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Though horizontal gene transfer (HGT) is widespread, genes and taxa experience biased rates of transferability. Curiously, independent transmission of homologous DNA to archaea, bacteria, eukaryotes, and viruses is extremely rare and often defies ecological and functional explanations. Here, we demonstrate that a bacterial lysozyme family integrated independently in all domains of life across diverse environments, generating the only glycosyl hydrolase 25 muramidases in plants and archaea. During coculture of a hydrothermal vent archaeon with a bacterial competitor, muramidase transcription is upregulated. Moreover, recombinant lysozyme exhibits broad-spectrum antibacterial action in a dose-dependent manner. Similar to bacterial transfer of antibiotic resistance genes, transfer of a potent antibacterial gene across the universal tree seemingly bestows a niche-transcending adaptation that trumps the barriers against parallel HGT to all domains. The discoveries also comprise the first characterization of an antibacterial gene in archaea and support the pursuit of antibiotics in this underexplored group.
eLife digest
Living things inherit most of their genetic material from their parents, so genes tend to be passed on from one generation to the next—from ancestors to descendants. Sometimes, however, DNA is transferred from one organism to another by other means. These events, collectively called horizontal gene transfer, are fairly common in nature; genes have been passed between different species as well as between different groups of organisms. For example, genes that confer resistance to antibacterial drugs have transferred from one species of bacteria to another, and other genes have also 'jumped' from bacteria to plants or animals.
Now Metcalf et al. have studied a gene that first arose in bacteria and that encodes an enzyme called a lysozyme. This enzyme breaks down the outer casing of a bacterial cell: a step that is required for a bacterium to reproduce and divide in two. When Metcalf et al. searched for relatives of the lysozyme gene, they found copies in many other species of bacteria and revealed that this gene has been repeatedly transferred between different bacteria. Members of the lysozyme gene family have also 'jumped out' of bacteria and into other organisms at least four times. Metcalf et al. found related lysozyme genes in a plant, an insect, many species of fungi, and a single-celled microbe (called an archaeon) that lives at hot, deep-sea vents.
A gene family being spread this widely across the tree of life has not been seen before. Nevertheless, as DNA is a common biological language to all living things, it is likely that all the different species that have received a lysozyme gene might use it for similar purposes.
Metcalf et al. reveal that the lysozyme could be being used as an antibacterial molecule. The archaeon lysozyme can kill a broad range of bacteria; and when the gene was transferred into Escherichia coli bacteria, only the bacteria that mutated the lysozyme gene to render it useless were able to survive. Metcalf et al. also revealed that the archaeon microbe produces more of the enzyme if bacteria are present, which allows it to outcompete these bacteria.
These findings suggest that there may be a number of horizontally transferred genes that have antibacterial activity against a wide range of bacteria. Searching for these genes—particularly in the largely underexplored group of archaea—might reveal new sources for antibiotic drugs to treat bacterial infections.
Main text
HGT is rampant among prokaryotes and phages and is an important mechanism for acquisition of new genes and functions (Popa and Dagan, 2011), including the shuttling of antibiotics and antibiotic resistance between bacteria (Clardy et al., 2009). Instances of interdomain horizontal transfer of diverse genes between two domains of life or between viruses and their hosts are also increasingly documented (Nelson et al., 1999; Husnik et al., 2013; Dunning Hotopp et al., 2007; Wu et al., 2013; Gladyshev et al., 2008; Bratke and McLysaght, 2008; Danchin et al., 2010). While a minority of these transfers have been functionally investigated, the biological activity, selective advantages, and ecological contexts of many interdomain HGT events remain poorly characterized (Dunning Hotopp 2011, Keeling and Palmer, 2008). In comparison to these intradomain or interdomain highways of HGT (Beiko et al., 2005), independent transmission of the same gene family to archaea, bacteria, eukaryotes, and viruses is extremely uncommon and subject to apparently rare events throughout the history of life (Moran et al., 2012; Lundin et al., 2010; Koonin et al., 2003; McClure, 2001; McDonald et al., 2012). When taken together, genome-enabled studies suggest that horizontal gene transfers (HGTs) are biased and experience a frequency gradient that decreases from within domain > between two domains > between all domains of life (Bruto et al., 2013; Zhaxybayeva and Doolittle, 2011; Puigbo et al., 2009; Andam and Gogarten, 2013, 2011). However, the fact that a large number of organisms from all domains of life have been artificially transformed with genes evolved in other taxa indicates that there is no fundamental obstruction to interdomain HGT when barriers to transfer are deliberately removed.
