Comparison of the Complete Genome Sequences of Bifidobacterium animalis subsp. lactis DSM 10140 and Bl-04

Bifidobacteria are important members of the human gut flora, especially in infants. Comparative genomic analysis of two Bifidobacterium animalis subsp. lactis strains revealed evolution by internal deletion of consecutive spacer-repeat units within a novel clustered regularly interspaced short palindromic repeat locus, which represented the largest differential content between the two genomes. Additionally, 47 single nucleotide polymorphisms were identified, consisting primarily of nonsynonymous mutations, indicating positive selection and/or recent divergence. A particular nonsynonymous mutation in a putative glucose transporter was linked to a negative phenotypic effect on the ability of the variant to catabolize glucose, consistent with a modification in the predicted protein transmembrane topology. Comparative genome sequence analysis of three Bifidobacterium species provided a core genome set of 1,117 orthologs complemented by a pan-genome of 2,445 genes. The genome sequences of the intestinal bacterium B. animalis subsp. lactis provide insights into rapid genome evolution and the genetic basis for adaptation to the human gut environment, notably with regard to catabolism of dietary carbohydrates, resistance to bile and acid, and interaction with the intestinal epithelium. The high degree of genome conservation observed between the two strains in terms of size, organization, and sequence is indicative of a genomically monomorphic subspecies and explains the inability to differentiate the strains by standard techniques such as pulsed-field gel electrophoresis.Actinobacteria, Firmicutes, Proteobacteria, and Bacteroidetes are dominant microbial phyla widely distributed in diverse ecosystems on the planet (10, 13, 20, 23, 33, 40, 51). Metagenomic analyses of the microbial landscape inhabiting various mammalian environments, notably the human gastrointestinal tract (GIT) and skin, have specifically identified Actinobacteria as an important and occasionally dominant phylum (18, 21, 33). Among the members of the large, diverse, and dynamic microbial community residing in the human GIT, Bifidobacterium is a dominant genus considered beneficial to humans and includes probiotic strains (live microorganisms which, when administered in adequate amounts, confer a health benefit on the host) (11). The population of bifidobacteria in the human intestine varies over time. Following vaginal delivery, the GIT of healthy newborns is typically colonized by bifidobacteria, especially in breast-fed infants, during the first few days of life (12). Interindividual variation, however, is remarkable in the human infant intestinal flora (41), and dominant genera are not always consistent across metagenomic analyses of the human gut flora (18, 30, 33, 41). Over time, the infant intestinal ecosystem becomes more complex as the diet becomes more diverse, with bifidobacteria typically remaining dominant until weaning (30).Bifidobacterium animalis subsp. lactis is a gram-positive lactic acid bacterium commonly found in the guts of healthy humans and has been identified in the infant gut biota, particularly in ileal, fecal, and mucosal samples (52, 56). Some strains of B. animalis subsp. lactis are able to survive in the GIT, to adhere to human epithelial cells in vitro, to modify fecal flora, to modulate the host immune response, or to prevent microbial gastroenteritis and colitis (4, 15, 20, 40, 52, 56). Additionally, B. animalis subsp. lactis has been reported to utilize nondigestible oligosaccharides, which may contribute to the organism''s ability to compete in the human gut. Carbohydrates resistant to enzymatic degradation and not absorbed in the upper intestinal tract are a primary source of energy for microbes residing in the large intestine. The benefits associated with probiotic strains of B. animalis subsp. lactis have resulted in their inclusion in the human diet via formulation into a large array of dietary supplements and foods, including dairy products such as yogurt. Deciphering the complete genome sequences of such microbes will provide additional insight into the genetic basis for survival and residence in the human gut, notably with regard to the ability to survive gastric passage and utilize available nutrients. Also, these genomes provide reference sequences for ongoing metagenomic analyses of the human environment, including the gut metagenome.Bifidobacterium animalis subsp. lactis is the most common bifidobacterium utilized as a probiotic in commercial dairy products in North America and Europe (22, 38). However, despite this commercial and probiotic significance, strain-level differentiation of B. animalis subsp. lactis strains has been hindered by the high genetic similarity of these organisms, as determined by pulsed-field gel electrophoresis and other nucleic acid-based techniques (6, 55, 56), and the lack of available genomic sequence information. The genome sequence of strain BB-12 (17) is not currently publicly available, and only a draft genome sequence in 28 contigs is available for strain HN019 (GenBank project 28807). The complete B. animalis subsp. lactis genome for strain AD011 (28) was only recently (2009) published. While this was an important first step, a single genome does not allow identification of unique targets for strain differentiation or comparative analyses within the subspecies.The objectives of this study were to determine the complete genome sequences of two B. animalis subsp. lactis strains, the type strain and a widely used commercial strain, to provide insights into the functionality of this species and into species identification and strain specialization.     

