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).     

Negative Correlation between Individual-Insect-Level Virulence and Colony-Level Virulence of Paenibacillus larvae,the Etiological Agent of American Foulbrood of Honeybees

Paenibacillus larvae is the etiological agent of American foulbrood (AFB) in honeybees. Recently, different genotypes of P. larvae (ERIC I to ERIC IV) were defined, and it was shown that these genotypes differ inter alia in their virulence on the larval level. On the colony level, bees mitigate AFB through the hygienic behavior of nurse bees. Therefore, we investigated how the hygienic behavior shapes P. larvae virulence on the colony level. Our results indicate that P. larvae virulence on the larval level and that on the colony level are negatively correlated.American foulbrood (AFB) is among the economically most important honeybee diseases. The etiological agent of AFB is the gram-positive, spore-forming bacterium Paenibacillus larvae (9). The extremely tenacious spores are the infectious form of this organism. These spores drive disease transmission within colonies (11), as well as between colonies as soon as they end up in the honey stores of an infected colony (12).The species P. larvae can be subdivided into four different genotypes designated ERIC I to ERIC IV based on results from repetitive-element PCR (20) using enterobacterial repetitive intergenic consensus (ERIC) primers (9, 10), with P. larvae ERIC I and ERIC II being the two practically most important genotypes (1, 2, 9, 10, 13, 16). The four genotypes were shown previously to differ in phenotype, including virulence on the larval level (8, 9). While larvae infected with genotypes ERIC II to ERIC IV were killed within only 6 to 7 days, it took P. larvae ERIC I around 12 to 14 days to kill all infected individuals. Therefore, genotype ERIC I was considered to be less virulent and the other three genotypes were considered to be highly virulent (7-9) on the larval level.P. larvae is an obligately killing pathogen which must kill its host to be transmitted. The virulence of such an obligate killer is thought to be determined primarily by two factors, (i) the probability of infecting a host and (ii) the time to host death (6). The problem of ensuring a high enough probability of infecting the next host is solved for P. larvae by (i) the tenacious exospores, which remain infectious for over half a century (17) and, therefore, can wait for decades for the next host to pass by, and (ii) a high pathogen reproduction rate (23) and, thus, the production of an extremely high number of spores within each infected larva.For evaluating the second factor determining P. larvae virulence, the time to host death, it is important to consider the two levels of honeybee hosts, the level of the individual larva dying from AFB and the level of the colony succumbing to AFB.The virulence of P. larvae genotypes on the larval level has been analyzed recently (8, 9). We have now determined the colony-level virulence for the two most common and practically important (10, 16) genotypes of P. larvae, ERIC I and ERIC II, significantly differing in virulence on the larval level (8). We will discuss how the time to larval death relates to the time to colony death and how the hygienic response shapes P. larvae virulence.     

Genetic Diversity and Multihost Pathogenicity of Clinical and Environmental Strains of Burkholderia cenocepacia