One significant question then is why do highways of intra- or interdomain transfers occur more frequently than transfers to all domains in the universal tree of life? There are at least two explanations. First, recurrent transfer of the same gene family may be limited by incompatible mechanics of gene transfer (e.g., transduction, transfection, plasmid exchange, isolation of eukaryotic genome in nucleus, separation of somatic and germline tissues in multicellular eukaryotes) between domains compared to within domains. However, the individual success of gene transfers between any two domains of life, for example archaea and bacteria (Nelson et al., 1999; van Wolferen et al., 2013), bacteria and eukaryote (Andersson, 2005; Bordenstein, 2007; Gladyshev et al., 2008; Danchin et al., 2010), and archaea and eukaryote (Andersson et al., 2003; Schonknecht et al., 2013), suggests that these barriers may be minimal. Second, the selective barriers against HGT of the same gene to multiple taxa and preservation of the gene through evolutionary time are multifaceted given the potential costs associated with HGT (Baltrus, 2013), and that each recipient may not benefit from the trait conferred. In the case of selfish genetic elements, HGT provides a strong benefit to the gene itself, yet these genes can be detrimental to the host organism and select for the evolution of countermeasures to gene propagation (Werren, 2011). Thus, it may be necessary for a transferred gene to confer a benefit to its new host in order to be stably maintained in the host genome over the long term. Given these evolutionary dynamics, there may be very few niche-transcending genes (Wiedenbeck and Cohan, 2011), defined as genes that are useful in different physiological capabilities, cellular structures, and ecological niches, which repeatedly increase fitness of each recipient across the whole diversity of life and can be stably and repeatedly transferred between very divergent organisms.
Among the few putative cases, there is a pore-forming toxin domain that appears to have been anciently transferred between diverse lineages (Moran et al., 2012). However, the distribution of the transfer across the tree of life is unclear because archaeal sequences were not included in phylogenetic analyses due to low support values. Other candidate genes encode proteins involved in nucleotide metabolism, intramembrane proteolysis, or membrane transport, but the transfer events defy clear interpretations due to their deep antiquity in evolutionary time and the confounding issues of ancient paralogy (Lundin et al., 2010; Koonin et al., 2003; McClure, 2001; McDonald et al., 2012). Moreover, these transfers are often not functionally validated in the recipient taxa.
Here we demonstrate for the first time, to our knowledge, that a functional antibacterial gene family scattered across the tree of life in diverse ecological contexts. This bacterial gene encodes a glycosyl hydrolase 25 (GH25) muramidase, a peptidoglycan-degrading lysozyme that hydrolyzes the 1,4-β-linkages between N-acetylmuramic acid and N-acetyl-D-glucosamine in the bacterial cell wall. Typically found in bacteria (Cantarel et al., 2009), the lytic enzyme classically functions in cell division and cell wall remodeling (Vollmer et al., 2008), while in bacteriophages they lyse bacterial peptidoglycan at the end of the phage life cycle (Fastrez, 1996). Although members of the GH25 muramidase family have been noted in other taxa (Korczynska et al., 2010; Nikoh et al., 2010), extensive analysis of their evolutionary history and functions have not been undertaken. We hypothesized that the transfer of antibacterial genes from bacteria to archaea and to eukaryotes bestows a niche-transcending adaptation that overcomes the barriers against repeated and evolutionarily stable HGT of the same type of gene across the tree of life.
