text:
New Research In
Physical Sciences
Social Sciences
Featured Portals
Articles by Topic
Biological Sciences
Featured Portals
Articles by Topic
- Agricultural Sciences
- Anthropology
- Applied Biological Sciences
- Biochemistry
- Biophysics and Computational Biology
- Cell Biology
- Developmental Biology
- Ecology
- Environmental Sciences
- Evolution
- Genetics
- Immunology and Inflammation
- Medical Sciences
- Microbiology
- Neuroscience
- Pharmacology
- Physiology
- Plant Biology
- Population Biology
- Psychological and Cognitive Sciences
- Sustainability Science
- Systems Biology
Evolutionary genomics of Staphylococcus aureus: Insights into the origin of methicillin-resistant strains and the toxic shock syndrome epidemic
Edited by John B. Robbins, National Institutes of Health, Bethesda, MD, and approved May 30, 2001 (received for review February 27, 2001)
An emerging theme in medical microbiology is that extensive variation exists in gene content among strains of many pathogenic bacterial species. However, this topic has not been investigated on a genome scale with strains recovered from patients with well-defined clinical conditions. Staphylococcus aureus is a major human pathogen and also causes economically important infections in cows and sheep. A DNA microarray representing >90% of the S. aureus genome was used to characterize genomic diversity, evolutionary relationships, and virulence gene distribution among 36 strains of divergent clonal lineages, including methicillin-resistant strains and organisms causing toxic shock syndrome. Genetic variation in S. aureus is very extensive, with ≈22% of the genome comprised of dispensable genetic material. Eighteen large regions of difference were identified, and 10 of these regions have genes that encode putative virulence factors or proteins mediating antibiotic resistance. We find that lateral gene transfer has played a fundamental role in the evolution of S. aureus. The mec gene has been horizontally transferred into distinct S. aureus chromosomal backgrounds at least five times, demonstrating that methicillin-resistant strains have evolved multiple independent times, rather than from a single ancestral strain. This finding resolves a long-standing controversy in S. aureus research. The epidemic of toxic shock syndrome that occurred in the 1970s was caused by a change in the host environment, rather than rapid geographic dissemination of a new hypervirulent strain. DNA microarray analysis of large samples of clinically characterized strains provides broad insights into evolution, pathogenesis, and disease emergence.
Microbial pathogens have great versatility in their ability to colonize distinct ecological niches and hosts and cause infections at different anatomic sites. The molecular processes responsible for disease and host specificity are poorly understood but are presumed to be caused in part by differences in gene content and allelic variation between strains. Although analysis of variation on the basis of differences at a limited number of genetic loci has been useful for estimating population structure and evolution (1, 2), it is now possible to index diversity in overall chromosomal gene content in isolates of a bacterial species. For example, whole-genome DNA microarray analysis has been used to compare the genomes of 13 variants of the tuberculosis vaccine strain, B. bacillus Calmette–Guérin, and Mycobacterium tuberculosis H37Rv (3). The data provided new information about the evolution of bacillus Calmette–Guérin strains used globally for vaccinating humans against tuberculosis and, importantly, suggested rational approaches to the design of improved diagnostics and vaccines. Recently, analysis of 15 strains of Helicobacter pylori with a whole-genome microarray was used to identify chromosomal diversity and new candidate virulence genes (4).
Staphylococcus aureus is a serious human pathogen responsible for life-threatening septicemia, endocarditis, and toxic shock syndrome (TSS). It also causes economically important mastitis in cows and sheep. Multilocus enzyme electrophoresis (MLEE) has been used to estimate overall genetic relationships among S. aureus strains and establish a population genetic framework for analysis of pathogenesis, virulence gene distribution, and evolution (1). The data have shown that relatively few extant clones are responsible for the majority of infections, and some of the lineages are nonrandomly associated with distinct human infections and mammalian hosts. The observation that a limited number of lineages are responsible for a large proportion of S. aureus infections implies that interclone variance in relative virulence is large and raises the possibility that variation in genome content in natural populations plays an important role. To investigate the molecular population genetics of S. aureus on a genome scale and obtain information bearing on host and disease specificity, isolates representing the major lineages identified by MLEE were analyzed with a DNA microarray formulated on the basis of the genome of strain COL. Broad insights were obtained into the virulence, population genetics, and evolution of a critical human pathogen.
Materials and Methods
Bacterial Strains.
Thirty-six S. aureus strains isolated from different human disease types and bovine and ovine mastitis were studied (Table 1). The strains were selected to represent the most abundant lineages identified by a population genetic study of 2,077 S. aureus isolates from worldwide sources (1). The sample included 11 methicillin-resistant S. aureus (MRSA) strains from diverse phylogenetic lineages (5). In addition, 15 isolates of electrophoretic type (ET) 234 were studied, including 9 strains cultured from the urogenital tract of women with TSS, 2 MRSA isolates of unknown disease origin, 1 organism isolated from the nasopharynx of an asymptomatic individual, and 3 TSS toxin-1-producing strains isolated before the TSS epidemic in the 1970s. The bovine and ovine strains were selected to represent the most abundant S. aureus lineages associated with intramammary infection in diverse localities (1, 6, 7).
DNA Microarray Construction, Hybridization, and Data Analysis.
DNA microarray experiments were carried out as described (published as supplemental data on the PNAS web site, www.pnas.org).
Southern Hybridization and PCR Analysis.
To confirm the microarray data, Southern blotting and PCR analysis were performed as described in the supplemental data.
Overall Genome Diversity in S. aureus.
DNA microarray analysis revealed extensive genome diversity within the S. aureus species. All 36 strains analyzed shared 2,198 ORFs, equivalent to ≈78% of all ORFs (n = 2,817). A maximum of 332 (12%) ORFs were absent in any one strain compared with the reference strain COL (Table 2, which is published as supplemental data on the PNAS web site, www.pnas.org). Many of these nonessential genes (n = 294) were located contiguously in regions 3–50 kb in size in strain COL. Eight of the regions contain genes encoding integrases or transposases.
The gene content of S. aureus strain COL was very similar to strains of ET 93, especially strain MSA3410 (Fig. 1); only 22 ORFs were absent in MSA3410 compared with strain COL. Eighteen of the 22 ORFs were phage-associated and were located in a single chromosomal region designated RD14 (see below).
Large Chromosomal Regions of Difference (RD).
Seventeen chromosomal regions consisting of more than three contiguous ORFs were absent in one or more strains compared with the reference strain COL (Fig. 2). One chromosomal region (RD13) was not absent in entirety in any strain but varied considerably in gene content among the 36 strains examined. Each of these 18 regions will be referred to as a "RD," a term used in studies of genome variation in M. tuberculosis (8). The RDs were numbered arbitrarily, and the size of each region given below refers to the length of the chromosomal segment in strain COL. RD8 and RD13 were characterized by PCR size variation, and RD1 and RD2 had large differences in gene content among the 36 strains examined (Fig. 3).
RD1 (≈40 kb; 35 ORFs) has several noteworthy features, including a cluster of six genes encoding serine protease homologues with 49–93% identity to each other, a group of 8 genes encoding proteins involved in bacteriocin production and resistance, contiguous genes for leukotoxins D and E, and genes encoding proteins with 49% amino acid identity to restriction modification type Ic proteins made by Streptococcus thermophilus. The bacteriocin genes were present in 9 of 36 strains analyzed, and the genes for leukotoxins D and E were present in 17 strains. The entire serine protease gene cluster was absent in three strains. RD1 was highly variable in gene content among the 36 strains examined, with 5 major variants identified.
RD2 (≈50 kb; 50 ORFs) is the staphylococcal chromosomal cassette mec (SCCmec) and was present only in MRSA strains. There was considerable variation in the gene content of RD2 among the 11 MRSA strains (Fig. 3). However, all MRSA strains had 13 of the ORFs present in RD2 in strain COL, including the mecA gene encoding an altered penicillin-binding protein that confers resistance to methicillin and related antimicrobial agents, the mecRI gene encoding a signal transducer, a gene (gdp) encoding a glycerophosphoryl diester phosphodiesterase homologue, a transposase gene associated with the insertion sequence-like element IS431mec, a gene encoding a cassette chromosome recombinase homologue, and 8 genes that encode hypothetical proteins of unknown function. Each of the 11 MRSA strains had 14–50 of the ORFs present in RD2 in strain COL. Only one strain (MSA3410), an MRSA strain of ET 9 (1), had the same gene complement in RD2 as strain COL.
RD3 (≈4 kb; 5 ORFs) contains two genes encoding proteins with homology to transposases and two genes that encode proteins with no homology to known proteins. RD3 was present in 29 of 36 strains examined.
RD4 (≈5 kb; 4 ORFs) has a gene encoding a cobalt ABC transporter homologue. RD4 was present in 25 of the 36 strains analyzed.
RD5 (≈5 kb; 7 ORFs) has genes encoding a surface protein with homology to fibronectin-binding protein (FnbA) of S. aureus, two proteins with homology to the staphylococcal accessory regulator of virulence (SarA), and a protein with homology to a multidrug-resistance protein made by Pyrococcus abyssi. RD5 was present in 15 of the 36 strains.
RD6 (≈6 kb; 7 ORFs) has genes that encode proteins that lack significant homology to known proteins. Fifteen of the 36 strains examined had RD6.
RD7 (≈6 kb; 8 ORFs) has one gene encoding a protein with homology to a hypothetical protein encoded by S. thermophilus insertion element IS1191 and 7 genes that encode proteins of unknown function. Only 5 of 36 strains examined had RD7.
RD8 (≈11 kb; 12 ORFs) is highly variable in size and gene content among the 36 strains examined (Fig. 3). Depending on the strain, RD8 has genes encoding an integrase-like protein, a putative ferrichrome-binding protein (febp), and proteins with homology to leukotoxin subunits F and S. PCR analysis found that RD8 varied from 6 to 11 kb among the 36 strains examined.
RD9 (≈14 kb; 15 ORFs) is a putative transposable element. RD9 has a transposase gene and a gene encoding a protein with 40% identity to TrsG, the transfer complex protein of S. aureus plasmid pG01. The other 13 ORFs in RD9 encode hypothetical proteins with no significant identity to proteins of known function. RD9 was present in 21 of the 36 strains investigated. Interestingly, microarray and Southern blot analysis indicated that strain MSA1601 contains more than one copy of RD9. Moreover, PCR amplification of the putative insertion site of RD9 indicated that this element has more than one chromosomal integration site among the 36 strains examined (data not shown).
RD10 (≈6 kb; 4 ORFs) contains genes that encode a putative oligopeptide transporter complex. This region was present in 33 of the 36 strains studied.
RD11 (≈3 kb; 5 ORFs) contains genes encoding proteins that lack significant homology to known proteins. RD11 was present in 7 of the 36 strains examined.
