of many other alternative CoRs remains elusive. We have analyzed HIV-1 envelope (Env) proteins from 66 individuals infected with the major subtypes of HIV-1 to determine if virus entry into highly permissive NP-2 cell lines expressing most known alternative CoRs differed by HIV-1 subtype. We also performed linear regression analysis to determine if virus entry via the major CoR CCR5 correlated with use of any alternative CoR and if this correlation differed by subtype. Virus pseudotyped with subtype B Env showed robust entry via CCR3 that was highly correlated with CCR5 entry efficiency. By contrast, viruses pseudotyped with subtype A and C Env proteins were able to use the recently described alternative CoR FPRL1 more efficiently than CCR3, and use of FPRL1 was correlated with CCR5 entry. Subtype D Env was unable to use either CCR3 or FPRL1 efficiently, a unique pattern of alternative CoR use. These results suggest that each subtype of circulating HIV-1 may be subject to somewhat different selective pressures for Env-mediated entry into target cells and suggest that CCR3 may be used as a surrogate CoR by subtype B while FPRL1 may be used as a surrogate CoR by subtypes A and C. These data may provide insight into development of resistance to CCR5-targeted entry inhibitors and alternative entry pathways for each HIV-1 subtype.Human immunodeficiency virus type 1 (HIV-1) infects target cells by binding first to CD4 and then to a coreceptor (CoR), of which C-C chemokine receptor 5 (CCR5) is the most common (6, 53). CXCR4 is an additional CoR for up to 50% of subtype B and D HIV-1 isolates at very late stages of disease (4, 7, 28, 35). Many other seven-membrane-spanning G-protein-coupled receptors (GPCRs) have been identified as alternative CoRs when expressed on various target cell lines in vitro, including CCR1 (76, 79), CCR2b (24), CCR3 (3, 5, 17, 32, 60), CCR8 (18, 34, 38), GPR1 (27, 65), GPR15/BOB (22), CXCR5 (39), CXCR6/Bonzo/STRL33/TYMSTR (9, 22, 25, 45, 46), APJ (26), CMKLR1/ChemR23 (49, 62), FPLR1 (67, 68), RDC1 (66), and D6 (55). HIV-2 and simian immunodeficiency virus SIVmac isolates more frequently show expanded use of these alternative CoRs than HIV-1 isolates (12, 30, 51, 74), and evidence that alternative CoRs other than CXCR4 mediate infection of primary target cells by HIV-1 isolates is sparse (18, 30, 53, 81). Genetic deficiency in CCR5 expression is highly protective against HIV-1 transmission (21, 36), establishing CCR5 as the primary CoR. The importance of alternative CoRs other than CXCR4 has remained elusive despite many studies (1, 30, 70, 81). Expansion of CoR use from CCR5 to include CXCR4 is frequently associated with the ability to use additional alternative CoRs for viral entry (8, 16, 20, 63, 79) in most but not all studies (29, 33, 40, 77, 78). This finding suggests that the sequence changes in HIV-1 env required for use of CXCR4 as an additional or alternative CoR (14, 15, 31, 37, 41, 57) are likely to increase the potential to use other alternative CoRs.We have used the highly permissive NP-2/CD4 human glioma cell line developed by Soda et al. (69) to classify virus entry via the alternative CoRs CCR1, CCR3, CCR8, GPR1, CXCR6, APJ, CMKLR1/ChemR23, FPRL1, and CXCR4. Full-length molecular clones of 66 env genes from most prevalent HIV-1 subtypes were used to generate infectious virus pseudotypes expressing a luciferase reporter construct (19, 57). Two types of analysis were performed: the level of virus entry mediated by each alternative CoR and linear regression of entry mediated by CCR5 versus all other alternative CoRs. We thus were able to identify patterns of alternative CoR use that were subtype specific and to determine if use of any alternative CoR was correlated or independent of CCR5-mediated entry. The results obtained have implications for the evolution of env function, and the analyses revealed important differences between subtype B Env function and all other HIV-1 subtypes.     

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.     