A collection of 54 clinical and agricultural isolates of Burkholderia cenocepacia was analyzed for genetic relatedness by using multilocus sequence typing (MLST), pathogenicity by using onion and nematode infection models, antifungal activity, and the distribution of three marker genes associated with virulence. The majority of clinical isolates were obtained from cystic fibrosis (CF) patients in Michigan, and the agricultural isolates were predominantly from Michigan onion fields. MLST analysis resolved 23 distinct sequence types (STs), 11 of which were novel. Twenty-six of 27 clinical isolates from Michigan were genotyped as ST-40, previously identified as the Midwest B. cenocepacia lineage. In contrast, the 12 agricultural isolates represented eight STs, including ST-122, that were identical to clinical isolates of the PHDC lineage. In general, pathogenicity to onions and the presence of the pehA endopolygalacturonase gene were detected only in one cluster of related strains consisting of agricultural isolates and the PHDC lineage. Surprisingly, these strains were highly pathogenic in the nematode Caenorhabditis elegans infection model, killing nematodes faster than the CF pathogen Pseudomonas aeruginosa PA14 on slow-kill medium. The other strains displayed a wide range of pathogenicity to C. elegans, notably the Midwest clonal lineage which displayed high, moderate, and low virulence. Most strains displayed moderate antifungal activity, although strains with high and low activities were also detected. We conclude that pathogenicity to multiple hosts may be a key factor contributing to the potential of B. cenocepacia to opportunistically infect humans both by increasing the prevalence of the organism in the environment, thereby increasing exposure to vulnerable hosts, and by the selection of virulence factors that function in multiple hosts.The betaproteobacterium Burkholderia cenocepacia, 1 of now 17 classified species belonging to the Burkholderia cepacia complex (BCC), is ubiquitous and extremely versatile in its metabolic capabilities and interactions with other organisms (38, 40, 57, 58). Strains of B. cenocepacia are pathogens of onion and banana plants, opportunistic pathogens of humans, symbionts of numerous plant rhizospheres, contaminants of pharmaceutical and industrial products, and inhabitants of soil and surface waters (14, 29, 33, 34, 37, 45). Originally described as a pathogen of onions (8), organisms of the BCC emerged in the past 3 decades as serious human pathogens, capable of causing devastating chronic lung infections in persons with cystic fibrosis (CF) or chronic granulomatous disease (21, 24, 28). Infections due to BCC are a serious concern to CF patients due to their inherent antibiotic resistance and high potential for patient-to-patient transmission (23). Although 16 of the BCC species have been recovered from respiratory secretions of CF patients in many countries (46, 58), B. cenocepacia has been the most common species isolated in North America, detected in 50% of 606, 83% of 447, and 45.6% of 1,218 patients in recent studies (35, 46, 52).The epidemiology of infectious disease caused by B. cenocepacia appears to involve patient-to-patient spread of genetically distinct lineages. B. cenocepacia lineages, such as ET12, Midwest, and PHDC, have been identified from large numbers of individuals in disease outbreaks in North America and Europe (11, 32, 54). A recently developed multilocus sequence typing (MLST) scheme has been shown to be a reliable epidemiologic tool for differentiating between the five subgroups (IIIA to IIIE) of B. cenocepacia, and strains representing three of these subgroups (IIIA, IIIB, and IIID) have been recovered from CF patients (2). Outside of the patient-to-patient transmission of clonal lineages, the mode of acquisition of strains causing sporadic cases of B. cenocepacia in CF patients remains unclear, although environmental sources are a logical reservoir for infection. Previously, an isolate of B. cenocepacia indistinguishable from the PHDC epidemic clonal lineage by using standard typing methods (e.g., repetitive-sequence-based PCR, randomly amplified polymorphic DNA, pulsed-field gel electrophoresis) was detected in an agricultural soil sample (34). Similarly, three distinct MLST sequence types containing both clinical and environmental (plant and soil) B. cenocepacia isolates were identified (1). These findings suggest that natural populations of B. cenocepacia in soil or associated with plants are a potential reservoir for the emergence of new human pathogenic lineages.Experimental models for the study of virulence potential and traits of B. cenocepacia include mouse and rat models with genetic defects allowing chronic lung infections to be established (e.g., see reference 48). Nematode (Caenorhabditis elegans), alfalfa (Medicago sativa), and onion (Allium cepa) models have also been routinely utilized for the identification of virulence factors (5, 29, 31). C. elegans has been extensively used to study the pathogenesis and virulence factors of a wide variety of bacterial and fungal pathogens (9, 15, 42, 51, 56). In several pathogens, including Pseudomonas (56) and Burkholderia (20), putative virulence factors important for the pathogenesis in mammalian systems (15, 51) have been identified using the C. elegans model. The C. elegans model might be limited in the detection of host-specific virulence factors; however, several attributes, such as small size and rapid development, make it an excellent whole animal model for pathogenesis research (16, 51).The evidence that individual strains of B. cenocepacia can be pathogenic to both plants and humans and are prevalent in various environmental niches has provoked particular interest in elucidating the clinical pathogenic potential of environmental isolates. The basis of this study was to examine whether genetically related B. cenocepacia strains exhibit shared characteristics that contribute to their pathogenicity in multiple hosts and to examine the potential for circulating environmental isolates to emerge as new clinical pathogens. Here, we tested the degree of virulence in animal (nematode) and plant (onion) infection models, the production of antifungal activity, and the genetic relatedness of clinical and environmental B. cenocepacia subgroup IIIB strains predominantly isolated from Michigan.     