RD12 (≈16 kb; 25 ORFs) is a putative pathogenicity island that has genes for staphylococcal enterotoxins b (seb), k (sek), a novel enterotoxin; an integrase; and β-lactamase (bla). RD12 was present in only 2 of the 36 strains studied. However, 4–21 of the RD12 ORFs were present somewhere in the chromosome in each of the 36 strains, indicating that related elements may exist in some strains.
RD13 (≈12 kb; 14 ORFs) contains 7 genes that encode proteins with homology to recently described staphylococcal exotoxin-like proteins (9), and 2 genes encoding a putative restriction and modification system. There was considerable variation among strains in the structure of RD13. Different combinations of the 7 exotoxin genes were identified (Fig. 3), and long-range PCR analysis found that RD13 varied in size from 12 to 17 kb among the 36 strains analyzed.
RD14 (≈44 kb; 72 ORFs) may represent a previously undescribed bacteriophage. PCR analysis of the putative integration site of RD14 confirmed that all test strains lacked the element at this chromosomal location. However, each of the 36 strains analyzed contained 8–63 of the ORFs present in RD14 in strain COL, indicating that related elements may exist elsewhere in the genome of some strains.
RD15 (≈4 kb; 4 genes) consists of 4 genes (cap5g, cap5h, cap5i, and cap5j) involved in synthesis of serotype 5 capsule. RD15 was present in 9 of 36 strains investigated, suggesting that these organisms produce serotype 5 capsule.
RD16 (≈12 kb; 12 ORFs) has a gene encoding a protein with 56% identity to a 67-kDa antigen made by Streptococcus pyogenes that crossreacts with myosin. RD16 was present in 35 of the 36 strains studied.
RD17 (≈10 kb; 10 ORFs), present in 28 of the 36 strains, contains genes that encode hypothetical proteins of unknown function.
RD18 (≈5 kb; 5 ORFs) is a plasmid related to pT181 of S. aureus. RD18 has genes that encode a tetracycline-resistance protein, a recombination protein, and a replication initiation protein. Microarray data indicated that RD18 was present as a multicopy plasmid in 3 of 36 strains examined.
Southern Hybridization and PCR Analysis.
All microarray data were confirmed by Southern blot analysis with one or more probes specific for ORFs in each RD. Two RDs with large size variation were identified by PCR among the 36 strains studied. Specifically, RD8 had size variants of 6–11 kb, and RD13 varied from 12 to 17 kb (data not shown).
Overall Chromosomal Relatedness of S. aureus Strains.
We used hierarchical clustering (4) to investigate chromosomal relationships among the 36 S. aureus strains on the basis of their gene content. The strains were clustered into distinct groups that correlate strongly with estimates of chromosomal relationships revealed by MLEE (1) (Fig. 3). Virtually all strains of closely related ETs identified by MLEE were grouped together (Fig. 1).
Diversity of S. aureus Strains Containing the mec Element.
The mec element (RD2) was present in all 11 MRSA strains as assessed by microarray and Southern blot analysis, although extensive diversity was present in the gene content of this region. The mec gene was present in at least five distinct S. aureus lineages that are highly differentiated in overall chromosomal gene content (Fig. 1).
Diversity of Isolates of Multilocus Electrophoretic Type 234, the Predominant Clone Causing TSS.
A previous study of 315 S. aureus isolates recovered from the urogenital tract of women with TSS by MLEE provided evidence that 90% of the organisms were clonally related by descent (10). To further assess the possibility of a close genetic relationship among urogenital TSS isolates, 15 isolates of ET 234 (cluster H1) (1) cultured from diverse geographic localities were studied. The isolates were similar in gene content, indicating close genetic affiliation of all 15 strains (Fig. 1). However, the RD content varied among the strains. For example, TSS isolate MSA2335 lacked RD1, and some of the 15 strains differed in the exotoxin gene complement in RD13. In addition, MSA1832, MSA2885, and MSA3412 lacked RD9, and 8 of the 15 strains contained 20–26 ORFs present in a contiguous region of RD14 in strain COL. These RD14 ORFs were absent in the other strains of ET 234 examined. Analysis of ORFs not contained within RDs revealed that a total of 208 were variably absent among the 15 ET 234 S. aureus isolates examined (supplemental Table 2). Hence, although ET 234 organisms were closely related, there was considerable heterogeneity in genome composition, indicating that the organisms have not evolved very recently from a common ancestor.
Diversity of Bovine and Ovine S. aureus Strains.
Molecular population genetic analysis has suggested the existence of bovine- and ovine-specialist strains of S. aureus (1, 6). To investigate genetic diversity among S. aureus isolates cultured from cows and determine whether these organisms had a distinct genome composition, strains were studied that represent the two most abundant lineages causing bovine mastitis (1, 6). Bovine strains RF122 (ET cluster F10) and MSA961 (ET cluster F4) were assigned to different clonal lineages by MLEE analysis (1). The two ovine mastitis strains studied (MSA535 and MSA551) were selected to represent the major clonal lineage (ET cluster F1) causing this disease (1). We found that the ovine strains and bovine strain RF122 were genetically allied (Fig. 1). In addition, bovine strain MSA961 was similar in gene content to human strain MSA817. Together, these data strongly support MLEE results, indicating that many bovine isolates are more closely allied to human isolates than to other bovine isolates (1, 6).
Species Genome Diversity.
We identified extensive genetic variation among strains representing the most abundant disease-causing clonal lineages of S. aureus (1), with as many as 332 ORFs absent in one strain compared with the reference strain COL. We also discovered that 2,198 (78%) of the 2,817 ORFs represented on the DNA microarray were present in all S. aureus strains studied, a result suggesting that these genes are essential for S. aureus cell maintenance. Some of these 2,198 genes encode known virulence factors, suggesting a critical role for these proteins in staphylococcal survival and pathogenesis. Conversely, ≈22% of S. aureus genes represent nonessential genetic information that is strain-specific. Some of these genes encode factors that facilitate colonization of specialized host or environmental niches. We note that a recent comparative genomics study of 15 H. pylori strains also reported that 22% of genes were not essential for core cell functions (4). In Escherichia coli, as many as 25% of genes have been reported to be strain specific (11). Taken together, the data suggest an emerging theme that a very large number of genes in the chromosome of several common human bacterial pathogens are devoted to contingency functions.
Importance of Horizontal Gene Transfer in the Evolution of S. aureus.
Many of the strain-variable ORFs identified in this study form 18 discrete RDs, including pathogenicity island-, phage-, plasmid-, transposon-, and insertion sequence-related elements. Only RD2 and RD15 have been previously characterized (12, 13). Our analysis found that several RDs have extensive variation in gene content and size. Of particular note are RD1 and RD13, which contain many putative virulence factor genes and genes for transposases. In this regard, a recently described operon containing enterotoxin genes (14) is present in the RD1 homologue of MRSA strain 252 (http://www.sanger.ac.uk). In addition, a pathogenicity island (SaPIbov) described in bovine S. aureus strain RF122 is present in the chromosome of RF122, adjacent to RD13 (15). It is clear that multiple deletion, integration, and recombination events have contributed to the variation in the RDs and the genome overall. At least 19 genes that would encode proteins with homology to mediators of lateral gene transfer (such as integrases and transposases) are present in strain COL; 14 of these 19 genes were located in RDs. Moreover, most RDs were widely distributed among strains of divergent clonal lineages that have not shared a recent ancestor. Taken together, it is clear that horizontal gene transfer has played a fundamental role in the evolution of pathogenic S. aureus, especially by assortive recombination of RDs containing virulence and antibiotic-resistance genes. It is also evident that the overall genomic relationships revealed by MLEE and whole-genome microarray analysis are largely cognate, indicating the presence of phylogenetic signal in the data. This means that chromosomal relationships inferred by these techniques are useful for study of many biomedically relevant characteristics.
Chromosomal Relatedness of S. aureus Strains from Different Host Species.
Previous studies have indicated that successful cross-species transfer of pathogenic S. aureus is infrequent (6). We found that a strain representing the most abundant clonal lineage causing bovine mastitis (RF122) was closely related to strains of the predominant ovine mastitis clonal type. This result suggests that a similar gene complement may be required to cause bovine and ovine intramammary infection. However, DNA microarray analysis allows comparison only of the gene content of strains to genes represented on the array. Hence, it is unlikely that we identified all strain-specific genes present in the strains examined. Identification of genes present in the 36 test strains but absent in strain COL is likely to provide new insight into the molecular mechanisms of pathogenic clone adaptation to specialized niches such as the mammary gland.
Evolution of MRSA Strains.
MRSA strains were first reported in 1961, soon after methicillin entered clinical use. Outbreaks of infections caused by MRSA were reported soon thereafter in Europe, and by the mid-1970s, MRSA had become a significant problem in the United States. MRSA are critical public health threats because they cause hospital-acquired infections that can be difficult and expensive to treat. Many MRSA strains are susceptible only to vancomycin, and this antibiotic is used extensively to treat patients infected with these organisms. Hence, there is also concern that MRSA may serve as a reservoir of organisms that may give rise to vancomycin-resistant strains that could not be killed with available antibiotics.
Two very different hypotheses have been put forth to explain the origin of MRSA strains (5, 16). The association of the mec gene with genetically diverse lineages of S. aureus, together with data indicating that the mec gene was horizontally transferable in the laboratory, led to the hypothesis that MRSA strains had evolved many independent times by lateral gene transfer of the mec element into phylogenetically distinct methicillin-susceptible precursor cell lines (5). In contrast, data obtained from study of MRSA by restriction fragment length polymorphism analysis with probes for mecA and Tn554 were interpreted to mean that MRSA organisms evolved from a susceptible clone that acquired the mec element and subsequently generated substantial chromosomal diversity (16). We discovered that MRSA strains were assigned to at least five distinct chromosomal genotypic groups that, relative to one another, are highly divergent, in some cases differing by greater than several hundred genes. Hence, the only reasonable interpretation of the data is that the MRSA strains have arisen multiple independent times by lateral transfer of the mec element into methicillin-susceptible precursors. Taken together, the microarray data unambiguously settle a longstanding controversy in the S. aureus field.
Origin of the TSS Epidemic.
Ninety percent of S. aureus strains recovered from women with urogenital-associated TSS are a distinct multilocus enzyme genotype, designated ET 234 (1, 10). Several lines of evidence (summarized in refs. 10 and 17) led to the hypothesis that the epidemic of TSS cases was caused by modification of the host population. However, the alternative hypothesis—an episode of periodic selection involving a fitness mutation occurring in a cell representing ET 234—could not be ruled out. Although the DNA microarray data confirmed that ET 234 organisms are genetically related and have shared a common ancestor, clearly they are not genetically identical, and the last ancestor has not been very recent in evolutionary time. Thus, the data indicate that the epidemic was caused by a change in the host environment (preadapted clone hypothesis) (10) and rule out the alternative explanation that rapid intercontinental dissemination of a new hypervirulent clone of S. aureus was responsible.