Diverse Enterotoxin Gene Profiles among Clonal Complexes of Staphylococcus aureus Isolates from the Bronx,New York

Staphylococcal enterotoxins (SE) can cause toxin-mediated disease, and those that function as superantigens are implicated in the pathogenesis of allergic diseases. The prevalence of 19 enterotoxin genes was determined by PCR in clinical S. aureus strains derived from wounds (108) and blood (99). We performed spa typing and multilocus sequence typing (MLST) to determine clonal origin, and for selected strains staphylococcal enterotoxin B (SEB) production was measured by enzyme-linked immunosorbent assay. Strains carried a median of five SE genes. For most SE genes, the prevalence rates among methicillin-resistant and methicillin-sensitive S. aureus isolates, as well as wound- and blood-derived isolates, did not differ. At least one SE gene was detected in all except two S. aureus isolates (>99%). Complete egc clusters were found in only 11% of S. aureus isolates, whereas the combination of sed, sej, and ser was detected in 24% of clinical strains. S. aureus strains exhibited distinct combinations of SE genes, even if their pulsed-field gel electrophoresis and MLST patterns demonstrated clonality. USA300 strains also showed considerable variability in SE content, although they contained a lower number of SE genes (mean, 3). By contrast, SE content was unchanged in five pairs of serial isolates. SEB production by individual strains varied up to 200-fold, and even up to 15-fold in a pair of serial isolates. In conclusion, our results illustrate the genetic diversity of S. aureus strains with respect to enterotoxin genes and suggest that horizontal transfer of mobile genetic elements encoding virulence genes occurs frequently.As a commensal, Staphylococcus aureus colonizes the nasal mucosa of 20 to 40% of humans (54), and as a pathogen it causes pyogenic diseases and toxin-mediated diseases (38). S. aureus produces many different virulence factors, including enterotoxins (SEs), which can cause defined toxic shock syndromes (4). The characterization of some of these toxins led to the discovery of superantigens (41), which bind to major histocompatibility complex class II molecules and Vβ chains of T-cell receptors, resulting in the activation of large numbers of T cells (20 to 30%) and massive cytokine production (10, 18). These superantigen-induced “cytokine storms” are responsible for the toxic effects seen in staphylococcal entertoxin B (SEB)- and toxic shock syndrome toxin (TSST)-associated shock syndromes in S. aureus infections (13, 40, 47). To date, 19 SEs have been identified based on sequence homologies, and studies have reported enterotoxin genes in up to 80% of all S. aureus strains (4, 21). Although many new enterotoxins have been identified, i.e., seg ser and seu (33, 37, 44, 49), their precise functions have not been characterized yet. The majority of experimental work with SEs is still done with SEB, toxic shock syndrome toxin 1, and SEA (27, 31), because these toxins are commercially available. Most SEs are located on mobile elements in bacterial genomes such as plasmids or pathogenicity islands and can thus be easily transferred horizontally between strains (5, 34, 35). Certain SE genes are grouped together. For instance seg, sei, sem, sen, and seo are commonly found in a gene cluster (egc) on genomic island νSAβ (34), and sel and sek are often found together with seb or sec on S. aureus pathogenicity islands. Other staphylococcal superantigen genes are encoded on plasmids (sed, sej, and ser) or are linked to the antibiotic resistance cassette SCCmec (seh) (44, 55). Phage φ3 carries either sea (strain Mu50), sep (N315), or sea sek seq (MW2) (1, 29).Although a few clinical studies have attempted to correlate shock and outcome with the presence of certain SEs in patients with S. aureus infections (17, 28), the contribution of these toxins to outcome is still unclear. Recent papers have proposed the SEs are immunomodulators and that colonization with S. aureus strains that produce SEB may contribute to the pathogenesis of asthma, chronic rhinitis, and dermatitis (2, 36, 46, 48, 56). The superantigen function of SEs in supernatants of S. aureus cultures can be neutralized by serum of colonized patients (21, 23). With new data emerging implicating SEs in the pathogenesis of chronic allergic syndromes, production of monoclonal antibodies and or vaccine strategies targeting SEs may be considered (6, 24, 26, 30) in the future. It is therefore important to characterize the prevalence of SE genes in clinical S. aureus strains.In this study, we analyzed SE content in both methicillin-resistant S. aureus (MRSA) and methicillin-sensitive S. aureus (MSSA) strains that were cultured from wounds (including USA300) and bloodstream infections of patients from a defined geographical area. In addition, SEB production was quantified by enzyme-linked immunosorbent assay (ELISA) in S. aureus strains carrying the seb gene, and spa typing confirmed clonal diversity among S. aureus isolates from different patients, as well as clonal stability in serial isolates, and multilocus sequence typing (MLST) done on a subset of less common spa types. We conclude that SE genes are abundant in S. aureus strains, albeit less abundant in USA300. SE content and combination are highly diverse and therefore more discriminatory than pulsed-field gel electrophoresis (PFGE) and MLST typing, albeit stable in serial isolates. Quantification of SEB production demonstrates that enterotoxin secretion can vary greatly among strains, even if they belong to the same S. aureus lineage. Given the complexities of SE prevalence, regulation, and possible function, we propose that the association of these toxins with chronic allergic diseases or outcome may be oversimplified at present. Precise characterizations of SE function and secretion patterns in individual S. aureus clones are warranted.     