Alignment of the c-Type Cytochrome OmcS along Pili of Geobacter sulfurreducens

Immunogold localization revealed that OmcS, a cytochrome that is required for Fe(III) oxide reduction by Geobacter sulfurreducens, was localized along the pili. The apparent spacing between OmcS molecules suggests that OmcS facilitates electron transfer from pili to Fe(III) oxides rather than promoting electron conduction along the length of the pili.There are multiple competing/complementary models for extracellular electron transfer in Fe(III)- and electrode-reducing microorganisms (8, 18, 20, 44). Which mechanisms prevail in different microorganisms or environmental conditions may greatly influence which microorganisms compete most successfully in sedimentary environments or on the surfaces of electrodes and can impact practical decisions on the best strategies to promote Fe(III) reduction for bioremediation applications (18, 19) or to enhance the power output of microbial fuel cells (18, 21).The three most commonly considered mechanisms for electron transfer to extracellular electron acceptors are (i) direct contact between redox-active proteins on the outer surfaces of the cells and the electron acceptor, (ii) electron transfer via soluble electron shuttling molecules, and (iii) the conduction of electrons along pili or other filamentous structures. Evidence for the first mechanism includes the necessity for direct cell-Fe(III) oxide contact in Geobacter species (34) and the finding that intensively studied Fe(III)- and electrode-reducing microorganisms, such as Geobacter sulfurreducens and Shewanella oneidensis MR-1, display redox-active proteins on their outer cell surfaces that could have access to extracellular electron acceptors (1, 2, 12, 15, 27, 28, 31-33). Deletion of the genes for these proteins often inhibits Fe(III) reduction (1, 4, 7, 15, 17, 28, 40) and electron transfer to electrodes (5, 7, 11, 33). In some instances, these proteins have been purified and shown to have the capacity to reduce Fe(III) and other potential electron acceptors in vitro (10, 13, 29, 38, 42, 43, 48, 49).Evidence for the second mechanism includes the ability of some microorganisms to reduce Fe(III) that they cannot directly contact, which can be associated with the accumulation of soluble substances that can promote electron shuttling (17, 22, 26, 35, 36, 47). In microbial fuel cell studies, an abundance of planktonic cells and/or the loss of current-producing capacity when the medium is replaced is consistent with the presence of an electron shuttle (3, 14, 26). Furthermore, a soluble electron shuttle is the most likely explanation for the electrochemical signatures of some microorganisms growing on an electrode surface (26, 46).Evidence for the third mechanism is more circumstantial (19). Filaments that have conductive properties have been identified in Shewanella (7) and Geobacter (41) species. To date, conductance has been measured only across the diameter of the filaments, not along the length. The evidence that the conductive filaments were involved in extracellular electron transfer in Shewanella was the finding that deletion of the genes for the c-type cytochromes OmcA and MtrC, which are necessary for extracellular electron transfer, resulted in nonconductive filaments, suggesting that the cytochromes were associated with the filaments (7). However, subsequent studies specifically designed to localize these cytochromes revealed that, although the cytochromes were extracellular, they were attached to the cells or in the exopolymeric matrix and not aligned along the pili (24, 25, 30, 40, 43). Subsequent reviews of electron transfer to Fe(III) in Shewanella oneidensis (44, 45) appear to have dropped the nanowire concept and focused on the first and second mechanisms.Geobacter sulfurreducens has a number of c-type cytochromes (15, 28) and multicopper proteins (12, 27) that have been demonstrated or proposed to be on the outer cell surface and are essential for extracellular electron transfer. Immunolocalization and proteolysis studies demonstrated that the cytochrome OmcB, which is essential for optimal Fe(III) reduction (15) and highly expressed during growth on electrodes (33), is embedded in the outer membrane (39), whereas the multicopper protein OmpB, which is also required for Fe(III) oxide reduction (27), is exposed on the outer cell surface (39).OmcS is one of the most abundant cytochromes that can readily be sheared from the outer surfaces of G. sulfurreducens cells (28). It is essential for the reduction of Fe(III) oxide (28) and for electron transfer to electrodes under some conditions (11). Therefore, the localization of this important protein was further investigated.     