Concluding Comment.
Our studies provide an overview of genome diversity present in natural populations of S. aureus, an important human and animal pathogen worldwide. Genome-scale DNA microarray analysis contributed improved understanding about the evolution and virulence gene distribution in this bacterium. Importantly, the data resolved the controversy pertaining to the evolutionary origin of MRSA strains and strongly supported the hypothesis that the TSS epidemic was caused by a change in the host cervicovaginal environment. Rapid indexing of genome-scale variation present in large samples of microbial isolates that are well characterized clinically will provide many new insights into pathogen virulence, host and disease specificity, and disease emergence and reemergence.
We thank the laboratory of S. Falkow for assistance with microarray procedures, and A. Elias, G. Somerville, and R. Belland for critical review of the manuscript.
↵‡ To whom reprint requests should be addressed at: Laboratory of Human Bacterial Pathogenesis, Rocky Mountain Laboratories, National Institute of Allergy and Infectious Diseases, National Institutes of Health, 903 South Fourth Street, Hamilton, MT 59840. E-mail: jmusser{at}niaid.nih.gov.
This paper was submitted directly (Track II) to the PNAS office.
- electrophoretic type;
- multilocus enzyme electrophoresis;
- methicillin-resistant S. aureus;
- region of difference;
- toxic shock syndrome
- Received February 27, 2001.
- Copyright © 2001, The National Academy of Sciences
- ↵
- Novick R P
- Musser J M,
- Selander R K
- ↵
- Enright M C,
- Day N P,
- Davies C E,
- Peacock S J,
- Spratt B G
- ↵
- Behr M A,
- Wilson M A,
- Gill W P,
- Salamon H,
- Schoolnik G K,
- Rane S,
- Small P M
- ↵
- Salama N,
- Guillemin K,
- McDaniel T K,
- Sherlock G,
- Tompkins L,
- Falkow S
- ↵
- Musser J M,
- Kapur V
- ↵
- Kapur V,
- Sischo W M,
- Greer R S,
- Whittam T S,
- Musser J M
- ↵
- ↵
- Mahairas G, G,
- Sabo P J,
- Hickey M J,
- Singh D C,
- Stover C K
- ↵
- Williams R J,
- Ward J M,
- Henderson B,
- Poole S,
- O'Hara B P,
- Wilson M,
- Nair S P
- ↵
- Musser J M,
- Schlievert P M,
- Chow A W,
- Ewan P,
- Kreiswirth B N,
- Rosdahl V T,
- Naidu A S,
- Witte W,
- Selander R K
- ↵
- Ochman H,
- Jones I B
- ↵
- Sau S,
- Bhasin N,
- Wann E R,
- Lee J C,
- Foster T J,
- Lee C Y
- ↵
- Ito T,
- Katayama Y,
- Hiramatsu K
- ↵
- Jarraud S,
- Peyrat M A,
- Lim A,
- Tristan A,
- Bes M,
- Mougel C,
- Etienne J,
- Vandenesch F,
- Bonneville M,
- Lina G
- ↵
- Fitzgerald J R,
- Monday S R,
- Foster T J,
- Bohach G A,
- Hartigan P J,
- Meaney W J,
- Smyth C J
- ↵
- Kreiswirth B,
- Kornblum J,
- Arbeit R D,
- Eisner W,
- Maslow J N,
- McGeer A,
- Low D E,
- Novick R P
- ↵
- Krause R M
- Musser J M,
- Krause R M
- ↵
- Shafer W M,
- Iandolo J J


text:
I'm a single parent of an autistic child, and in the early stages of setting up my own small business. Goes without saying: money is super tight. Yet my wardrobe is stuffed full of beautiful dresses, fluffy petticoats, and shoes (glorious shoes!), and I rarely step out without a shiny red lip.
So it goes without saying that I quite often receive frankly intrusive questions about how I manage to afford my wardrobe. If I dare to mention that a new dress has just arrived in the mail, I'll quite often be met with an eye-roll and rudely snapped "Aren't you supposed to be an impoverished single mother?"
As someone once said to me: people always presume poor people shouldn't have nice things.
Darlings. This simply isn't the case. We all deserve a little glamour in our lives – especially if we're poor!
I'm poor. But my wardrobe is still fabulous!
Here's how I do it.
First: don't think you need to rush out and buy everything at once. It takes time, thought, and planning. Which is great, as it gives you more time to save money!
I started accumulating all these bits and pieces about four years ago when I started burlesque dancing (which also coincided with some fairly significant weight loss and the need for a new wardrobe). I was trying to redefine my style (which, at the time, was largely post-pregnancy-full-time-post-grad-tracksuits-I-hate-myself), and started by reading a lot of blogs and getting an idea for what I liked. I kept images of 'inspirational' outfits I liked. When I started to get a really clear idea of what I liked, I made my first purchase: a Vivien of Holloway dress and petticoat.
I was sensible about it. I bought the dress second hand on Ebay for less than half the cost price. I bought a middle-range petticoat to go with it. The dress was black with red polka dots, the petticoat was red. I already had a pair of black heels for burlesque dancing. It was a fabulous outfit, cost less than $100, and at first I kept it for special occasions.
Not long after, I bought my second VoH dress. I made sure it went with my petticoat (a red petticoat for a black/red dress and then a blue/white dress). I had a red belt that went with both. You see what I'm saying here? Then – and now – when I purchase anything, I make sure I already have coordinates in my wardrobe. It means I don't need to buy anything else just to go with it, and it doesn't languish unworn in my wardrobe because 'I have nothing to wear it with!'
In short: do your research, and don't impulse buy. Know what's in your wardrobe, and if you need to, keep a written list of what you need to round out what's already in there. If most of your wardrobe is in blues, reds, and blacks … red shoes are a solid investment.
Always keep an eye on Ebay. Living in Australia, the cost of returning clothes to the US is absolutely prohibitive, so anything I buy that doesn't fit goes straight to Ebay – and I'm not the only one who does this. PUG, Vivien of Holloway, Bernie Dexter, Trashy Diva, Stop Staring … there's a constant supply of new and near new dresses on Ebay. Sometimes you won't save much on the cost of the dress – but you can save a lot on postage! Also for Australian/NZ ladies, you're less likely to encounter the epic Australian mark-ups you'll find in Australian stores.
Join the Facebook page of your favourite stores, sign up for newsletters, and register for a member account online. Promotions and discounts will be posted in these places first. Some shops will offer you a discount on your birthday, others will offer a discount just for being a member (What Katie Did comes to mind). Always Google for a promo code before you go through the checkout! If you're not in a hurry for a particular outfit, it can be worthwhile to wait for a promo code to become available.
On the topic of Facebook: increasingly there are groups dedicated to swapping and selling pinup clothing and accessories. Some are brand specific, others are more general. This is a great way to swap a dress that didn't fit or hasn't been worn, or to outright sell an item.
The likes of Bernie Dexter, Pinup Couture, Trashy Diva, Hell Bunny and so on have dozens if not more retailers online, and the prices vary wildly (I've seen Bernie Dexter dresses as low as $98, as high as $190, though the RRP is $156). RESEARCH AND COMPARE PRICES! Shop around! Also: don't be scared to ask about price matching. You can also ask about discounted/free shipping if you're spending a significant amount of money, particularly with smaller businesses who really do rely on each and every sale – at Betty Le Bonbon, we offer discounts to repeat customers and anyone placing a particularly large order. It's our way of saying thanks to people who support us.
Speaking of shipping: In the case of some stores (such as Pinup Girl Clothing), it's cheapest to purchase directly from them. Postage is expensive, but if you spend over X amount, postage is free – for international customers, if you spend $300, you get free shipping. $300 is a lot of money – so I maintain a wish list, and I save my money until I have enough to place an order.
Don't buy cheap knockoffs that are poorly made and last only a few washes. There are a lot of them on Ebay, and while it can be tempting when you're poor, you'll just be left with a rag after a handful of washes. There's a couple of stores I can think of who seem to have zero shame in cashing in on well-established brands, branding and designs (Queen of Holloway is particularly notorious for ripping off other designers, and they've even pinched images of bloggers in the original brands and used the images to advertise their knock off clothing). The clothing is crap … and you're supporting intellectual theft and dickery. Please don't support dickery.
Bide your time! As I mentioned above, keep a written list of what you want to add to your wardrobe. When you have a reasonable list, see if you're able to get a few of those items from one shop to save on postage. Pinup Girl Clothing offer a range of items, not just clothing. Perhaps you're planning to buy a dress – why not see if they've got a black cardigan, red ballet flats and purple hairpin you've got on your list? You can also try ASOS, Modcloth, Unique Vintage or various other online stores who offer more than just clothing.
Where possible, support small business women! Not only are you supporting an independent business, but you can build up a rapport with the owner, and there's a good chance she'll support you in return (helping you to source dresses in your size, offering payment plans, giving you a head's up on sales etc).
Finally: I'm absolutely ruthless when it comes to culling my wardrobe down. If I haven't worn an item in a year, it goes on Ebay. If something arrives and it doesn't fit, it also goes straight to Ebay – I don't shove it to the back of the wardrobe for 'when I lose weight', or hold onto it and hope one day it'll inexplicably look better when I wear it. I'm regularly going through my wardrobe and know which items are no longer working for me. When people ask me if I'm wearing a new dress and start carrying on about me being poor, what they're not realizing is that I probably sold another dress from my wardrobe in order to fund the new one.
What are your top tips for saving money when shopping for your wardrobe?
For more great posts by Jasmine, check out Betty Le Bonbon's Cheek Boutique's Blog! www.bettylebonbon.com

subset: commoncrawl


text:
Blue Season Bali | Scuba Diving in Bali | Company Overview
Blue Season Bali is the Bali scuba diving industry's premier dive center. Our services are structured around nine core service products ranging from Discover Scuba Diving experiences and PADI courses to scuba diving internships, technical diving instructor training programs and Eco Internships. If you want top quality scuba diving services in Bali, you have found your solution here at Bali's top-rated five-star PADI Career Development Center – Blue Season Bali.
Winner – Outstanding Dive Business Award, PADI Asia Pacific
Winner – Instructor Development Award, PADI Asia Pacific
Finalist – Project AWARE Environmental Award, PADI Asia Pacific
Please learn more about our Bali diving services below:
Bali Diving
Diving in Bali has so much to offer from Shipwrecks in Tulamben (North Bali diving) to Sunfish diving in Nusa Penida (South Bali diving). Dive Bali's Macro dive sites like PJ and Seraya to see Mimic Octopus, Harlequin Shrimps, Hairy Frogfish and other rare and unusual critter life. Bali diving is suitable for all levels of divers from complete beginner divers to experienced professional divers. We also offer technical diving day trips to explore the deep diving Bali has to offer.