Complete Genome Sequence of Probiotic Bifidobacterium animalis subsp. lactis Strain V9

Bifidobacterium animalis subsp. lactis strain V9 is a Chinese commercial bifidobacteria with several probiotic functions. It was isolated from a healthy Mongolian child in China. We present here the complete genome sequence of V9 and compare it to 3 other published genome sequences of B. animalis subsp. lactis strains. The result indicates the lack of polymorphism among strains of this subspecies from different continents.Bifidobacterium animalis subsp. lactis strain V9 was isolated from the feces of a healthy Mongolian child in China (5). It has shown a high level of tolerance to gastric acid and bile acids (5). This strain has been implemented in the industrial production of dairy starter cultures by Inner Mongolia Yili Industrial Group Company Limited, the largest dairy corporation in China.Whole-genome sequencing of B. animalis subsp. lactis V9 was performed with a combined strategy of 454 sequencing (8) and Solexa paired-end sequencing technology (2). Genomic libraries containing 7-kb inserts were constructed, and 325,824 paired-end reads and 67,177 single-end reads were generated using the GS FLX system, giving 36.0-fold coverage of the genome. A total of 96.0% of the reads were assembled into four large scaffolds, including 163 nonredundant contigs, using the 454 Newbler assembler (454 Life Sciences, Branford, CT). A total of 8,953,102 reads (2-kb library) were generated to reach a depth of 335-fold coverage with an Illumina Solexa Genome Analyzer IIx and mapped to the scaffolds using the Burrows-Wheeler Alignment (BWA) tool (7). The gaps between scaffolds were filled by sequencing PCR products using an ABI 3730 capillary sequencer. The analysis of the genome was performed as described previously (3, 4).The complete genome sequence of V9 contains a circular 1,944,050-bp chromosome, with a GC content of 60.5%. The genome size is slightly larger than the sequenced genome sizes of B. animalis subsp. lactis strains DSM 10140^T (1), Bl-04 (1), and AD011 (6) due to a unique insertion of 4,037 bp. The V9 genome contains 1,636 genes in total, including 1,572 coding genes, 4 rRNA operons, and 52 tRNAs.Comparison of the four B. animalis subsp. lactis genomes revealed nearly perfect synteny. AD011 is the most diverged strain, with more single nucleotide polymorphisms (SNPs) and indels than the other three strains. There are 197 SNPs in AD011, with 70 synonymous and 16 nonsynonymous SNPs, which means that there is only 1 SNP per 10 kb, indicating the high consistency within this subspecies. The other three strains are almost identical, with only 25 SNPs in V9, 13 SNPs in Bl-04, and 44 SNPs in DSM 10140^T. Strain V9 was isolated from the feces of a Mongolian child in Inner Mongolia, China, where traditional fermented milk has been consumed for thousands of years, and the other three strains were originally isolated from fecal samples (1, 6) or yogurt (1) in the United States of America, France, and Korea. The result indicated the lack of polymorphism among multiple lineages from different continents (1).Interestingly, compared to the other three sequenced B. animalis subsp. lactis strains, V9 has a large insertion, which encodes one putative transposase (BalV_1091) and two sugar metabolism-related proteins, an alpha-1,4-glucosidase (BalV_1092) and an ABC transporter solute-binding protein (BalV_1093). This insertion is a copy of the region at positions 1,860,164 to 1,864,073, which is commonly shared by all four B. animalis subsp. lactis strains.     

Distribution of In Vitro Fermentation Ability of Lacto-N-Biose I,a Major Building Block of Human Milk Oligosaccharides,in Bifidobacterial Strains