Generation of Single-Copy Transposon Insertions in Clostridium perfringens by Electroporation of Phage Mu DNA Transposition Complexes

Transposon mutagenesis is a tool that is widely used for the identification of genes involved in the virulence of bacteria. Until now, transposon mutagenesis in Clostridium perfringens has been restricted to the use of Tn916-based methods with laboratory reference strains. This system yields primarily multiple transposon insertions in a single genome, thus compromising its use for the identification of virulence genes. The current study describes a new protocol for transposon mutagenesis in C. perfringens, which is based on the bacteriophage Mu transposition system. The protocol was successfully used to generate a single-insertion mutant library both for a laboratory strain and for a field isolate. Thus, it can be used as a tool in large-scale screening to identify virulence genes of C. perfringens.Clostridium perfringens is a gram-positive, anaerobic bacterium that forms heat-resistant spores. It is widespread in the soil and commonly found in the gastrointestinal tract of mammals. It has been implicated in several medical conditions in humans, ranging from mild food poisoning to necrotic enteritis and gas gangrene. C. perfringens strains also cause a variety of important diseases in domestic animals, including several enteric syndromes, such as enterotoxemia in cattle, sheep, and pigs, necrotic enteritis in poultry, and typhocolitis in equines (17, 40).Understanding the pathogenesis of these infections is of crucial importance for the development of new tools for the prevention and control of C. perfringens-related diseases. Genetic modification is a valuable approach to identify new virulence factors and to study their role in the pathogenesis of C. perfringens.Since the 1980s, several tools for manipulation of C. perfringens at the molecular level have been developed (1, 5, 28, 35, 38). Among these tools, transposon mutagenesis is a method that is widely used for identification of virulence genes. Until now, the only reproducible method for transposon mutagenesis in C. perfringens was based on Tn916, a tetracycline resistance-encoding conjugative transposon originally isolated from Enterococcus faecalis (10, 11, 13). Tn916 has been used extensively for transposon mutagenesis due to its broad host range and has been proven to be valuable for the identification of genes in C. perfringens (3, 7, 22). Nevertheless, this method has major disadvantages; multiple Tn916 insertion events occur with an incidence of 65% to 75%, severely complicating identification of genes responsible for phenotype changes (3, 7, 19). Furthermore, Tn916 is still active after insertion, resulting in unstable mutants (6, 39, 42). To our knowledge, generation of Tn916-derived transposon mutants in C. perfringens field strains has never been described.Although a variety of transposon mutagenesis methods are available for gram-positive bacteria (4, 37, 41, 43), the inherent species nonspecificity, as well as the lack of mobility of the integrated transposon, makes the bacteriophage Mu-based transposon delivery system a system of choice for a variety of species (16, 26, 46). The Mu transposition approach includes in vitro assembly of a complex between the transposon DNA and the transposase enzyme, the transpososome, followed by delivery of the transpososome into the recipient cells. Once inside a cell, the Mu transpososome becomes activated in the presence of divalent cations, resulting in genomic integration of the delivered transposon. The bacteriophage Mu transposition system is also functional in vitro (15, 32, 33), in contrast to the Tn916 mutagenesis strategy, which is restricted to transposon mobilization in vivo following conjugation or electroporation. Under the optimal in vitro conditions, the Mu transposition reaction requires only the MuA transposase, a mini-Mu transposon, and target DNA as macromolecular components (15).In this study, a novel protocol is described for transposon mutagenesis in C. perfringens that exploits the bacteriophage Mu transposition system. To our knowledge, this report is the first report describing a mutagenesis method generating single-insertion transposon mutants in laboratory and field isolates of C. perfringens. This method is important for the identification of C. perfringens virulence factors involved in the numerous diseases caused by this bacterium.     

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.