Read more about our Bali Diving day trips .
Bali Dive Safaris
Dive Safari Bali - The best way to truly experience the different Bali diving locations is to take part in one of our Bali Dive Safaris . Our Bali dive safaris are multi-day dive programs designed to maximize your in-water time, minimize traveling time and allow you to experience more of the local Balinese culture. Our standard overnight dive safari's combine a host of different locations around Bali. If you want to dive it all, choose a Bali Dive Safari .
Read more about our Bali Dive Safaris .
Bali Sunfish Diving (Mola-mola)
Sunfish Bali - Blue Season Bali runs seasonal Mola Mola Bali Sunfish dive programs every year for those Bali divers wishing to get a chance to dive with the wonderful Bali Sunfish. Bali Mola Mola season gets underway from July and lasts until late October. Book your Bali Sunfish diving holiday early to avoid disappointment! Space on the Mola Mola Bali Sunfish diving trips are limited and our boats are regularly fully booked during the Sunfish diving season.
Read more about our Bali Sunfish Diving .
Blue Season Bali @ The Menjangan
Blue Season Bali is proud to announce the opening of our latest Bali Diving operation based in the Menjangan National Park in North West Bali. In conjunction with The Menjangan Hotel Blue Season Bali is operating a full service PADI 5 Star Dive facility, running daily dive programs to the Menjangan National park and beyond.
Read more about our Menjangan Diving Resort .
Blue Season Bali @ Zen Resort Bali
Blue Season Zen Dive is a Bali Dive Resort that will take you to another dimension. Experience world class Bali diving whilst enjoying an unforgettable dive holiday experience. Luxurious sea-view villas, delightful tropical gardens, holistic health and beauty treatments, integrated ayurveda, yoga, pranayama & meditation, will rejuvenate your mind, body and soul.
Read more about our Bali Diving Resort .
PADI Dive Course Bali
PADI Dive courses : Scuba diving lessons are required to make scuba diving in Bali safe and enjoyable. You can learn to dive in Bali and gain your PADI Diving license with Bali's most acclaimed five-star scuba diving center. Our PADI Dive courses start from the PADI Discover Scuba Diving program and go all the way to the PADI Divemaster and PADI Instructor Level courses. Our experienced educational team trains more scuba diving professionals than any other company in the region. Why not let them introduce you to the wonderful world of diving in Bali ?
Read more about our PADI Dive Courses in Bali .
PADI ECO Internships Bali
PADI ECO Internships Bali - Do you care about the environment? Are you interested in learning more about our ocean planet? Go Eco and join Niels, our in-house marine biologist, and the Blue Season Bali team with our weekly ECO Internships in Bali. Designed to give you the ultimate diving holiday whilst doing your bit for the underwater environment, these ECO Internships offer you, the ECO enthusiast, the scuba diving experience of a lifetime.
Read more about our PADI ECO Internships .
PADI Scuba Diving Internships Bali
PADI Diving Internships Bali - Change your life and enter into a professional scuba diving internship with Blue Season Bali. You can ditch the 9 to 5 and become a PADI Divemaster or PADI instructor. Learn the ropes alongside our Diving Internship team and find the ultimate job in paradise as a PADI Diving professional. We have divemaster internships and PADI instructor internships available, run exclusively by our full time PADI Course Directors and Master Instructors.
Read more about our Scuba Diving Internships .
PADI IDC Bali (PADI Instructor Courses Bali)
PADI Instructor Course (PADI IDC) – Enter the world of the PADI Professional and become a PADI Dive Instructor in Bali. Our PADI IDC courses allows you to turn the ocean into your office. Our Platinum PADI Course Director will guide you through the Instructor Development Course, and with our unsurpassed pass rate for PADI IDC Instructor Internships, you know you will have the best chance of success.
Read more about the PADI IDC in Bali .
Jalan Danau Tamblingan 59x, Sanur, Bali, Indonesia.
Telephone: +62 361 270852 / 282574
Fax: +62 361 270842
Email: firstname.lastname@example.com
Web: www.blueseasonbali.com


text:
New Research In
Physical Sciences
Social Sciences
Featured Portals
Articles by Topic
Biological Sciences
Featured Portals
Articles by Topic
- Agricultural Sciences
- Anthropology
- Applied Biological Sciences
- Biochemistry
- Biophysics and Computational Biology
- Cell Biology
- Developmental Biology
- Ecology
- Environmental Sciences
- Evolution
- Genetics
- Immunology and Inflammation
- Medical Sciences
- Microbiology
- Neuroscience
- Pharmacology
- Physiology
- Plant Biology
- Population Biology
- Psychological and Cognitive Sciences
- Sustainability Science
- Systems Biology
Gnotobiotic mouse model of phage–bacterial host dynamics in the human gut
Contributed by Jeffrey I. Gordon, October 22, 2013 (sent for review August 29, 2013)
A consortium of sequenced human gut bacteria was introduced into germ-free mice followed by a "staged" phage attack with virus-like particles purified from the fecal microbiota of five healthy adult humans. Unique phages were identified attacking microbiota members in nonsimultaneous fashion. Some host bacterial species acquired resistance to phage attack through ecological or epigenetic mechanisms. Changes in community structure observed after attack were transient. Spontaneous induction of prophages present in seven bacterial taxa was modest, occurring independently of the phage attack. Together, these results reveal a largely temperate phage–bacterial host dynamic and illustrate how gnotobiotic mouse models can help characterize ecological relationships in the gut by taking into account its most abundant but least understood component, viruses.
Bacterial viruses (phages) are the most abundant biological group on Earth and are more genetically diverse than their bacterial prey/hosts. To characterize their role as agents shaping gut microbial community structure, adult germ-free mice were colonized with a consortium of 15 sequenced human bacterial symbionts, 13 of which harbored one or more predicted prophages. One member, Bacteroides cellulosilyticus WH2, was represented by a library of isogenic transposon mutants that covered 90% of its genes. Once assembled, the community was subjected to a staged phage attack with a pool of live or heat-killed virus-like particles (VLPs) purified from the fecal microbiota of five healthy humans. Shotgun sequencing of DNA from the input pooled VLP preparation plus shotgun sequencing of gut microbiota samples and purified fecal VLPs from the gnotobiotic mice revealed a reproducible nonsimultaneous pattern of attack extending over a 25-d period that involved five phages, none described previously. This system allowed us to (i) correlate increases in specific phages present in the pooled VLPs with reductions in the representation of particular bacterial taxa, (ii) provide evidence that phage resistance occurred because of ecological or epigenetic factors, (iii) track the origin of each of the five phages among the five human donors plus the extent of their genome variation between and within recipient mice, and (iv) establish the dramatic in vivo fitness advantage that a locus within a B. cellulosilyticus prophage confers upon its host. Together, these results provide a defined community-wide view of phage–bacterial host dynamics in the gut.
The human gut is home to tens of trillions of microbial cells representing all three domains of life, although most are bacteria. These organisms collaborate and compete for functional niches and physical locations (habitats). Together, they form a continuously functioning microbial metabolic "organ." The microbial diversity, interpersonal variation, and dynamism of the human gut microbiota make the task of identifying the factors that define community configurations extremely challenging.
In some ecosystems, phages maintain high bacterial strain level diversity through lysis of their host strains (constant diversity dynamics model; refs 1, 2). The resulting emptied niche is filled with either an evolved resistant bacterial strain or a taxonomically closely related bacterial species. These dynamics have been observed in open marine environments (1). In contrast, a recent study of 37 healthy adults indicated that a person's fecal microbiota was remarkably stable, with 60% of bacterial strains retained over the course of 5 y (3). Stability followed a power law dynamic that when extrapolated suggests that most strains in an individual's gut community are retained for decades (3). In a metagenomic analysis of virus-like particles (VLPs) purified from the fecal microbiota of healthy adult monozygotic twins and their mothers, sampled over the course of a year, viral community structure exhibited high interpersonal variation. In contrast, the viral (phage) population within an individual was very stable over time, both at the level of sequence conservation and relative abundance (4). These observations, as well as other reports (5⇓–7), suggest that temperate lifestyles, rather than a predator–prey relationship, dominate the phage–host bacterial cell dynamic in the distal guts of healthy humans.
To improve our understanding of viral–bacterial host dynamics, we constructed a gnotobiotic mouse model containing a simplified defined artificial community composed of 15 prominent human gut-derived bacterial taxa whose genomes had been sequenced (Dataset S1). This 15-member artificial community was used as bait for a staged attack that involved oral gavage of VLPs purified from human fecal samples. This system allowed us to (i) test whether phage populations would mount a simultaneous attack on susceptible members of the microbial community or whether such an attack would be nonsimultaneous (i.e., have an identifiable sequence), (ii) document the capture of previously unknown viruses present in the VLP preparations by members of the artificial community, and (iii) track induction of native prophages.
Results and Discussion
Attacking a 15-Member Artificial Human Gut Microbiota with VLPs Isolated from the Fecal Microbiota of Healthy Adult Humans.
Our experimental design consisted of three groups of germ-free C57BL/6J mice (n = 5 per group). Each group was kept in a separate gnotobiotic isolator, where each mouse was individually caged. The first group was gavaged with the 15-member artificial community at 8 wk of age. Three weeks later, they were each gavaged with a pool of VLPs (p-VLP) isolated from fecal samples obtained from five healthy humans ("live p-VLP group"). A "heat-killed p-VLP group" was also colonized with the artificial community but 3 wk later received a heat-killed version of the VLPs used for the first group. The third group did not receive a gavage of bacteria ("germ-free group") but was gavaged with the same live p-VLP pool given to the first group. Fecal samples were collected from members of each treatment group at frequent intervals (Fig. S1).
Neither the bacterial gavage nor the p-VLP inoculum contained components that appeared to compromise gut barrier/immune function or perturb overall health status. At the time of sacrifice, none of the treatment groups exhibited any significant differences in total body weight or adiposity (epididymal fat pad weight as a percentage of total body weight; P = 0.3957 and P = 0.4794, respectively; Kruskal–Wallis test; Dataset S1). FACS analysis did not reveal any differences between the groups in the CD4+ and CD8+ T-cell compartments of their spleens or mesenteric lymph nodes (MLNs), as judged by CD44 and CD62L T-cell activation markers, Ki-67+ (proliferation marker), and FoxP3+ (Treg cells marker). For the germ-free group, Illumina shotgun sequencing of ileal and colonic contents obtained at the time of sacrifice disclosed that the p-VLP inoculum did not contain bacterial taxa (or bacterial spores) that could establish themselves in the guts of recipient animals (Dataset S1).