Results and discussion
A bacterial GH25 muramidase is present in all domains of life
During a homology search, we uncovered 75 nonredundant sequences (E-values ≤ 10−12) of a bacterial GH25 muramidase in disparate taxa across the tree of life, indicating possible HGT of a bacterial gene to both eukaryotic and archaeal species as well as to phages. Putative HGT events were identified in the genomes of the plant Selaginella moellendorffii (Banks et al., 2011), the deep-sea hydrothermal vent archaeon Aciduliprofundum boonei (Reysenbach et al., 2006), the pea aphid Acyrthosiphon pisum (International Aphid Genomics Consortium 2010, Nikoh et al., 2010), and several species of fungi such as Aspergillus oryzae (Machida et al., 2005). We verified the presence of the lysozyme gene in natural populations of selected HGT recipients by PCR and sequencing of the GH25 muramidase domain (Figure 1—figure supplement 1), including Aciduliprofundum field samples harvested from hydrothermal vents worldwide. We detected lysozyme genes in 9 out of 12 field isolates of Aciduliprofundum from deep-sea vents in the Atlantic and Pacific oceans, 5 out of 6 species in the plant genus Selaginella, and 8 out of 9 aphid species in the subfamily Aphidinae (Supplementary file 1). However, it is possible that PCR-negative strains actually do possess lysozyme genes and were simply more divergent than could be detected with our primers. At the protein level, sequenced field isolate lysozymes were relatively similar to each other in each clade, with 74% pairwise identity (385aa alignment) amongst Aciduliprofundum sequences, 85.1% (203aa alignment) amongst three intact Selaginella sequences, and 87.3% identity (93aa alignment) amongst Aphidinae sequences, with more variable divergence between clades (full identity matrix available in Supplementary file 2). We also found lysozymes in two additional WO phages as part of an ongoing next generation sequencing project of Wolbachia viruses (unpublished data).
To rule out spurious bacterial contamination in these genomes and to confirm genomic integration of the lysozyme gene, we employed direct sequencing of PCR products amplified using primers inside the lysozyme gene paired with primers outside the gene for Aciduliprofundum field samples and S. moellendorffii. Incorporation of the lysozyme gene was verified in all cases tested (Figure 1—figure supplement 2). Additionally, Aciduliprofundum samples were grown in strict monocultures as determined by 16S amplicon monitoring. Integration of the A. pisum lysozyme has been previously established (Nikoh et al., 2010). Flanking genes in the recipient genomes were non-bacterial on either side of the transferred lysozyme in each case (Figure 1, Supplementary file 3), with two exceptions. A bordering gene in A. boonei, ADP-ribose-1"-monophosphatase (App-1), possesses both bacterial and archaeal homologs and a phylogenetic analysis suggests HGT unrelated to the lysozyme transfer (Figure 1—figure supplement 3A). This transfer was likely between archaea and Thermotogae bacteria. The second exception is a GH2 hydrolase gene adjacent to the lysozyme in A. oryzae. This hydrolase has bacterial homologs in the phylum Actinobacteria, and recapitulates the same phylogenetic pattern seen in the GH25 muramidase (see below, Figure 1—figure supplement 3B). Thus, it is likely that the lysozyme and GH2 hydrolase were transferred to fungi in a single event. GH2 hydrolases have a number of carbohydrate-degrading enzymatic abilities (Cantarel et al., 2009), including β-galactosidase, β-mannosidase, and β-glucuronidase activities, suggesting that this gene may benefit the fungi by adding additional digestive/nutritive capabilities in Dikarya.
Non-bacterial GH25 muramidases arose from HGT
To establish parallel HGT, i.e., the independent transfer of the same gene family to multiple lineages, we conducted a phylogenetic analysis on 86 GH25 muramidase sequences using Bayesian and maximum likelihood inference methods (Figure 2A). We combined non-redundant Aciduliprofundum, Selaginella, and WO sequences obtained from PCR and Sanger sequencing with blastp results to reconstruct the phylogeny. Putative instances of HGT are diagrammed in Figure 3. Additionally, transferred lysozymes in nonbacterial taxa were used as queries to identify homologs and make a second set of phylogenetic trees to confirm the HGT (Figure 2B–D, Figure 2—figure supplements 1 and 2, Supplementary file 4). Three key results emerge from these phylogenetic analyses: (i) at least three independent instances of interdomain HGT of the bacterial GH25 muramidase occurred in nonbacterial taxa (Aciduliprofundum, Selaginella, and Insecta) as well as a number of transfers to bacteriophages, (ii) vertical transmission of the transferred gene ensues in some descendant taxa (i.e., Aciduliprofundum and Selaginella), and (iii) frequent HGT of the muramidase between bacterial clades accompanies the interdomain transfer, indicating unusually frequent and broad-ranging HGT of this niche-transcending gene family.