Cytoskeletal Asymmetrical Dumbbell Structure of a Gliding Mycoplasma,Mycoplasma gallisepticum,Revealed by Negative-Staining Electron Microscopy

Several mycoplasma species feature a membrane protrusion at a cell pole, and unknown mechanisms provide gliding motility in the direction of the pole defined by the protrusion. Mycoplasma gallisepticum, an avian pathogen, is known to form a membrane protrusion composed of bleb and infrableb and to glide. Here, we analyzed the gliding motility of M. gallisepticum cells in detail. They glided in the direction of the bleb at an average speed of 0.4 μm/s and remained attached around the bleb to a glass surface, suggesting that the gliding mechanism is similar to that of a related species, Mycoplasma pneumoniae. Next, to elucidate the cytoskeletal structure of M. gallisepticum, we stripped the envelopes by treatment with Triton X-100 under various conditions and observed the remaining structure by negative-staining transmission electron microscopy. A unique cytoskeletal structure, about 300 nm long and 100 nm wide, was found in the bleb and infrableb. The structure, resembling an asymmetrical dumbbell, is composed of five major parts from the distal end: a cap, a small oval, a rod, a large oval, and a bowl. Sonication likely divided the asymmetrical dumbbell into a core and other structures. The cytoskeletal structures of M. gallisepticum were compared with those of M. pneumoniae in detail, and the possible protein components of these structures were considered.Mycoplasmas are commensal and occasionally pathogenic bacteria that lack a peptidoglycan layer (50). Several species feature a membrane protrusion at a pole; for Mycoplasma mobile, this protrusion is called the head, and for Mycoplasma pneumoniae, it is called the attachment organelle (25, 34-37, 52, 54, 58). These species bind to solid surfaces, such as glass and animal cell surfaces, and exhibit gliding motility in the direction of the protrusion (34-37). This motility is believed to be essential for the mycoplasmas'' pathogenicity (4, 22, 27, 36). Recently, the proteins directly involved in the gliding mechanisms of mycoplasmas were identified and were found to have no similarities to those of known motility systems, including bacterial flagellum, pilus, and slime motility systems (25, 34-37).Mycoplasma gallisepticum is an avian pathogen that causes serious damage to the production of eggs for human consumption (50). The cells are pear-shaped and have a membrane protrusion, consisting of the so-called bleb and infrableb (29), and gliding motility (8, 14, 22). Their putative cytoskeletal structures may maintain this characteristic morphology because M. gallisepticum, like other mycoplasma species, does not have a cell wall (50). In sectioning electron microscopy (EM) studies of M. gallisepticum, an intracellular electron-dense structure in the bleb and infrableb was observed, suggesting the existence of a cytoskeletal structure (7, 24, 29, 37, 58). Recently, the existence of such a structure has been confirmed by scanning EM of the structure remaining after Triton X-100 extraction (13), although the details are still unclear.A human pathogen, M. pneumoniae, has a rod-shaped cytoskeletal structure in the attachment organelle (9, 15, 16, 31, 37, 57). M. gallisepticum is related to M. pneumoniae (63, 64), as represented by 90.3% identity between the 16S rRNA sequences, and it has some open reading frames (ORFs) homologous to the component proteins of the cytoskeletal structures of M. pneumoniae (6, 17, 48). Therefore, the cytoskeletal structures of M. gallisepticum are expected to be similar to those of M. pneumoniae, as scanning EM images also suggest (13).The fastest-gliding species, M. mobile, is more distantly related to M. gallisepticum; it has novel cytoskeletal structures that have been analyzed through negative-staining transmission EM after extraction by Triton X-100 with image averaging (45). This method of transmission EM following Triton X-100 extraction clearly showed a cytoskeletal “jellyfish” structure. In this structure, a solid oval “bell,” about 235 nm wide and 155 nm long, is filled with a 12-nm hexagonal lattice. Connected to this bell structure are dozens of flexible “tentacles” that are covered with particles 20 nm in diameter at intervals of about 30 nm. The particles appear to have 180° rotational symmetry and a dimple at the center. The involvement of this cytoskeletal structure in the gliding mechanism was suggested by its cellular localization and by analyses of mutants lacking proteins essential for gliding.In the present study, we applied this method to M. gallisepticum and analyzed its unique cytoskeletal structure, and we then compared it with that of M. pneumoniae.     