Microbial biomass, defined as nanograms DNA/milligram wet weight of feces, increased linearly and abruptly in the 4 d following introduction of the artificial community in both the live and heat-killed p-VLP groups (Fig. S2A). Fecal DNA concentrations correlated significantly with fecal bacterial cell counts (determined by flow cytometry; Spearman correlation, 0.843; Fig. S2B).
Because the genome sequences of the 15 bacterial taxa were known, we used community profiling by sequencing (COPRO-Seq; ref. 8), a method based on short read (50 nt) shotgun sequencing of total fecal community DNA, to quantify the relative abundance of each taxon as a function of time after initial colonization and after the staged VLP attack [2,544,433 ± 96,255 (mean ± SEM) reads per sample; Dataset S1]. Principal coordinates analysis of a Hellinger distance matrix constructed from the COPRO-Seq datasets showed that most of the variation in composition over time occurred during the period of initial community assembly (Fig. S3A). Changes in the relative abundance of community members also occurred following gavage of the live but not the heat-killed p-VLP preparation (Fig. 1 A–C and Fig. S3 B–N).
A Nonsimultaneous Pattern of Change in the Abundances of Five Phages.
To identify which exogenously administered VLP-associated viruses might be causing the observed structural rearrangements in community configuration, we modified our previously reported method for purifying VLPs (4) so that it could be applied to mouse fecal samples. We then sequenced DNA isolated from the purified VLP preparations [n = 27; two fecal pellets/VLP preparation, each amplified by multiple displacement amplification (MDA); 49,819 ± 6,983 (mean ± SEM) pyrosequencer reads per sample; Fig. S1 and Dataset S1]. To discriminate between activation of endogenous prophage in members of the artificial community versus exogenous viruses derived from the p-VLP preparation, reads generated from either the input VLPs or total mouse fecal DNA were mapped to the sequenced genomes of bacterial community members and to the mouse genome. We used reads without significant matches to either dataset to characterize viral genomes not represented in the starting 15-member artificial community.
In total, five viral genomes, none of which have been described previously, were assembled and annotated from these analyses. These viruses were detected in the gut communities of mice that had received the live p-VLP preparation but not in the heat-killed p-VLP group (Fig. 1 D and E, Fig. S4 A–H, and Dataset S1). Rather than finding a concurrent attack on all susceptible members of the model human gut microbiota, we observed a nonsimultaneous pattern of change in the abundances of these viruses with corresponding changes in the representation of community members.
A DNA virus with a circular 37-kb genome, human synthetic community phage 01 (ϕHSC01), was the first to significantly increase in abundance. It not only encodes typical phage proteins (e.g., terminase, tail protein, DNA polymerase; Dataset S1), but also a protein containing a Bacteroidetes-associated carbohydrate-binding often N-terminal (BACON) domain (Profam ID: PF13004) postulated to target glycoproteins and possibly host mucin (9). From the time of its first detection in feces 24 h after animals were gavaged with the live p-VLP preparation, the marked increase in abundance of ϕHSC01 over the course of the next 2 d correlated with a decrease in the abundance of Bacteroides caccae (R2 = −0.446; P = 3.2 × 10−8 after Bonferroni correction; Fig. 1 B and D). No other community member showed a statistically significant inverse correlation, suggesting that this bacterium is a host for ϕHSC01. The drop in the relative abundance of B. caccae was abrupt, occurring over the course of 1 d between days 2 and 3 after VLP gavage. The 74.5 ± 3.7% decrease relative to pretreatment levels was followed by a recovery to 75.8 ± 5.2% of the pre-VLP gavage values within 3–6 d (Fig. 1). This fourfold decrease was independently validated using quantitative (q)PCR (from 3.4 × 105 to 7.8 × 104 genome equivalents/mg of fecal pellet; see SI Methods). The spike in viral abundance, just like the coincident reduction in B. caccae abundance, was remarkably consistent in terms of its magnitude and time course among the individually caged members of this treatment group.
Evidence That Phage Resistance Occurs Because of Ecological or Epigenetic Mechanisms.
To determine whether B. caccae's recovery after viral attack was based on acquisition of identifiable fixed changes in its genome, we performed deep shotgun sequencing of total fecal community DNA isolated from samples obtained 9–19 d after bacterial gavage and 9–25 d after gavage of live and heat-killed p-VLPs. Pooling sequencing reads from these four groups of samples allowed us to assemble the B. caccae genome at an average coverage of 30-fold per treatment group, giving us enough resolution to identify mutations that could be responsible for conferring viral resistance. The results did not reveal deletions, insertions, or SNPs that were unique to the live p-VLP treatment group after the viral gavage and fixed in more than 10% of the B. caccae population (SI Results). Incorporation of short fragments of viral DNA within a locus flanked by short repeats [Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR, elements)] leads to bacterial resistance to viruses whose genomes have sequence similarity to the incorporated fragments (10). B. caccae does not contain any discernible CRISPR loci or associated proteins (Dataset S1). Moreover, no other prominent community member accumulated new spacers during the experiment (SI Results).
One interpretation of these findings is that phage "resistance" occurred because of ecological rather than genetic mechanisms. In this conceptualization, the gut environment consists of a number of microhabitats, some of which are occupied by B. caccae in ways that make it inaccessible to viral attack; the rise of B. caccae following the staged phage attack is not due to emerging resistance to the virus but rather an expansion of an unexposed population after the virus is washed out of the ecosystem. An alternative but not mutually exclusive possibility is that resistance is acquired due to phase variants or down-regulation of a phage receptor, making B. caccae resistant to viral attack without any discernible mutations in its genome (epigenetic mechanism). Another explanation is that mutations occur in one or more regions of the genome that are difficult to sequence and/or assemble.
Nonsimultaneous Detection of Viruses and Community Rearrangements.
The second virus to show an increase in abundance was ϕHSC02. All four predicted proteins encoded by this phage with a 6.2-kb genome (Fig. 1E) exhibit significant similarity to the Alpavirinae, a recently described subfamily identified from Bacteroidetes prophages, that belongs to the Microviridae (previously considered to be exclusively lytic) (11, 12). The changes in relative abundance of ϕHSC02 best correlated with a change (reduction) in the abundance of Bacteroides ovatus. Expansion of this virus and the attendant decrease in B. ovatus were first detected 2 d after the "crash" of B. caccae (i.e., within 5 d of gavage with the live p-VLP preparation) and coincided with the onset of recovery of the B. caccae population (Fig. 1 B–E).
The six- to eightfold reductions in relative representations of these two Bacteroides species took place during the 7-d period after gavage with live p-VLPs and were followed by a rise in abundance over a 7- to 8-d interval (Fig. 1 B and D). As these organisms increased their representation, we documented transient decreases in Bacteroides cellulosilyticus, as well as the two Bacteroides thetaiotaomicron strains present in the community. At the same time, levels of Parabacteroides distasonis, Clostridium symbiosum, Clostridium scindens, and Ruminococcus obeum rose. These successional changes in bacterial abundance were limited to the group of mice that had received the live p-VLPs (Fig. S3).
The rise and fall of these organisms in the live p-VLP group occurred during a time period when three other previously undescribed viruses appeared: ϕHSC03 (153.4 kb), ϕHSC04 (104.2 kb), and ϕHSC05 (95.7 kb). These viruses, initially detected 7 d after gavage of the live p-VLP preparation, subsequently increased in abundance to approximately equivalent levels and persisted during the remaining 14 d of the experiment (Fig. S4 C–H). Unlike the distinctive negative correlation between ϕHSC01 and B. caccae abundance, and subsequently ϕHSC02 and B. ovatus abundance, the simultaneous appearance and rise of ϕHSC03, ϕHSC04, and ϕHSC05, their subsequent persistence, and the coincident complex patterns of change in the abundances of bacterial community members during this later period of the experiment made it difficult for us to assign candidate bacterial hosts to these three previously undescribed phages. Therefore, the same live p-VLPs used for the in vivo staged phage attack were also used for in vitro "attacks" of monocultures of each of the species present in the 15-member artificial community (SI Methods). There was no enrichment of any of the five previously undescribed viruses in any of the cultures (n = 2 independent experiments), suggesting that the bacterial host susceptibilities and requirements for infection with these phages are not recapitulated under these in vitro conditions (SI Results and Dataset S1). This observation highlights the value of gnotobiotic animal models for isolation of previously undescribed gut viruses.
Tracking the Origin of Each of the Five Phages Among the Human Donors as Well as Their Genome Variation Between and Within Recipient Mice.
To determine whether ϕHSC01–ϕHSC05 were distributed among all of the human VLP donors or whether they were unique to particular individuals, we generated a hybrid assembly using reads from the original VLP-derived viromes from each of the five donors as well as from the p-VLP preparation used for gavage (SI Methods). The hybrid assembly yielded 159 contigs greater than 2 kb. Based on read distribution and percent identity in contigs, we concluded that ϕHSC01 and ϕHSC04 originated from a twin in family 2 (F2T1.2), whereas ϕHSC02 and ϕHSC03 originated from the twin in family 4 (F4T1.2) (Fig. S5A). ϕHSC05 was observed in four of the five individuals used to construct the VLP pool. Mapping reads from each human donor fecal virome to ϕHSC05 revealed that the virus recovered from mice likely came from individual F3T1.2 because the average percent identity of reads from this person's virome mapping to the ϕHSC05 genome was equivalent to the percent identity obtained from VLPs isolated from mouse fecal samples (Fig. S5B). Nonetheless, analyzing VLP DNA purified from fecal samples obtained from each recipient mouse 9 d and 17 d after gavage of the live p-VLPs, and from cecal samples obtained at sacrifice, revealed animal-to-animal variations in ϕHSC05 genome structure (Fig. S5C). These genomic variations could have been present in the human donor fecal virome and distributed to different mouse recipients. However, we also observed variations in ϕHSC05 genome structure within mice over time (Fig. S5C), raising the possibility of a red queen dynamic (evolution of the viral genome in response to evolution of the bacterial host over time). It is important to note that none of the other four phages (ϕHSC01–4) showed this type of variation between animals, or within individual animals as a function of time (Fig. S5 D–G), indicating that ϕHSC05's genome evolution is not a general feature in mice harboring this artificial community. Together, our findings not only illustrate the utility of gnotobiotic mice for identifying candidate bacterial hosts for human gut-associated phage, but also for identifying differences in properties among related virotypes derived from different human gut viromes.
Distribution and Persistence of Phages Within the Gut.