To statistically validate parallel HGT across the tree of life, we performed a Shimodaira–Hasegawa test (SH-test) (Shimodaira and Hasegawa, 1999) by comparing our consensus tree (Figure 2A) against a hypothetical tree with a binary constraint in which bacterial sequences are monophyletic and separate from monophyletic nonbacterial sequences. In this hypothetical tree consistent with the tree of life, lineage relationships within the bacterial and nonbacterial groups were permissively set as unconstrained. Results indicate that the hypothetical tree is significantly worse than the HGT tree, as expected (p<0.01, D(LH) = −133.9, SD = 31.5). We repeated this analysis with the hypothetical tree compared to 100 randomly sampled HGT trees from maximum likelihood bootstrapping and found the hypothetical tree was also worse than each of these trees (p<0.01). Finally, we performed SH tests between the HGT tree and either 1) a three-domain constraint tree or 2) a monophyletic eukaryote branch constraint tree, and again found that the constraint trees were significantly worse than the HGT tree (p<0.01). Thus, the null hypothesis of vertical descent is rejected, even under the most permissive conditions.
We observed that each interdomain HGT event (Figure 2) occurred between taxa that coexist in the same ecological niche, a likely prerequisite for HGT. For instance, the A. boonei lysozyme is in a clade dominated by Firmicutes whose members can be common in deep ocean sediments (Orcutt et al., 2011a). Indeed, Bacillus species have even been found in hydrothermal vents of the same fields in which Aciduliprofundum strains were isolated (Reysenbach et al., 2000). The A. pisum lysozyme clade includes Wolbachia prophages and Proteobacteria, which are common endosymbionts of aphids and other insects (Gomez-Valero et al., 2004; Augustinos et al., 2011; Wang et al., 2014). The S. moellendorffii plant lysozyme is closely related to Actinobacteria, which are dominant microbes in soil (Bulgarelli et al., 2013). These associations, while not proof of HGT, establish interactions that may have facilitated the transfers, although any number of intermediate gene carriers is possible. While the phylogenetic pattern of the GH25 muramidase found in fungi is consistent with HGT (Figure 2A, Figure 2—figure supplement 2), the transfer occurred anciently in fungal evolution prior to the divergence of Dikarya, as the domain is present in both Basidiomycota and Ascomycota, but not other fungal phyla. As is the case with most putative ancient transfers, the deep branches of the tree are poorly supported and a definitive donor taxon cannot be established. Additionally, the possibility of multiple ancient transfers between bacteria and fungi or among fungi and plants cannot be excluded. However, a nucleotide-level phylogeny also supports HGT from an ancestral Actinobacterium (Figure 2—figure supplement 3).