This study investigated the potential utilization of lacto-N-biose I (LNB) by individual strains of bifidobacteria. LNB is a building block for the human milk oligosaccharides, which have been suggested to be a factor for selective growth of bifidobacteria. A total of 208 strains comprising 10 species and 4 subspecies were analyzed for the presence of the galacto-N-biose/lacto-N-biose I phosphorylase (GLNBP) gene (lnpA) and examined for growth when LNB was used as the sole carbohydrate source. While all strains of Bifidobacterium longum subsp. longum, B. longum subsp. infantis, B. breve, and B. bifidum were able to grow on LNB, none of the strains of B. adolescentis, B. catenulatum, B. dentium, B. angulatum, B. animalis subsp. lactis, and B. thermophilum showed any growth. In addition, some strains of B. pseudocatenulatum, B. animalis subsp. animalis, and B. pseudolongum exhibited the ability to utilize LNB. With the exception for B. pseudocatenulatum, the presence of lnpA coincided with LNB utilization in almost all strains. These results indicate that bifidobacterial species, which are the predominant species found in infant intestines, are potential utilizers of LNB. These findings support the hypothesis that GLNBP plays a key role in the colonization of bifidobacteria in the infant intestine.Bifidobacteria are gram-positive anaerobic bacteria that naturally colonize the human intestinal tract and are believed to be beneficial to human health (21, 30). Breastfeeding has been shown to be associated with an infant fecal microbiota dominated by bifidobacteria, whereas the fecal microbiota of infants who are consuming alternative diets has been described as being mixed and adult-like (12, 21). It has been suggested that the selective growth of bifidobacteria observed in breast-fed newborns is related to the oligosaccharides and other factors that are contained in human milk (human milk oligosaccharides [HMOs]) (3, 4, 10, 11, 16, 17, 34). Kitaoka et al. (15) have recently found that bifidobacteria possess a unique metabolic pathway that is specific for lacto-N-biose I (LNB; Galβ1-3GlcNAc) and galacto-N-biose (GNB; Galβ1-3GalNAc). LNB is a building block for the type 1 HMOs [such as lacto-N-tetraose (Galβ1-3GlcNAcβ1-3Galβ1-4Glc), lacto-N-fucopentaose I (Fucα1-2Galβ1-3GlcNAcβ1-3Galβ1-4Glc), and lacto-N-difucohexaose I (Fucα1-2Galβ1-3[Fucα1-4]GlcNAcβ1-3Galβ1-4Glc)], and GNB is a core structure of the mucin sugar that is present in the human intestine and milk (18, 27). The GNB/LNB pathway, as previously illustrated by Wada et al. (33), involves proteins/enzymes that are required for the uptake and degradation of disaccharides such as the GNB/LNB transporter (29, 32), galacto-N-biose/lacto-N-biose I phosphorylase (GLNBP; LnpA) (15, 24) (renamed from lacto-N-biose phosphorylase after the finding of phosphorylases specific to GNB [23] and LNB [22]), N-acetylhexosamine 1-kinase (NahK) (25), UDP-glucose-hexose 1-phosphate uridylyltransferase (GalT), and UDP-galactose epimerase (GalE). Some bifidobacteria have been demonstrated to be enzymatically equipped to release LNB from HMOs that have a type 1 structure (lacto-N biosidase; LnbB) (33) or GNB from the core 1-type O-glycans in mucin glycoproteins (endo-α-N-acetylgalatosaminidase) (6, 13, 14). It has been suggested that the presence of the LnbB and GNB/LNB pathways in some bifidobacterial strains could provide a nutritional advantage for these organisms, thereby increasing their populations within the ecosystem of these breast-fed newborns (33).The species that predominantly colonize the infant intestine are the bifidobacterial species B. breve, B. longum subsp. infantis, B. longum subsp. longum, and B. bifidum (21, 28). On the other hand, strains of B. adolescentis, B. catenulatum, B. pseudocatenulatum, and B. longum subsp. longum are frequently isolated from the adult intestine (19), and strains of B. animalis subsp. animalis, B. animalis subsp. lactis, B. thermophilum and B. pseudolongum have been shown to naturally colonize the guts of animals (1, 2, 7, 8). However, it is unclear whether there is a relationship between the differential colonization of the bifidobacterial species and the presence of the GNB/LNB pathway. In the present study, we investigated the ability of individual bifidobacterial strains in the in vitro fermentation of LNB and in addition, we also tried to determine whether or not the GLNBP gene (lnpA), which is a key enzyme of the GNB/LNB pathway, was present.     

Identification and Typing of Lactococcus lactis by Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry

Currently, the genus Lactococcus is classified into six species: Lactococcus chungangensis, L. garvieae, L. lactis, L. piscium, L. plantarum, and L. raffinolactis. Among these six species, L. lactis is especially important because of its use in the manufacture of probiotic dairy products. L. lactis consists of three subspecies: L. lactis subsp. cremoris, L. lactis subsp. hordniae, and L. lactis subsp. lactis. However, these subspecies have not yet been reliably discriminated. To date, mainly phenotypic identification has been used, with a few genotypic identifications. We discriminated species or subspecies in the genus Lactococcus not only by proteomics identification using matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) but also by phenotypic and genotypic identification. The proteomics identification using differences in the mass spectra of ribosomal proteins was nearly identical to that by genotypic identification (i.e., by analyses of 16S rRNA and recA gene sequences and amplified fragment length polymorphism). The three ribosomal subunits 30S/L31, 50S/L31, and 50S/L35 were the best markers for discriminating L. lactis subsp. cremoris from L. lactis subsp. lactis. Proteomics identification using MALDI-TOF MS was therefore a powerful method for discriminating and identifying these bacteria. In addition, this method was faster and more reliable than others that we examined.Lactococci are lactic acid bacteria (LAB) that are important contributors to the production of fermented dairy products, and some species produce antimicrobial compounds. Most species in the genus Lactococcus have been isolated from food-related sources and plants and are generally regarded as safe. Probiotic foods use these LAB, and there have been various studies of the relationship between these foods and the maintenance of human intestinal health (32). Lactococcus was first established as a genus distinct from the genus Streptococcus in 1985 (29).Currently, six species and three subspecies in the genus Lactococcus have been validated. Lactococcus plantarum has been isolated mainly from plants; L. garvieae has been isolated from fish, animals, and milk, and L. piscium has been isolated from salmon. Lactococcus lactis is most commonly found in raw milk, cheese, and other dairy products; L. raffinolactis has been found in raw milk and cheese, and L. chunagangensis has been isolated from wastewater. Among the six species, L. lactis is considered one of the most important in food production because it is used to manufacture fermented milk, butter, and cheese. Because of this importance, the whole genomes of three strains of L. lactis—L. lactis subsp. cremoris SK11 (10), L. lactis subsp. cremoris MG 1363 (37), and L. lactis subsp. lactis IL1403 (2)—have been sequenced.Since L. lactis was first described by Orla-Jensen in 1919 (21), there have been various classifications. To date, L. lactis has been classified into three subspecies: L. lactis subsp. cremoris, L. lactis subsp. hordniae, and L. lactis subsp. lactis. However, this classification was based on only a few phenotypic characteristics and is considered imperfect because of its inherent disadvantages of sensitivity to culture conditions or bacterial growth phase. Discriminating between L. lactis subsp. cremoris and L. lactis subsp. lactis is particularly difficult but is very important in industrial applications, because the activities of the two subspecies in cheese manufacture differ. In addition, when newly isolated bacterial strains are registered in public culture collections, these strains have to be identified and discriminated at the subspecies level. Normally, these two subspecies are identified on the basis of the following phenotypic features: (i) the ability to ferment maltose and ribose, (ii) growth in 4% NaCl (pH 9.2) at 40°C, (iii) the ability to produce ammonia from arginine, and (iv) the presence of glutamate decarboxylase activity (18-20). However, determining the results of the phenotypic identification is difficult because they are sometimes ambiguous and time sensitive, as demonstrated by the sugar fermentation tests described below, which gave different results over time. In addition, the results of phenotypic identifications in previous reports were not identical each other (9, 28, 35).From an evolutionary viewpoint, it is reasonable to classify subspecies by using the divergence of housekeeping genes that are well preserved at the genus or species level. 16S rRNA gene sequencing is the most common technique currently used to identify species. At the subspecies level, however, 16S rRNA gene sequence identity is often very high, and these sequences therefore cannot be used for identification purposes (14, 24, 27, 36). Recently, for LAB, the partial sequences of the recA (recombinase A), pheS (phenylalanyl tRNA synthetase alpha subunit), and rpoA (DNA-directed RNA polymerase alpha chain) genes have been effectively used for species or subspecies identification (5, 7, 17), and the analysis of 16S rRNA gene sequences in combination with housekeeping gene sequences has been used to identify subspecies.In recent years, a number of important experiments have used matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS) for rapid bacterial identification, including clostridia (15), LAB (34), Listeria (1), mycobacteria (12), salmonellae (6), viridans group streptococci (8), and other nonfermenting bacteria (16). In these studies, MALDI-TOF MS spectra were obtained from intact cells without biomarker purification or chromatographic separation. MALDI-TOF MS is a good tool for the analysis of biopolymers because of its soft ionization, and it plays a central role in proteomic research. Because of their simplicity, speed, and accuracy, MS methods have been successfully applied to biomarker discovery and the characterization of various bacterial agents. Although DNA sequencing is the current standard for molecular characterization of bacteria, molecular methods cannot be easily applied for rapid classification and identification.Our aim was to examine whether a proteomic approach using MALDI-TOF MS was effective for rapid bacterial identification, especially of two of the subspecies of L. lactis.     

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).     