Intestinal transit time in mice is on the order of several hours (13). We collected intestinal contents from the proximal and distal small intestine, cecum, and colon, as well as a fecal sample from each mouse in the live and heat-killed p-VLP treatment groups at the time of their sacrifice (25 d after the staged p-VLP attack). All gut samples were processed for COPRO-Seq analysis, and an aliquot of cecal contents was used to isolate VLPs for subsequent shotgun sequencing of viral DNA (Dataset S1). The results revealed no detectable phages in any of the gut segments from mice that received the heat-killed p-VLP preparation (Fig. S6A). In the live p-VLP treatment group, neither ϕHSC03, ϕHSC04, nor ϕHSC05 exhibited significant differences in their relative abundances between the distal small intestine and distal colon, and between luminal contents and feces (Fig. S6A). Moreover, at the time of sacrifice, there were no significant biogeographical differences in the relative abundance of bacterial species within or between members of the two treatment groups (Fig. S6B).
The fact that ϕHSC03, ϕHSC04, and ϕHSC05 first appeared in members of the live p-VLP treatment group 7 d after the single gavage of p-VLPs suggests that an intra- and/or extracellular compartment/reservoir exists that harbors components of the administered human fecal phage population. Pseudolysogeny, a state where phages exist in a host bacterial cell without multiplying or synchronizing their replication with the host (14) could represent one potential mechanism for persistence. Hypervariable domains, including C-type lectins, have been identified in gut-associated phages (15), suggesting that extracellular sequestration with binding to mucus or epithelial cell surface glycans could represent another potential mechanism. Barr et al. found that enrichment of phages in mucus occurs through binding of Ig-like domains exposed on phage capsids to carbohydrate residues present in the mucin glycoprotein component of mucus, thereby creating a form of antimicrobial defense that could protect mucosal surfaces (16). COPRO-Seq analysis of cecal samples obtained from mice in the germ-free treatment group that lacked the 15-member artificial community and were gavaged with the live p-VLP preparation alone revealed no detectable phages in the cecum at the time of sacrifice (Dataset S1), supporting the notion that persistence of ϕHSC03–ϕHSC05 may be dependent upon the presence of bacteria. [Note that members of the gut microbiota, including Bacteroides spp. represented in the artificial community, are known to impact the mucus layer and mucosal glycans through a variety of means (17⇓–19).] Although we cannot completely rule out the possibility that ϕHSC03 and ϕHSC04 were first detected at later time points because there had been a selection for mutants that could replicate better, this seems unlikely; their late appearance was a common feature in mice receiving the live p-VLP preparation, and, as noted above, there was no obvious variation in their viral genomes between animals and within a given mouse over time (Fig. S5 F and G).
Prophage Activation in the 15-Member Artificial Community.
Thirteen of the 15 bacterial taxa in the artificial community had predicted prophages in their genomes (Dataset S1). To verify these predictions and to assess the capacity of these prophages to undergo induction, we used reads obtained from shotgun pyrosequencing of DNA isolated from two sources: (i) VLPs purified from fecal samples collected at weekly intervals from animals gavaged with live p-VLP preparation and (ii) VLPs purified from cecal samples obtained at sacrifice. Instead of mapping randomly throughout bacterial genomes (implying a background level of bacterial DNA contamination in the purified VLPs), VLP reads mapped to one or more of the predicted prophages. In this way, we identified 10 prophages derived from seven bacterial genomes that had the capacity to undergo induction in vivo (Fig. S7).
B. cellulosilyticus WH2 has two prophages, one of which (prophage 1) exhibited the greatest fold-induction among these 10 prophages. Prophage 1 has a lambdoid genome architecture (Fig. 2A) with syntenic arrangements identified in other Bacteroides genomes (Fig. S8A). Its induction was observed in all mice 5–9 d after initial gavage of the 15-member artificial community prior to introduction of either live or heat-killed p-VLPs. Induction occurred at the end of the period of initial bacterial community assembly, right after microbial biomass reached its peak (Fig. S2A), suggesting a potential role of bacterial density in the induction process. Induction of prophage 1 correlated with a decrease in the relative abundance of its bacterial host (Fig. 2 B and C). The other B. cellulosilyticus prophage (prophage 2) did not exhibit significant levels of induction at any time point surveyed during the experiment (Fig. 2D and Fig. S8B; also see SI Results showing that prophage 2 can be induced in vitro).
B. cellulosilyticus WH2 was represented in the 15-member artificial community by a library of 93,458 isogenic mutants, with each mutant strain containing a single randomly inserted modified mariner transposon (Tn) (91.5% of predicted ORFs had insertions covering the first 80% of each gene with an average of 13.9 insertions per ORF). Because the modified Tn had engineered recognition sites for the type II restriction endonuclease MmeI at its ends, 16 bp of flanking chromosomal DNA could be excised together with the Tn after MmeI digestion of community DNA and sequenced (20). This makes it possible to use high throughput sequencing to define the precise location and abundance of each transposon mutant in the library (SI Methods). Comparing the number of reads for each mutant in an "output" population after a given selection to the number of reads generated from an "input" population provides information about the effect each transposon insertion had on the fitness of the organism under the selection condition applied (20, 21).
Applying this Tn INsertion Sequencing (INSeq) analysis to DNA prepared from fecal samples collected before, during, and after prophage 1 induction showed a dramatic enrichment for transposons located within a ∼600bp intergenic region positioned between the ORFs encoding the prophage's putative Rha protein (22) and cI repressor at the time of its induction 5–9 d after introduction of the 15-member artificial community (Fig. S9 A and B and SI Results). The intergenic region upstream of cI in phage lambda is an extremely well-studied transcriptional regulatory region; the right operator (OR) with its three sites that competitively bind the repressor and Cro proteins, constitutes a carefully regulated switch between lysogenic and lytic cycles (23, 24). Upon RecA activation, the repressor is cleaved and the prophage is induced (25). Thus, accumulation of mutations in this region could have important consequences on the regulation and lifecycle of the lambdoid prophage 1 and its bacterial host.
In some mice, enrichment of strains with Tn inserts in the cI-rha intergenic region was observed as early as 2 d after gavage of the 15-member artificial community (before prophage induction). Enrichment did not reflect clonal expansion of a single mutant strain within a given animal, but rather expansion of 1 or more of 10 independent mutants, each harboring a single transposon insertion within this intergenic region. The number and sites of these insertion mutants varied between animals (Fig. S9A). Moreover, no Tn insertions were observed within the ORF encoding the putative cI repressor or in the region 100 bp immediately upstream of the ORF in the input library, nor in any of the output fecal samples (Fig. S9A), suggesting an essential role for the repressor and the upstream OR region in bacterial host fitness (Fig. S9 C and D).
Control experiments were carried out for 20 d (the time before p-VLP gavage) using the same 15-member artificial community but where B. cellulosilyticus WH2 was represented by the wild-type strain rather than by a library of Tn mutants. Although community assembly and structure were highly similar to that observed when the artificial community contained the Tn mutant library (compare Fig. S8C with Fig. 1A), neither prophage 1 nor prophage 2 were induced, and no drop in B. cellulosilyticus WH2 abundance was observed (Fig. S8D). These findings suggest that Tn mutations in the cI-rha intergenic region facilitate prophage induction in the mouse model.
We defined the time course of clonal expansion of bacteria containing Tn inserts in this intergenic region to quantify the fitness effects of disrupting this part of the prophage genome. Reads mapping to this locus represented 77.9 ± 4.6% (mean ± SEM) of all Tn reads in fecal samples collected between 5 d and 9 d after bacterial gavage (range, 61.3–96.9%). A sliding window analysis was performed to determine if any other 600-bp region of the B. cellulosilyticus WH2 genome containing a Tn insertion went through a clonal expansion analogous to that documented for the cI-rha intergenic region during the first 31 d of the experiment in mice belonging to the live and heat-killed p-VLP treatment groups. The results revealed that on average any given 600-bp window with a Tn decreased its abundance over time, usually to less than 0.001% of the B. cellulosilyticus WH2 population. Only 5–10% of the windows other than the cI-rha intergenic region exhibited any enrichment over time, with less than 0.1% of the windows reaching levels >1% of the population (Fig. S9B). However, all of these other enrichments occurred 11 d or more following gavage when the bacterial host population was recovering from prophage induction (Fig. S9B). Importantly, strains with the Tn-containing cI-rha intergenic region selected for before and during prophage 1 induction subsequently maintained high relative abundance (∼4%) in the B. cellulosilyticus population in both the live and heat-killed p-VLP treatment groups (Fig. S9B).
These results indicate that prophage 1 induction is restricted in time (i.e., nonrecurring over the course of the experiment) and insensitive to the attack of other members of the artificial community by exogenous human fecal phage. Together, the data demonstrate how disruption of an intergenic region, located just upstream of the predicted OR region that functions as a switch between lysogenic and lytic cycles in other lambda phages, and between a putative repressor and antiterminator, is capable of conferring a fitness advantage to its bacterial host strain before and independently of prophage induction.
Gnotobiotic mice containing defined consortia of sequenced human gut bacterial symbionts provide a tractable system that is more realistic than in vitro approaches for characterizing phage–bacterial host dynamics. These mice disclosed that: (i) a deliberately executed phage attack with a mixture of diverse human phages did not result in a simultaneous attack of all susceptible members of the artificial human gut microbiota, but rather was manifested by a succession of changes in the abundance of a subset of its bacterial taxa; (ii) phage resistance can occur through an ecological or epigenetic mechanism (i.e., without changes in bacterial CRISPR elements or bacterial genes encoding cell surface markers); (iii) one phage that was widely distributed among the five human donors but only reached low abundance in mice exhibited variations in its genome sequence in gnotobiotic animals over time, raising the possibility of red queen dynamics in this case; and (iv) prophages contain important in vivo fitness determinants for their host strains; these determinants can reside in regulatory regions responsible for prophage induction (as illustrated by B. cellulosilyticus WH2).
The model gut microbial community that these animals harbored was remarkably resilient with several fold changes in the relative abundance of different taxa occurring for only brief periods of time. Nonetheless, the identified targets of phage attack (B. caccae and B. ovatus) did not fully return to preattack levels in the artificial community or to levels seen in mice receiving the heat-killed p-VLPs that were sampled at corresponding time points, suggesting modest long-lasting effects. The transient nature of the changes in community structure observed after p-VLP attack, and the fact that prophage induction was modest in most bacterial hosts, support the view that the phage–bacterial host dynamic in this simplified defined gut ecosystem is predominately temperate rather than lytic (4).