Interestingly, the lysozyme gene in the aphid A. pisum consists of a fusion of a bacterial GH25 muramidase domain and a eukaryotic carboxypeptidase domain. The gene includes five introns (Nikoh et al., 2010), none of which interrupt the GH25 domain, consistent with a relatively recent HGT event and the absence of the gene from most sequenced insects (Figure 1). The lysozyme in the fungus A. oryzae, meanwhile, contains only a single intron, but it does interrupt the GH25 domain, consistent with the domain's long association with fungi from the subkingdom Dikarya (Figure 1). We found no evidence of a GH25 muramidase in 323 sequenced archaeal genomes spanning all the major phyla and sister taxa to A. boonei (Reysenbach et al., 2006; Flores et al., 2012). This lack of homology does not appear to be due to insufficient representation of archaeal diversity, as the 323 members span all of the major phyla: Crenarchaeota (56), Euryarchaeota (205), Nanoarchaeota (10), and Thaumarchaeota (39). Indeed, if vertical descent were assumed for a recent Bayesian phylogeny of archaea with sequenced genomes (Brochier-Armanet et al., 2011), this would require at least 10 independent losses of the lysozyme gene, an assumption that is certainly less parsimonious than a single HGT event. Moreover, the relative divergence of the small subunit rRNA gene in A. boonei compared to the putative bacterial HGT donors is greater than the relative divergence of the lysozyme gene (Figure 4), a finding that is inconsistent with both genes being transmitted by vertical descent. Also, there are no other homologs beyond those presented in this study in 132 plant genomes, and only one insect species with additional homologs out of 109 insect genomes. Thus, if the lysozyme were present in the last common ancestor of all domains, it would require the unlikely loss of the gene in dozens of lineages while maintaining it in an exceedingly small number of species. In summary, the presence of a GH25 muramidase in nonbacterial species represents a series of recurrent, independent horizontal gene transfer events derived from diverse, ecologically associated bacteria.
A. boonei GH25 muramidase is antibacterial
We next undertook a series of experiments to test the hypothesis that the transferred muramidase functions as an antibacterial molecule. Since HGT frequently results in pseudogenized and nonfunctional genes (Kondrashov et al., 2006; Nikoh et al., 2010, 2008; Dunning Hotopp et al., 2007), we first investigated the amino acid sequences for preserved antibacterial action of the transferred lysozymes in nonbacterial genomes. We aligned all 86 GH25 muramidase sequences to identify conserved sites (Figure 5A). We then mapped the conserved amino acids to a three-dimensional structure prediction of the A. boonei GH25 muramidase domain (Figure 5B). Highly conserved residues (>85% identity between all taxa) invariably mapped to the previously identified active site pocket (Martinez-Fleites et al., 2009). Conservation was also evident for structure predictions of other GH25 muramidases in the phylogeny such as S. moellendorffii (Figure 5C).
Second, we cloned, expressed, and purified the GH25 muramidase domain from the archaeon A. boonei as well as from closely related homologs in Paenibacillus polymyxa and PhiBP. We obtained each muramidase in a pure elution (Figure 6—figure supplement 1) and tested for antibacterial action against a range of bacterial species. As predicted, the A. boonei GH25 muramidase efficiently killed several species of bacteria in the phylum Firmicutes - the putative donor group of the gene (Figure 6A). The bacterial inhibition by A. boonei GH25 muramidase was more potent than the positive control, chicken egg white lysozyme, and was dose-dependent (Figure 6B). Bacterial and phage muramidases did not elicit antibacterial killing, similar to cyan fluorescent protein and buffer-only negative controls. Bacteria typically use a large protein complex to limit their lysozymes' activity to the septum during cell division (Uehara and Bernhardt, 2011), and PhiBP phage has a documented spectrum of activity limited only to a P. polymyxa strain unavailable for our analyses (Halgasova et al., 2010). As expected, the A. boonei GH25 muramidase did not exhibit antibacterial activity against Gram-negative species or Gram-positive species outside of the families Bacillaceae and Paenibacillaceae, which was equivalent to the killing range of chicken egg white lysozyme with the exception of the Actinobacterium Micrococcus luteus (Figure 6—figure supplement 2).
Third, the A. boonei muramidase domain is part of a larger gene (1725 bp) composed of other domains that may broaden or constrain the range of antibacterial activity. To test the full-length gene's function in the absence of genetic tools in this system, we cloned the entire gene into an expression plasmid in E. coli and discovered that bacterial colonies grew poorly, with tiny, slow-growing colonies on solid media, and substantial cell death coinciding with a small amount of leaky expression in liquid culture (Figure 7A). However, two colonies grew to normal size and upon sequencing, we determined that their expression plasmids were disrupted by insertions of 774 bp (mutant 1) and 768 bp (mutant 2) of a native IS1 family transposase from E. coli at 21 bp or 266 bp from the start of the lysozyme gene, respectively. These insertions also resulted in a number of premature stop codons in the lysozyme reading frame, disrupting production of the full-length gene (Figure 7B). Thus, E. coli death requires intact and full-length lysozyme, and toxicity is not due to the expression construct itself. In sum, expression of the complete lysozyme resulted in E. coli death, while cloned genes with insertion sequences and premature stop codons abolished the lytic capacity of these proteins from within E. coli cells, providing evidence for an expanded host range to the antibacterial action.