African 1, an Epidemiologically Important Clonal Complex of Mycobacterium bovis Dominant in Mali,Nigeria, Cameroon,and Chad

We have identified a clonal complex of Mycobacterium bovis present at high frequency in cattle in population samples from several sub-Saharan west-central African countries. This closely related group of bacteria is defined by a specific chromosomal deletion (RDAf1) and can be identified by the absence of spacer 30 in the standard spoligotype typing scheme. We have named this group of strains the African 1 (Af1) clonal complex and have defined the spoligotype signature of this clonal complex as being the same as the M. bovis BCG vaccine strain but with the deletion of spacer 30. Strains of the Af1 clonal complex were found at high frequency in population samples of M. bovis from cattle in Mali, Cameroon, Nigeria, and Chad, and using a combination of variable-number tandem repeat typing and spoligotyping, we show that the population of M. bovis in each of these countries is distinct, suggesting that the recent mixing of strains between countries is not common in this area of Africa. Strains with the Af1-specific deletion (RDAf1) were not identified in M. bovis isolates from Algeria, Burundi, Ethiopia, Madagascar, Mozambique, South Africa, Tanzania, and Uganda. Furthermore, the spoligotype signature of the Af1 clonal complex has not been identified in population samples of bovine tuberculosis from Europe, Iran, and South America. These observations suggest that the Af1 clonal complex is geographically localized, albeit to several African countries, and we suggest that the dominance of the clonal complex in this region is the result of an original introduction into cows naïve to bovine tuberculosis.Mycobacterium bovis causes bovine tuberculosis (TB), an important disease of domesticated cattle that has a major economic and health impact throughout the world (61, 64, 65). The pathogen is a member of the Mycobacterium tuberculosis complex, which includes many species and subspecies that cause similar pathologies in a variety of mammalian hosts. The most notable member of the complex is M. tuberculosis, the most important bacterial pathogen of humans. In contrast to M. tuberculosis, which is largely host restricted to humans, M. bovis is primarily maintained in bovids, in particular, domesticated cattle, although the pathogen can frequently be recovered from other mammals, including humans (61). Bovine TB is found in cattle throughout the world and has been reported on every continent where cattle are farmed (3).Bovine TB has been reduced or eliminated from domestic cattle in many developed countries by the application of a test-and-cull policy that removes infected cattle (3, 8, 16, 17, 61, 64, 65). However, in Africa, although bovine TB is known to be common in both cattle and wildlife, control policies have not been enforced in many countries due to cost implications, lack of capacity, and infrastructure limitations (8, 16, 17, 57). In 1998, Cosivi et al. reported of bovine TB, “Of all nations in Africa, only seven apply disease control measures as part of a test-and-slaughter policy and consider bovine TB a notifiable disease; the remaining 48 control the disease inadequately or not at all” (16). In the intervening years, the situation is not thought to have improved (8); however, preliminary surveys of bovine TB have been carried out in some African countries (4, 7, 12, 37, 44, 49, 53, 54, 56).The most common epidemiological molecular-typing method applied to strains of M. bovis is spoligotyping. This method identifies polymorphism in the presence of spacer units in the direct-repeat (DR) region in strains of the M. tuberculosis complex (36, 67). The DR is composed of multiple, virtually identical 36-bp regions interspersed with unique DNA spacer sequences of similar size (direct variant repeat [DVR] units). Spacer sequences are unique to the DR region, and copies are not located elsewhere in the chromosome (68). The DR region may contain over 60 DVR units; however, 43 of the spacer units were selected from the spacer sequences of the M. tuberculosis reference strain H37Rv and M. bovis BCG strain P3 and are used in the standard application of spoligotyping to strains of the M. tuberculosis complex (29, 36). The DR region is polymorphic because of the loss (deletion) of single or multiple spacers, and each spoligotype pattern from strains of M. bovis is given an identifier (http://www.Mbovis.org).Several studies of the DR regions in closely related strains of M. tuberculosis have concluded that the evolutionary trend for this region is primarily loss of single DVRs or multiple contiguous DVRs (22, 29, 68); duplication of DVR units or point mutations in spacer sequences were found to be rare. The loss of discrete units observed by Groenen et al. (29) led them to suggest that the mechanism for spacer loss was homologous recombination between repeat units. However, a study by Warren et al. (69) suggested that for strains of M. tuberculosis, insertion of IS6110 sequences into the DR region and recombination between adjacent IS6110 elements were more important mechanisms for the loss of spacer units.The population structure of the M. tuberculosis group of organisms is apparently highly clonal, without any transfer and recombination of chromosomal sequences between strains (15, 30, 60, 61). In a strictly clonal population, the loss by deletion of unique chromosomal DNA cannot be replaced by recombination from another strain, and the deleted region will act as a molecular marker for the strain and all its descendants. Deletions of specific chromosomal regions (regions of difference [RDs] or large sequence polymorphisms) have been very successful at identifying phylogenetic relationships in the M. tuberculosis complex (11, 25, 26, 35, 48, 50, 61, 62, 66). However, because the loss of spoligotype spacer sequences is so frequent, identical spoligotype patterns can occur independently in unrelated lineages (homoplasy), and therefore, the deletion of spoligotype spacers may be an unreliable indicator of phylogenetic relationship (61, 69).In samples of M. bovis strains from Cameroon, Nigeria, Chad, and Mali, spoligotyping was used to show that many of the strains had similar spoligotype patterns that lacked spacer 30, and it has been suggested that strains from these four countries are phylogenetically related (12, 18, 49, 53). We have extended the previous observations of spoligotype similarities between strains from these countries and confirmed the existence of a unique clonal complex of M. bovis, all descended from a single strain in which a specific deletion of chromosomal DNA occurred. We have named this clonal complex of M. bovis strains African 1 (Af1), and we show that this clonal complex is dominant in these four west-central African countries but rare in eastern and southern Africa. Extended genotyping, using variable-number tandem repeats (VNTR), of strains with the most common spoligotype patterns suggests that each of these four west-central African countries has a unique population structure. Evolutionary scenarios that may have led to the present day distribution of the Af1 clonal complex are discussed.     