Our findings extend previous work analyzing the dynamics of well-characterized T4 and T7 phages in gnotobiotic mice monocolonized with Escherichia coli, where their in vitro behavior was a very limited predictor of their in vivo behavior (26). Using a more complex defined microbiota, we also observe a diverse range of viral dynamics: (i) exogenous viruses rising in abundance soon after their introduction (with a corresponding decrease in abundance of their putative bacterial hosts) followed by depletion of these viruses from the community without any obvious trace of genetic resistance or adaptation; (ii) exogenous viruses that survive at undetectable levels for almost a week before increasing their abundances, and in one case, displaying genetic variability over time; and (iii) basal levels of induction of 10 prophages with only one prophage achieving a level that produced detectable alteration in the abundance of its bacterial host. Duerkop et al. also showed that prophage induction differed in vitro and in vivo, and how under the appropriate conditions induction could provide a fitness benefit to the bacterial host (27), further highlighting the importance of a temperate lifestyle in the gut, and why prophages are so widely distributed in gut bacterial genomes without necessarily being induced at significant levels. Our findings are also consistent with the previous finding that T7 phages are capable of surviving at undetectable levels for 1 wk in germ-free animals before they rise in abundance after gavage of a bacterial host (26); this capacity to maintain infectivity has potential implications for preventive phage therapy.
We envision a future where gnotobiotic mouse models allow "personalized" assessment of phage–bacterial interactions. Complex mixtures of VLPs, isolated from previously frozen fecal samples obtained from human donors representing various ages, physiological or disease states, or geographical regions/cultural traditions of interest, can be introduced into recipient mice harboring a defined collection of human gut community members. The community can be used as a "filter" to identify and assemble the genomes of phages present in the human donor viromes and link them to bacterial hosts. Miniaturization of methods for preparing VLPs from mouse fecal pellets provides a way for purifying these phages and at the same time verifying that they have lytic activity. The system is capable of distinguishing very closely related virotypes present in multiple human gut microbiota based on their differential ability to establish themselves in recipient gnotobiotic mice. These attributes not only provide a discovery pipeline that complements metagenomic surveys of the human gut virome by identifying phages "buried" in large gut microbiome datasets, but facilitate identification of phages that can be used as experimental tools to deliberately manipulate model microbial communities or that can be considered as candidate therapeutic agents.
Protocols for the recruitment of human subjects and sampling of their fecal microbiota were approved by the Washington University Human Research Protection Office. All experiments involving mice were performed with protocols approved by the Washington University Animal Studies Committee. Procedures for gnotobiotic mouse husbandry, introduction of VLPs purified from human fecal samples into mice, sampling fecal microbiota from gnotobiotic mice, isolation of total DNA from mouse feces and intestinal contents, quantification of microbial cells in fecal samples by flow cytometry, preparation of DNA libraries for Illumina HiSeq or MiSeq sequencing, preparation of VLP DNA from mouse fecal samples, 454 pyrosequencing of VLP-derived DNA, COPRO-Seq analysis, assembly and annotation of viral genomes, cross-contig comparisons, INSeq analysis of fitness determinants present in the B. cellulosilyticus WH2 prophage, PCR quantification of B. caccae abundance in the fecal microbiota of gnotobiotic mice, CRISPR analysis, PCR determination of Tn insertions in uninduced and induced B. cellulosilyticus WH2 prophage 1, in vitro induction of B. cellulosilyticus WH2 prophages, and in vitro assays for bacterial host tropism of phages represented in the pooled human fecal VLP preparation are described in SI Methods.
We thank Dave O'Donnell and Maria Karlsson for their assistance with gnotobiotic mouse husbandry, Philip Ahern for help with immune characterization of tissues, Martin Meier for technical support with robotics, and members of the J.I.G. laboratory for their many helpful suggestions during the course of this study. This work was supported by grants from the National Institutes of Health (DK30292, DK078669, and DK078669S1) and the Crohn's and Colitis Foundation of America. A.R. is the recipient of an International Fulbright Science and Technology Program award.
↵1Present address: Departamento de Ciencias Biológicas, Universidad de los Andes, Bogotá 111711, Colombia.
- ↵2To whom correspondence should be addressed. E-mail: jgordon{at}wustl.edu.
Author contributions: A.R. and J.I.G. designed research; A.R., M.W., and N.P.M. performed research; A.R., F.L.R., and J.I.G. analyzed data; and A.R. and J.I.G. wrote the paper.
The authors declare no conflict of interest.
Data deposition: The data reported in this paper have been deposited in the European Nucleotide Archive (Project ID PRJEB4370).
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1319470110/-/DCSupplemental.
Freely available online through the PNAS open access option.
- ↵
- ↵
- ↵
- Faith JJ,
- et al.
- ↵
- ↵
- Minot S,
- et al.
- ↵
- ↵
- Minot S,
- et al.
- ↵
- McNulty NP,
- et al.
- ↵
- ↵
- Barrangou R,
- et al.
- ↵
- ↵
- ↵
- ↵
- ↵
- Minot S,
- Grunberg S,
- Wu GD,
- Lewis JD,
- Bushman FD
- ↵
- Barr JJ,
- et al.
- ↵
- Vaishnava S,
- et al.
- ↵
- ↵
- ↵
- ↵
- ↵
- Henthorn KS,
- Friedman DI
- ↵
- ↵
- Ptashne M
- ↵
- ↵
- ↵
- Duerkop BA,
- Clements CV,
- Rollins D,
- Rodrigues JL,
- Hooper LV
Citation Manager Formats
Article Classifications
- Biological Sciences
- Microbiology


text: Add to Cart Quick view Pond Airlift 1.5 LP Solar 5w 200 GPH - 1,500 Gallons MSRP: Now: $199.00 Was: What is an airlift? An airlift is, as the name suggests, moving (lifting) of water by air. Air bubbles rise up in the water and thereby displace the water. (ie) the bubbles push the water up. Keep any pool or pond alive and clean by cheaply and cost... MSRP: Now: $199.00 Was: Add to Cart Quick view


text:
Medicinal science has come so far, remember when Lind figured out that citrus fruits prevents scurvy? Or when Lister was like "yo, we should sterilise stuff before we cut human bodies open so they stop dying from infections." and everyone was like "that might work hey." Or when Fleming discovered penicillin?
Is this because you were born post the 1800's and in your lifetime you weren't exposed to scurvy, the black plague, or unsterilised surgeries?
Remember when you would go in for a pap smear and the examination bed would be up against the wall so when you had to open wide for an examination your left knee couldn't go as far down as your right knee? And the GP was telling you to relax, even though you're contorting your body into this unnatural position of having one leg at an acute angle and the other one kind of bent but also laying flatter than the other one because you aren't able to push the wall out the way?
So the GP is trying to pry open your vagina with a sinister sounding tool which you KNOW is a speculum but the sound its making is akin to something you've heard in a workshop or a building site. Now the doctor is getting frustrated because the medieval contraption wedging your vagina open is too big and that's probably why it's not seamlessly entering you.
You're a little offended as well as physically uncomfortable now because the doctor has informed you that they need to select a smaller apparatus, as if this is your fault.
'How big did you initially think my vagina would be? And how can you tell with my pants on?' are the questions you want to ask, but don't, because what if the answer is something similar to: 'I can tell by your chunky thighs and irregular gait that your vagina will clearly need our biggest spec to open it up.'
Once the doc has successfully wrestled your vagina open and brushed the delicate walls of your cervix for a sample of cells, you're allowed to lower your legs and put your underwear back on, then think about how long your knee will be numb for as it has tried to fruitlessly move a solid wall 10 inches away from you for the last 7 hours (3 minutes).
Remember when doctors realised this and moved the bed away from the wall?
Is this because that never happened?!
Perhaps I'm exaggerating, maybe there are some examination rooms that exist in the world that have the bed in the middle of the room instead of on the side of it. I'm sure they exist… the same way that I know the Illuminati exist and are just biding their time before they take over the world.
But so far, I am yet to see any true evidence of this.
I hope if you're reading this and you haven't experienced the joy of a cervical screening, you're not immediately put off by them. They are very useful! They detect and can ultimately help to prevent cervical cancer.
The thing is, despite how invasive and uncomfortable it may feel to be subjected to the mercy of a speculum and a cranky GP, who has done nothing for the day except prescribe antibiotics and write sick notes for people who want a long weekend, these screenings are important.
The vagina is the eyes to the cervix and the cervix is where cancer can dwell. So don't think of your vagina being invaded, think of it as your cervix being serviced like you would get your car serviced.
You know how your parents come to visit and ask, "Did you get your car serviced?" and you lie and say, "Yes of course." Then your dad opens the glove compartment to check the car service history and you scream "OH WAIT! No I didn't! I had it booked but something came up and it's scheduled for next month!".
Then you feel bad about lying to your dad who has done nothing but support you and love you unconditionally. In fact, you feel so guilty about it that you book your car in for a service straight away and vow to do so every time you're supposed to.
Our bodies don't have a service record in our glove compartments for someone to guilt trip you with. However, the next time you put off having a cervix exam, I want you to remember this essay and remember that there is a chunky thighed, somewhat prudish, dramatic gal who supports you and loves you unconditionally who wants you to get your cervix lightly brushed once every 3 years.


text:
Hi, I'm Joe Kim
About me
About me
I enjoy many personal and professional passions. Here, you'll get a brief glimpse at some of those things. I've always loved being in the outdoors. I love mountains, nature, and tranquility.
My diverse background has led me into work that blends aspects of engineering, medicine, public health, technology, systems thinking, quality improvement, continuing education, implementation science, health services research, leadership, organizational dynamics, and business.


text:
Flight Management
Upload information and flight details of each attendee, indicating: Departure time, arrival time, airport of origin and destination, flight number and other details.
In order for this feature to be available for use, it must be enabled in Event Tools
How can I add flight details to an attendee?
Manually insert flight details
To add flight information, click on: People > Flights > Add Flight.
Mandatory fields are: Person, Airline, Flight number, Gate, Seat, Ticket type, Issue date, Departure airport, Arrival airport, Departure date, Arrival date.
Insert bulk flights via spreadsheet
Click on Edit > Import > Download Demo File.
When the file downloads, enter the information requested, save the file and upload.
Mandatory fields are: username, email, airportBegin, airportEnd, dateBegin, dateEnd, dateIssue
Click on Import > Green File Icon >Import >End.
How to edit registered information
To edit information for flights already registered, select the user / flight you want, then click EDIT
Then in the window to the right, you can edit the information.
Press END to save the edit.
To remove registered information.
To remove an attendees flight details, press EDIT, click on the checkbox of the information you would like to delete and press REMOVE in the top right corner.
Viewing Flight Information
Flight information for each Attendee can be sent via email or viewed on a dedicated button on the App.
E-mail: The organizer must go to People> Attendees , click the attendees name and then click View Voucher. A new tab will open with the attendees voucher. The organizer must download and send it to the Attendee. For more information, please read Attendee List and Digital Ticket Voucher .
App: In this option, the Attendee must log in to the application and press MENU, then press VOUCHER to view the flight information
How to update information already registered?
If you keep the same registered flight locations for the same user, you can update dates + times and additional information by uploading a new worksheet to the platform.