Fourth, if horizontally transferred lysozymes serve as antibacterials to fend off bacterial niche competitors, two predictions follow: the lysozyme will be upregulated in response to bacterial competition and upregulation may correlate with a relative growth advantage in coculture. We thus cultured A. boonei cells in anaerobic marine media (Reysenbach et al., 2006) with and without cohabiting Mesoaciditoga lauensis (phylum Thermotogae) that was isolated from the same hydrothermal vent field as Aciduliprofundum in the Eastern Lau Spreading Center (Reysenbach et al., 2013). As expected, we observed a significant increase in A. boonei lysozyme expression at four (up to 127% increase) and 12 hr (up to 43% increase) of coculture with M. lauensis in comparison to negative control cultures of the singular A. boonei (Figure 8A). Ideally, A. boonei wild type and lysozyme knockouts would be employed to test relative fitness and bacterial inhibition. However, genetic manipulation of A. boonei is not currently feasible.
Growth experiments of A. boonei and M. lauensis were continued for 72 hr, during which there was a relative Malthusian fitness (Lenski et al., 1991) increase for A. boonei in coculture vs. monoculture (Figure 8B) across the exponential growth phase. This difference is marginally non-significant, perhaps due to low sample sizes (p = 0.11, N = 5, MWU two-tailed test). When the species are cultured separately for 72 hr, M. lauensis cell abundance is greater than that of A. boonei during 14 out of the 19 sampling points (Figure 8C, blue circles), indicating that bacteria outperform archaea in monoculture conditions. However, when the two species are cocultured, the cell abundances reverse and A. boonei outperforms M. lauensis for 14 out of the 19 time points (Figure 8D, red circles). This competitive frequency difference is significant (Chi-square test, p = 0.0035), complementing the Malthusian fitness increase. Additionally, for each monoculture time point, there are 4.43% fewer A. boonei cells on average than M. lauensis, while in coculture there are 6.22% more A. boonei cells per time point (Mann Whitney U. p = 0.023). Thus, A. boonei outcompetes its bacterial competitor in coculture despite a higher monoculture growth rate for the bacteria, although whether lysozyme upregulation is directly responsible for this effect cannot be definitively proven. Other possible explanations for this finding include A. boonei scavenging of bacterial waste products, possessing a superior ability to obtain a rate-limiting nutrient, or deploying alternative antibacterial defense mechanisms.
The universal tree represents the evolutionary relationships between cellular domains and establishes the modern foundation for benchmarking the magnitude of HGT across life. Indeed, HGTs have been described between each domain including archaea and bacteria (Nelson et al., 1999; van Wolferen et al., 2013), bacteria and eukaryote (Andersson, 2005; Bordenstein, 2007; Gladyshev et al., 2008; Danchin et al., 2010), and archaea and eukaryote (Andersson et al., 2003; Schonknecht et al., 2013). Despite these cases and others (Brown, 2003; Zhaxybayeva and Doolittle, 2011), HGTs are not without limits and often succumb to the selective costs of genomic rearrangements, cytotoxic effects, disruptive insertions, and functional inefficiencies upon integration (Baltrus, 2013).
It follows then that HGTs do not occur at equal rates across the universal tree, but rather experience preferential routes in which the costs of HGT are easier to overcome. The resulting pattern of HGT can be understood as a gradient of decreasing frequency from within domain > between two domains > between all domains of life (Bruto et al., 2013; Zhaxybayeva and Doolittle, 2011; Puigbo et al., 2009; Andam and Gogarten, 2011, 2013). In support of this pattern, the overwhelming evidence of gene transfers between bacteria is counterbalanced by the extreme lack of parallel gene transfers across all extant groups of life. As these parallel transfers are usually ancient and occur in non-model organisms (Lundin et al., 2010; Koonin et al., 2003; McClure, 2001; McDonald et al., 2012; Moran et al., 2012), they can defy clear interpretations due to their deep antiquity and lack of functional validation.