Broad Conservation of Milk Utilization Genes in Bifidobacterium longum subsp. infantis as Revealed by Comparative Genomic Hybridization

Human milk oligosaccharides (HMOs) are the third-largest solid component of milk. Their structural complexity renders them nondigestible to the host but liable to hydrolytic enzymes of the infant colonic microbiota. Bifidobacteria and, frequently, Bifidobacterium longum strains predominate the colonic microbiota of exclusively breast-fed infants. Among the three recognized subspecies of B. longum, B. longum subsp. infantis achieves high levels of cell growth on HMOs and is associated with early colonization of the infant gut. The B. longum subsp. infantis ATCC 15697 genome features five distinct gene clusters with the predicted capacity to bind, cleave, and import milk oligosaccharides. Comparative genomic hybridizations (CGHs) were used to associate genotypic biomarkers among 15 B. longum strains exhibiting various HMO utilization phenotypes and host associations. Multilocus sequence typing provided taxonomic subspecies designations and grouped the strains between B. longum subsp. infantis and B. longum subsp. longum. CGH analysis determined that HMO utilization gene regions are exclusively conserved across all B. longum subsp. infantis strains capable of growth on HMOs and have diverged in B. longum subsp. longum strains that cannot grow on HMOs. These regions contain fucosidases, sialidases, glycosyl hydrolases, ABC transporters, and family 1 solute binding proteins and are likely needed for efficient metabolism of HMOs. Urea metabolism genes and their activity were exclusively conserved in B. longum subsp. infantis. These results imply that the B. longum has at least two distinct subspecies: B. longum subsp. infantis, specialized to utilize milk carbon, and B. longum subsp. longum, specialized for plant-derived carbon metabolism.The newborn infant not only tolerates but requires colonization by commensal microbes for its own development and health (3). The relevance of the gut microbiome in health and disease is reflected by its influence in a number of important physiological processes, from physical maturation of the developing immune system (28) to the altered energy homeostasis associated with obesity (51, 52).Human milk provides all the nutrients needed to satisfy the neonate energy expenditure and a cadre of molecules with nonnutritional but biologically relevant functions (6). Neonatal health is likely dependent on the timely and complex interactions among bioactive components in human milk, the mucosal immune system, and specialized gut microbial communities (30). Human milk contains complex prebiotic oligosaccharides that stimulated the growth of select bifidobacteria (24, 25) and are believed to modulate mucosal immunity and protect the newborn against pathogens (23, 33, 41). These complex oligosaccharides, which are abundantly present in human milk (their structures are reviewed by Ninonuevo et al. [31] and LoCascio et al. [24]), arrive intact in the infant colon (5) and modulate the composition of neonatal gastrointestinal (GI) microbial communities.Bifidobacteria and, frequently, Bifidobacterium longum strains often predominate the colonic microbiota of exclusively breast-fed infants (10, 11). Among the three subspecies of B. longum, only B. longum subsp. infantis grows robustly on human milk oligosaccharides (HMOs) (24, 25). The availability of the complete genome sequences of B. longum subsp. infantis ATCC 15697 (40) and two other B. longum subsp. longum strains (22, 39) made possible the analysis of whole-genome diversity across the B. longum species. Analysis of the B. longum subsp. infantis ATCC 15697 genome has identified regions predicted to enable the metabolism of HMOs (40); however, their distribution across the B. longum spp. remains unknown. We predict that these regions are exclusively conserved in B. longum strains adapted to colonization of the infant gut microbiome and are therefore capable of robust growth on HMOs. In this work, whole-genome microarray comparisons (comparative genomic hybridizations [CGHs]) were used to associate genotypic biomarkers among 15 B. longum strains exhibiting various HMO utilization phenotypes and host associations.     