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.     

Draft Genome Sequences of Yersinia pestis Isolates from Natural Foci of Endemic Plague in China

To gain insights into the evolutionary origin, emergence, and pathogenicity of the etiologic agent of plague, we have sequenced the genomes of four Yersinia pestis strains isolated from the zoonotic rodent reservoir in foci of endemic plague in China. These resources enable in-depth studies of Y. pestis sequence variations and detailed whole-genome comparisons of very closely related genomes from the supposed site of the origin and the emergence of global pandemics of plague.Here we report on the genomes of Yersinia pestis strains B42003004, K1973002, E1979001, and F1991016, which represent a sample of the genetic diversity found in four foci of endemic plague in China (24). Y. pestis bv. orientalis strain F1991016 was isolated in 1991 from Cangyuan County, China, from a rat (Rattus flavipectus), and Y. pestis bv. antiqua strain E1979001 was isolated in 1979 from Jianchuan, China, from a vole (Eothenomys miletus). Both Y. pestis strains K1973002 and B42003004 of biovars medievalis and antiqua, respectively, originate from marmota species (Marmota himalayana Hetian 1973; Marmota baibacina Wenquan 2003) (24). Genome analyses of these key isolates outline the details of microevolution of the plague bacterium, as these isolates represent important evolutionary milestones of the species, which is thought to have originated in Central Asia as a clonal descendant of Yersinia pseudotuberculosis (1). Genomic DNA was subjected to whole-genome shotgun sequencing and closure strategies as previously described (15). Plasmid (pHOS2) and fosmid (pCC1fos) libraries were constructed, with insert sizes of 4 to 6 kb and 30 to 40 kb, respectively. An average of 67,000 high-quality Sanger reads (total, 268,160) was obtained with an 860-bp average read length. The genomes with an average 12-fold read coverage depth were assembled using a Celera Assembler (11) and manually annotated using Manatee (http://manatee.sourceforge.net/). Genomic architectures were compared using Mauve (5, 18), and proteomes were analyzed with the BLAST score ratio tool (17).The young evolutionary history of the species and resulting homogenous population structure is reflected in a high degree of proteome conservation between the sequenced isolates and the modern strain CO92 (16). Y. pestis pathogenicity is anchored in its mobile inventory, and typically, isolates harbor three virulence plasmids, the species-specific plasminogen activator and murine toxin plasmids and the low-calcium-response plasmid pCD (23). Their pCD-borne lcrV antigen shows a genetic makeup identical to that of CO92 (2, 16). The insertion sequence element expansion clearly distinguishes these Central Asian isolates from the progenitor Y. pseudotuberculosis (3, 8). Comprehensive analyses reveal a lack of genome-wide synteny and suggest massive intrachromosomal rearrangements, a characteristic feature of Y. pestis genome evolution (6, 8). Besides insertion sequence element abundance, we observed isolate-specific propagation patterns that not only shaped the reorganization of the genomic architecture but also are known to drive microevolutionary adaptation in Y. pestis (4, 9, 14, 21, 24). Based upon the phenotypic and genotypic features that differentiate these isolates (13, 20, 24), B42003004 belongs to the most ancient Y. pestis lineage known to exist in China; hence, it is phylogenetically thought to be closest to the species progenitor Y. pseudotuberculosis (22). We studied metabolic genes that determine their biovar classification and investigated the underlying genetic determinants (24). Isolate K1973002 is defective in the nitrate reductase napA gene, similar to strain KIM (7), and represents the results of the evolutionary processes implicated in the biovar conversion from antiqua to medievalis. Isolate F1991016 carries an in-frame deletion in the glycerol-3-phosphate dehydrogenase glpD gene (19), similar to strain CO92 (16), and characteristic of the antiqua-to-orientalis conversion. The observed genetic traits strengthen the hypothesis that biovars medievalis and orientalis arose through parallel evolution from a glycerol- and nitrate-positive antiqua progenitor due to the acquisition of independent mutations (1, 10, 14). Variable-number tandem-nucleotide-repeat alleles (12) (allele K, K1973002; allele K, B42003004; allele P, E1979001; allele G, F1991016) are not biovar specific and are not discriminative enough to differentiate these isolates, which clearly supports a population-based phylogeny, as introduced by Achtman et al. (1).The whole-genome draft sequences of these evolutionary key isolates of Y. pestis will facilitate additional bioinformatic and phylogenetic analyses. The availability of high-quality Sanger sequences is crucial to resolve the genetically homogenous population structure and to shed light on Y. pestis speciation. Understanding the plasticity and genome dynamics further aids in forensic and epidemiological analyses by setting up the basis for an accurate and robust typing system for plague surveillance and promotes diagnostics development and control measures.     