But remember, you should always keep the user key the same. Example:
If you have registered with the XYZ email, you must keep the same until the end of the project or perform the deletion and add the correct one again. If the user does not hit or exist, the platform does not assimilate information or create new users.
How do I add flight information to an email?
To add dynamic content to an email you can add the attendee's Digital Ticket Voucher which will include information such as: a unique QR code for the attendee, attendee's name and information, hospitality information (flights/rooms/shuttles), as well as acting as a ticket to go through access controls.
To learn more about it, click here.


text:
The vision of the British Dyslexia Association is a dyslexia friendly society enabling all dyslexic people to reach their potential. Mission: To •campaign and lobby for long lasting and sustainable change for the benefit of dyslexic people •provide impartial and objective advice and support to dyslexic people and those with whom they come into contact •set the standards for and accredit dyslexia knowledge and professional expertise •deliver innovative solutions which break new ground in the field of dyslexia •disseminate and share best practice regionally, nationally and internationally •promote research.
Ten percent (10%) of the British population are dyslexic; 4% severely so. Dyslexia is identified as a disability as defined in the Equality Act 2010. Many of the dyslexic people across the UK, whether adults or children, are unable to fulfil their potential as a large percentage of the population still do not understand what dyslexia is, the difficulties which the condition presents and do not know how best to support them. Dyslexia is not an obvious difficulty; it is hidden. As a result, dyslexic people have to overcome numerous barriers to make a full contribution to society.
The British Dyslexia Association (BDA) campaigns for a dyslexia friendly society where barriers to dyslexic people do not exist. The BDA works to ensure that ALL people with dyslexia fulfil their potential. To achieve this we need to create change, set standards and support and enable people. It is the voice of dyslexic people; it listens to their views, represents their agendas and presses for long lasting sustainable change.
The Helpline is completely free and confidential for all dyslexia related calls and emails. The Helpline is manned by a small number of staff and a team of trained volunteers. All information, advice and signposting is completely impartial enabling the caller to make important choices and decisions for themselves.
Contacting Helpline by Phone
To talk to Helpline staff, phone 0845 251 9002. Opening times: 10am - 4pm Mondays to Fridays. Closed Wednesday afternoons.
Contact us
We publish a newletter four times a year that is free and sent directly to your inbox. To sign up for this, click here.
We can also be found on Facebook, Twitter, Google+ and LinkedIn.


text:
Executive Suites
This is a suite in which you can mix business with pleasure. A set of double doors separates your bedroom and private bathroom from your lounge and its own adjoining bathroom. Both rooms have their own entrances. When you shut the double doors you create an independent space with its own seating and dining area for business meetings or entertaining.
Features include
- Savoir beds
- Wireless high speed internet access
- Luxury toiletries
- Evening Turndown service
- Express checkout
- Exquisite bed linens
- Feather duvet and pillow selection
- Hairdryer
- Individually controlled air-conditioning and central heating
- Opulent marble bathrooms with separate showers
- Office supplies and stationery
- Plush bathrobes and ladies and gents slippers
- In-room electronic safe
- Nespresso machine
- Tea and coffee making facilities
- Bottled mineral water
- Finest handmade king size mattresses
- Sky television offering over 1,100 television and radio channels


text:
1800-102-1100 (Airtel)
080-40103000 (Toll Paid)
Hi friends, I am sharing various Customer Care Toll Free Numbers for all Service Centers, We are also starting Telugu Tech Updates, System Trouble Shooting Guide, System Hardware Tips & Tricks Topics, Product Reviews, We are going to posting now. It will help to you Resolve Computer or Laptop Issues.
Need to activate my office, but i am not having product key. Neither retailer is providing it nor microsoft team is helping to get it activated. Suggest and help ..!
contact me I will activate your copy....email@example.com
hey bro i need u rhelp to activate my windows 8.1 i have all the keys. plz contact me firstname.lastname@example.com
hello everyone,
Iam saisharath i buyed new nokia lumia 720 unfortunately while creating microsoft account my bro entered in place of birth date year he entered today's so, when iam logging in it is showing that it is a child consent you need a parent permission please help me out...........
Subject: Defective Microsoft Product (Wireless Mouse) : Images attached
From: sayan mondal
To: firstname.lastname@example.com
Cc: firstname.lastname@example.com, sayan mondal
Content-Type: multipart/mixed; boundary=089e0116166016627c04ec37d149
Hi Sobha,
As per your request today morning I am sending you the pictures of the
defective wireless mouse.
Plese have a look on these and find the below details (Images attached :
Details :
Product : Microsoft Wireless mouse.
Model : GMF-00006
Model No. : 14271447
S/N No : 0239851405442 EC
Complaint : The mouse is not working.
Purchased on : 19/10/2013
Price : 899/-
Purchased from : Reliance Digital, Phoenix Market City,Mahadev Pura,
Customer Name : Sayan Mondal
Contact : 7259269151
Please replace the product.
Sayan Mondal
Hello everyone ,
i am vinay, recently i have updated my windows to 8.1 bt frm thn onwards facing problem in some apps as whn i tried to open apps like NEWS, Solatire,etc,,,, they are automatically getting minimised, i have done all the troubleshootings bt there is no use and i am really frustated of tht .. so guys please help me with this..............
I want to upgrade server 2008 r2 foundation to server 2012 r2.
how to do and what extra cost i have to bear ?
Anybody please tell me when windows 10 Is coming in india or in india pre-installed in pc , laptops etc
please please reply me
i wanted to buy the windows 8.1 for 3to 4pc some one will help me contact me in firstname.lastname@example.com
I updated my windows 8.1 to windows 10. later on after using for around 2 weeks due to some issues I reverted it back to 8.1. thereafter to overcome those issues i resetted my laptop to factory settings, And lost my genuine windows 8.1 and ms office suite 2013.
how cn i get them back ?? please help.
In my PC .,from outlook mails are not going through.
I am not getting what is the problem.when i am trying to send any mail.,it is staying in out box itself.Please tell me the way to resolve the problem.
thanks in Advance.,
Contact me in "email@example.com
Hi, can any one tell me how save X-lite SIP accounts to all the domain users in dhcp clients, instead of configuring in
individually to domain users. If possible from GPO.
Hi, Am Gokul am face issue in windows 8.1, am using mts wifi data card, after inserting data my pc gets hangs and restarts am not sure why this problem causes. Can any one suggest me please.. Thannks
Backwash windows phone
After updating my laptop to windows 10
My laptop faced some problems like it is no longer wifi enabled.
I mean there is no wifi present in my lappy.
Also it is showing the blue screen of death after every few couple of days i.e.
There might be a problem with your PC
system thread error.
Anyone having any suggestions please reply.
i have installed windows 10 in my laptop but while upgrading i missed the key so how can i upgrade my windows with missing key.please help me
My Phone Lumia 510 in i have create new live id for enter for the live account then display error message : Windows Live isn't available at the moments. Please wait a few moments and try again.
No use of calling toll free no. 18001021100 to discuss about surface pro3 problem. From last couple of days I tried reaching them, patiently lisnining to music for more than 30 mns and going off. Very disgusting service.
What is d mail id of diz company i have toh logde a complaint of a laptop distributor wo puts pirated windows and etc things


text:
Railway engineers stir on December 15 demanding Group-B status
BHUBANESWAR: East Coast Railway Engineers Association (ECoREA) has decided to hold demonstration in front of zonal and divisional railway headquarters on December 15 demanding Group-B status to junior engineers and senior section engineers working in the Railways.
ECoREA president Bobin Mohanty said they do not get promotion from grade C to grade B. Most of them retire without getting a promotion, he alleged.
"We have been demanding before the Railways to solve our issues, but nobody pays heed to our requests. The All India Railway Engineers Federation (AIREF) is supporting us. Other zones will also conduct such demonstrations to get justice," he added.
He said 32 members of parliament (MPs) have brought the issue to the notice of Prime Minister Narendra Modi. "The PMO has also recommended the Railway Ministry to consider their demand. But the Railway Board does not take any positive step. It has demoralized 80 thousand engineers like me," he added.
Association finance secretary S B Satpathy said they will also raise the safety issues of the railways during their demonstration. "Our engineer friends are doing the crucial works related to safety of track and trains. If the railways wants to invest fund for strengthening of safety, then it should give us promotion and separate us from labourers," he added.
They will stage token dharna in front of the ECoR headquarters at Bhubaneswar, offices of divisional railways managers (DRM) at Khurda Road, Sambalpur and Waltair on December 15.
Source: TOI


text:
Worship at St Cross
9.00 am – Said Eucharist every Sunday
10.00 am – All Age Worship on the 1st Sunday (Cafe Church every three months)
Sung Eucharist on all other Sundays (with Young Church on the 3rd Sunday)
6.30 pm – Celtic Eucharist on the 1st Sunday
10.00 am - Said Eucharist every Wednesday
also on the 2nd Sunday of the month
6.30 pm - BCP Choral Evensong at a church in the Knutsford Deanery
and on the 5th Sunday we share worship with the Knutsford Methodists, either at the Methodist Church or St Cross.
There are other occasional services, such as Taizé, healing services, and the celebration of Feast Days and Festivals. Check the diary or the notice boards for more information.


text:
Author(s): Mitch Albom
Every family is a ghost story ...' As a child, Charley Benetto was told by his father, 'You can be a mama's boy or a daddy's boy, but you can't be both.' So he chooses his father, only to see him disappear when Charley is on the verge of adolescence. Decades later, Charley is a broken man. His life has been destroyed by alcohol and regret. He loses his job. He leaves his family. He hits rock bottom after discovering he won't be invited to his only daughter's wedding. And he decides to take his own life. Charley makes a midnight ride to his small hometown: his final journey. But as he staggers into his old house, he makes an astonishing discovery. His mother - who died eight years earlier - is there, and welcomes Charley home as if nothing had ever happened. What follows is the one seemingly ordinary day so many of us yearn for: a chance to make good with a lost parent, to explain the family secrets and to seek forgiveness.
* Online trade and consumer campaigns to ensure best possible word-of-mouth recommendation: www.albom.com and www.foronemoreday.co.uk * Separate Irish campaign * Submitted for trade promotions, including Paperbacks of the Year * Reading copies available
*'Mitch Albom sees the magical in the ordinary' Cecilia Ahern *'Mitch Albom, in this new book, once again demonstrates why he is one of my favourite writers ... FOR ONE MORE DAY will make you smile. It will make you wistful. It will make you blink back tears of nostalgia' James McBride, author of THE COLOR OF WATER *'Another very touching page-turner' STAR magazine *'A warm and tender tale' IRISH EVENING HERALD
Mitch Albom writes for the Detroit Free Press and has been voted America's No. 1 sports columnist ten times by the Associated Press Sports Editors. A former professional musician, he hosts a daily radio show on WJR in Detroit.