Bile-Inducible Efflux Transporter from Bifidobacterium longum NCC2705, Conferring Bile Resistance

Bifidobacteria are normal inhabitants of the human gut. Some strains of this genus are considered health promoting or probiotic, being included in numerous food products. In order to exert their health benefits, these bacteria must overcome biological barriers, including bile salts, to colonize and survive in specific parts of the intestinal tract. The role of multidrug resistance (MDR) transporters in bile resistance of probiotic bacteria and the effect of bile on probiotic gene expression are not fully understood. In the present study, the effect of subinhibitory concentrations of bile on the expression levels of predicted MDR genes from three different bifidobacterial strains, belonging to Bifidobacterium longum subsp. longum, Bifidobacterium breve, and Bifidobacterium animalis subsp. lactis, was tested. In this way, two putative MDR genes whose expression was induced by bile, BL0920 from B. longum and its homolog, Bbr0838, from B. breve, were identified. The expression of the BL0920 gene in Escherichia coli was shown to confer resistance to bile, likely to be mediated by active efflux from the cells. To the best of our knowledge, this represents the first identified bifidobacterial bile efflux pump whose expression is induced by bile.In the gut, commensal bacteria, including those that have health-promoting or probiotic activity, are challenged by the presence of several toxic compounds of intestinal origin, such as bile salts. Bile is secreted into the duodenum to an estimated concentration of 0.2 to 2% for a given bile salt (14). Bile salts are detergent-like compounds with strong antimicrobial activity (2). Therefore, intestinal microorganisms have developed strategies to tolerate physiological concentrations of bile salts during passage through, or colonization of, the gut.Bifidobacteria are common inhabitants of the human gastrointestinal tract (GIT) (19, 39). Some strains of this genus are known to have probiotic activity and are used as functional ingredients in food products around the world (40). The health-promoting effects attributed to these microorganisms are numerous (20, 42). To exert beneficial actions, these bacteria must overcome biological barriers including acid in the stomach and bile in the intestine in order to, at least temporarily, colonize specific parts of the GIT. Thus, understanding the mechanisms of resistance of a given commensal or probiotic microorganism to toxic substances, such as bile salts, is important in the context of its physiology in the GIT. Although bifidobacterial bile tolerance mechanisms are currently poorly understood, bifidobacteria with increased resistance to bile salts have been obtained by progressive adaptation to gradually increasing concentrations of these compounds (27, 29). Through the analysis of such bile-tolerant derivatives, it was shown that the acquisition of bile resistance coincides with changes in membrane protein profiles (27) and carbohydrate metabolism (35, 38), while also conferring cross-resistance to other environmental stresses (29, 36). This indicates that the bifidobacterial response to bile entails a complex cellular activation/repression process, which impacts on general metabolic pathways (37).Specific bile resistance mechanisms have been described in intestinal bacteria, with bile efflux and bile salt hydrolysis being the most prevalent (31). In this respect, multidrug resistance (MDR) transporters seem to play a crucial role in conferring a bile resistance phenotype. MDR proteins are present in all organisms and frequently confer resistance against several structurally unrelated toxic compounds (31). Research on these transporters has mainly been focused on their role in antibiotic resistance. However, given their ubiquitous presence this does not seem to be their primary function. In fact, recent studies support a role for various MDR transporters in allowing microorganisms to survive, establish, and persist in their (human) host (31).It has been shown elsewhere that MDR proteins confer resistance to bile in different enteric bacteria (32). Bile was found to upregulate the expression of the multidrug efflux system cmeABC in Campylobacter jejuni (22), and inhibition of this pump was found to reduce the colonization ability of the microorganism by reducing bile resistance (21). In vitro and in vivo induction of acrAB expression by bile was observed in Vibrio cholerae (6), and also other MDR proteins appear to be induced in this microorganism (5). In the intestinal bacterium Bacteroides fragilis, the expression of different MDR pumps was also found to be upregulated by bile (34).Several MDR systems have been identified in gram-positive bacteria (including probiotic bacteria) (8, 28), but their role in conferring resistance to intestinal toxic compounds, such as bile salts, and the effect of bile on gene expression have not received much scientific attention. Transporters able to extrude bile salts have been found in gram-positive bacteria such as Lactococcus lactis (44, 45) and Lactobacillus johnsonii (7). In Lactobacillus plantarum a membrane protein whose expression is induced by bile, both in vitro and in vivo, was previously identified (3). Proteins conferring resistance to bile, and whose expression is induced by it, have also been reported in other lactobacilli (30, 43). Just a couple of studies have been published on MDR transporters in bifidobacteria. Margolles and coworkers identified and characterized two MDR transporters from Bifidobacterium breve, BbmAB and BbmR, conferring resistance to antimicrobials (25, 26). In Bifidobacterium longum an MDR transporter, Ctr, was found to export cholate from the cell, conferring resistance to this compound when cloned in a heterologous bacterial host (33). However, there is still a knowledge gap with regard to possible effects of bile on MDR gene expression and the potential role of MDR transporters in bile resistance in bifidobacteria.In the present study, the effect of subinhibitory concentrations of bile on the expression levels of genes encoding bifidobacterial MDR protein homologs was tested. For this purpose, known or putative MDR genes were selected from the genomes of different Bifidobacterium strains belonging to the species B. longum, B. breve, and Bifidobacterium animalis. A putative MDR-encoding gene, present as a homolog in both B. breve and B. longum and whose expression was strongly induced by bile, was identified, and the B. longum gene was then characterized.     