Enterocin X,a Novel Two-Peptide Bacteriocin from Enterococcus faecium KU-B5, Has an Antibacterial Spectrum Entirely Different from Those of Its Component Peptides

Enterocin X, composed of two antibacterial peptides (Xα and Xβ), is a novel class IIb bacteriocin from Enterococcus faecium KU-B5. When combined, Xα and Xβ display variably enhanced or reduced antibacterial activity toward a panel of indicators compared to each peptide individually. In E. faecium strains that produce enterocins A and B, such as KU-B5, only one additional bacteriocin had previously been known.Bacteriocins are gene-encoded antibacterial peptides and proteins. Because of their natural ability to preserve food, they are of particular interest to researchers in the food industry. Bacteriocins are grouped into three main classes according to their physical properties and compositions (11, 12). Of these, class IIb bacteriocins are thermostable non-lanthionine-containing two-peptide bacteriocins whose full antibacterial activity requires the interaction of two complementary peptides (8, 19). Therefore, two-peptide bacteriocins are considered to function together as one antibacterial entity (14).Enterocins A and B, first discovered and identified about 12 years ago (2, 3), are frequently present in Enterococcus faecium strains from various sources (3, 5, 6, 9, 13, 16). So far, no other bacteriocins have been identified in these strains, except the enterocin P-like bacteriocin from E. faecium JCM 5804^T (18). Here, we describe the characterization and genetic identification of enterocin X in E. faecium KU-B5. Enterocin X (identified after the enterocin P-like bacteriocin was discovered) is a newly found class IIb bacteriocin in E. faecium strains that produce enterocins A and B.     

Mixed Infections of Pepino Mosaic Virus Strains Modulate the Evolutionary Dynamics of this Emergent Virus

Pepino mosaic virus (PepMV) is an emerging pathogen that causes severe economic losses in tomato crops (Solanum lycopersicum L.) in the Northern hemisphere, despite persistent attempts of control. In fact, it is considered one of the most significant viral diseases for tomato production worldwide, and it may constitute a good model for the analysis of virus emergence in crops. We have combined a population genetics approach with an analysis of in planta properties of virus strains to explain an observed epidemiological pattern. Hybridization analysis showed that PepMV populations are composed of isolates of two types (PepMV-CH2 and PepMV-EU) that cocirculate. The CH2 type isolates are predominant; however, EU isolates have not been displaced but persist mainly in mixed infections. Two molecularly cloned isolates belonging to each type have been used to examine the dynamics of in planta single infections and coinfection, revealing that the CH2 type has a higher fitness than the EU type. Coinfections expand the range of susceptible hosts, and coinfected plants remain symptomless several weeks after infection, so a potentially important problem for disease prevention and management. These results provide an explanation of the observed epidemiological pattern in terms of genetic and ecological interactions among the different viral strains. Thus, mixed infections appear to be contributing to shaping the genetic structure and dynamics of PepMV populations.Pepino mosaic virus (PepMV; genus Potexvirus, family Flexiviridae) was identified in 1974 as the agent responsible for a viral disease of pepino crops (Solanum muricatum) in Peru (30). PepMV in tomato (Solanum lycopersicum) was first reported in The Netherlands in 1999 (74) but has since spread rapidly in Europe (3, 11, 38, 48, 51, 57) and beyond (20, 35, 36, 42, 68), causing epidemics and severe economic losses (27, 29, 36, 51, 67, 69). The PepMV host range is limited mainly to the Solanaceae (59), and the virus is easily transmitted from plant to plant by contact (30), vectored by bumblebees (65), or seedborne-transmitted (37). PepMV infections in tomato are associated with a wide range of leaf symptoms: mild and severe mosaics, bubbling, laminal distortions, and stunting (26, 27, 51). Fruit symptoms occur with or without leaf symptoms, and the main impact of PepMV is on fruit quality (irregular lycopene distribution [26]) but not on yield (69). Therefore, PepMV is currently considered a dangerous pathogen and is included in the European Plant Protection Organization alert list (15) as one of the most important tomato viruses worldwide (27, 51, 57, 68, 69).The PepMV genome consists of a single, positive-sense, ∼6,400-nucleotide (nt) RNA strand containing five open reading frames (ORFs). ORF1 encodes the putative viral polymerase (RdRp) (3). ORFs 2, 3, and 4 encode the triple gene block (TGB) proteins TGBp1, TGBp2, and TGBp3, which are essential for virus movement (46, 75, 78). Potato virus X TGBp1 is a multifunctional protein that induces plasmodesmal gating, moves from cell to cell, has ATPase and RNA helicase activities, binds viral RNAs, and acts as suppressor of RNA silencing (39, 76-78). ORF5 encodes the coat protein (CP) which, in addition to its structural role, is required for cell-to-cell and long-distance movement (12). Finally, two short untranslated sequences flank the coding regions, and there is a poly(A) tail at the 3′ end of the genomic RNA (3, 11, 48).Previous studies have shown that Spanish PepMV populations sampled between 2000 and 2004 were genetically very homogeneous (∼99% nucleotide identity), most comprising isolates highly similar to the so-called European tomato strain (PepMV-EU). However, a few isolates sampled in 2004 in the Murcia region (Southeastern Spain) were distinct and highly similar to the US2 strain reported in the United States (51). U.S. isolates (US1 and US2) and a Chilean isolate from infected tomato seeds (CH2) share only 79 to 86% nucleotide identity with European (EU) isolates (36, 42). The CH2 type has been reported recently in greenhouses for tomato production in Poland (29) and Belgium (27). In this last study, CH2 was predominant in single infections and also frequent in mixed infections with isolates of the EU type (27). However, all PepMV types (EU, US1, US2, and CH2) have been found in United States, where the PepMV-EU type has been the most prevalent, and mixed infections were found in samples collected from Arizona, Colorado, and Texas (35).Several studies of plant virus populations have reported a reduced genetic diversity of populations separated in time or space (19, 40, 56) with high virus genetic stability (23). Nevertheless, how genetic and ecological factors modulate the evolutionary dynamics of viruses and determine epidemiological patterns is still poorly understood (25, 47).We have characterized the population genetic structure of PepMV in infected samples of commercial tomato crops in the Murcia region (southeastern Spain) between 2005 and 2008. Phylogenetic analysis was performed, and genetic diversity values among PepMV isolates were estimated to determine the structure of the population and the strength and direction of selection. In addition, the biological properties (host range, fitness, and virulence) of two cloned isolates of the CH2 and EU types were studied to understand the evolutionary dynamics of natural PepMV populations.