Multiplex PCR for Detection of Botulinum Neurotoxin-Producing Clostridia in Clinical,Food, and Environmental Samples

Botulinum neurotoxin (BoNT), the most toxic substance known, is produced by the spore-forming bacterium Clostridium botulinum and, in rare cases, also by some strains of Clostridium butyricum and Clostridium baratii. The standard procedure for definitive detection of BoNT-producing clostridia is a culture method combined with neurotoxin detection using a standard mouse bioassay (SMB). The SMB is highly sensitive and specific, but it is expensive and time-consuming and there are ethical concerns due to use of laboratory animals. PCR provides a rapid alternative for initial screening for BoNT-producing clostridia. In this study, a previously described multiplex PCR assay was modified to detect all type A, B, E, and F neurotoxin genes in isolated strains and in clinical, food, environmental samples. This assay includes an internal amplification control. The effectiveness of the multiplex PCR method for detecting clostridia possessing type A, B, E, and F neurotoxin genes was evaluated by direct comparison with the SMB. This method showed 100% inclusivity and 100% exclusivity when 182 BoNT-producing clostridia and 21 other bacterial strains were used. The relative accuracy of the multiplex PCR and SMB was evaluated using 532 clinical, food, and environmental samples and was estimated to be 99.2%. The multiplex PCR was also used to investigate 110 freshly collected food and environmental samples, and 4 of the 110 samples (3.6%) were positive for BoNT-encoding genes.Botulinum neurotoxins (BoNTs) are the most toxic agents known, and as little as 30 ng neurotoxin is potentially lethal to humans (36). These toxins are responsible for botulism, a disease characterized by flaccid paralysis. Seven antigenically distinct BoNTs are known (types A to G), and BoNT types A, B, E, and F are the principal types associated with human botulism (37). Significant sequence diversity and antigenically variable subtypes have recently been reported for the type A, B, and E neurotoxin genes (14, 22, 23, 42).Apart from the species Clostridium botulinum, which itself consists of four phylogenetically distinct groups of organisms, some strains of other clostridia, namely Clostridium butyricum and Clostridium baratii, are also known to produce BoNTs (2, 4, 7, 13, 20, 26, 34, 44). Also, strains that produce two toxins and strains carrying silent toxin genes have been reported (8, 22, 24, 39). Due to the great physiological variation of the BoNT-producing clostridia, their isolation and identification cannot depend solely on biochemical characteristics (32). Indeed, the standard culture methods take into consideration only C. botulinum and not C. baratii and C. butyricum, and identification and confirmation require detection of BoNT by a standard mouse bioassay (SMB) (12). The SMB is highly sensitive and specific but also expensive, time-consuming, and undesirable because of the use of experimental animals. Detection of neurotoxin gene fragments by PCR is a rapid alternative method for detection and typing of BoNT-producing clostridia (3). Different PCR methods have been described for detecting neurotoxin type A-, B-, E-, and F-producing clostridia (9, 15-18, 21, 40, 41).A previously described multiplex PCR method able to simultaneously detect type A, B, E, and F neurotoxin genes is a useful tool for rapid detection of the BoNT-producing clostridia (31). While this method generally has a high level of inclusivity for detection of type B, E, and F neurotoxin genes, limitations for detection of the recently described subtype A2, A3, and A4 strains have been identified (6, 28). To increase the efficiency of this multiplex PCR method, new primers were designed to detect genes for all identified type A neurotoxin subtypes (19). Additionally, an internal amplification control (IAC) was added according to ISO 22174/2005. The specificity and selectivity of this multiplex PCR method were evaluated in comparison with an SMB (12) using target and nontarget strains, and the robustness was assessed using clinical, food, and environmental samples. Moreover, to evaluate the applicability of this multiplex PCR method, a survey with food and environmental samples was performed in a German food control laboratory.     

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.