One feature that parallel HGTs have in common is that the gene's phenotype must transcend different physiological capabilities, cellular structures, and ecological niches to repeatedly increase the fitness of each recipient across the whole diversity of life. While not traditionally used in the context of parallel HGT across all cellular domains, the term niche-transcending gene appropriately captures these conditions (Wiedenbeck and Cohan, 2011). The lysozyme gene family we describe in archaea, bacteria, eukaryotes, and viruses provides one such example because the adaptive benefit of an antibacterial muramidase has repeatedly surmounted the obstacles against recurrent HGT. Indeed, horizontally transferred homologs of the GH25 muramidase exhibit differential tissue expression in A. pisum (Nikoh et al., 2010) and bacteriolytic activity in the fungus Aspergillus nidulans (AN6470.2) (Bauer et al., 2006). Thus, the horizontally transferred homologs in eukaryotes confer the same transcriptional and enzymatic activity as in the archaea.
The muramidase in a thermophilic archaeon is of special note as archaea do not possess murein cell walls (Albers and Meyer, 2011), and genes encoding an antibacterial peptide have never before been identified (Cantarel et al., 2009). Members of the genus Aciduliprofundum are widespread thermoacidophiles in deep-sea hydrothermal vent chimney biofilms (Flores et al., 2012) in which bacteria are frequent inhabitants (Orcutt et al., 2011a; Miroshnichenko and Bonch-Osmolovskaya 2006), including the M. lauensis species tested above. Archaea have been largely ignored in the context of antibiotic discovery, likely because of the conjecture that archaea do not compete with bacteria in nature. However, given that they coexist with diverse bacterial species in the environment (Oren, 2002; Kato and Watanabe, 2010; Orcutt et al., 2011b) and can compete for similar resources, there may be significant, unexploited potential for antibiotics in this domain. Only a handful of antimicrobial peptides produced by archaea have been characterized, and those are active only against other archaea (O'Connor and Shand 2002) despite the fact that archaea are known to inhibit bacteria in diverse environments (Atanasova et al., 2013; Shand and Levya, 2008). It is also possible that since Aciduliprofundum strains metabolize peptides, the lysozyme enables a nutritive strategy in which lysed bacteria provide nutrients for the archaeon to scavenge.
Based on this work, we suspect that systematic surveys of archaeal gene products will likely uncover a broad range of antibacterial activities, and may eventually offer novel peptide or small molecule therapeutics. Such antibacterial products may have naturally evolved thermostability that would increase their attractiveness as therapeutics. GH25 muramidases have been demonstrated as effective antibacterials against biofilms of Streptococcus pneumoniae (Domenech et al., 2011) and related enzymes have proven efficacious in mouse models of bacterial mucosal colonization (Fenton et al., 2010), sepsis (Loeffler et al., 2003), and endocarditis (Entenza et al., 2005).
In summary, we infer that the evolutionary path to this parallel HGT was paved by the universal drive for nonbacterial taxa to compete in a bacterial world. We predict that similar to the cascade of antibiotic gene transfer discoveries that followed their initial reporting, parallel transfers of genes to all cellular domains and viruses might regularly have antimicrobial functions.
Materials and methods
Unless otherwise stated, reagents were obtained from Fisher Scientific (Waltham, WI).
PCR and sequencing
PCR was performed using GoTaq DNA Polymerase (Promega, Madison, WI) with primers listed in Supplementary file 1. PCR products were electrophoresed using 1% agarose gels in sodium boric acid buffer. Following electrophoresis, gels were dyed with GelRed (Phenix Research, Candler, NC) and imaged on an Alpha Innotech GelRed Imager (Alpha Innotech, San Leandro, CA). Amplified bands were excised from the gels and purified with an SV Wizard Gel Cleanup kit (Promega). Following purification, DNA concentration was measured using the Qubit DNA high sensitivity kit (Life Technologies, Grand Island, NY) and sequencing reactions were performed by Genewiz (South Plainfield, NJ).
The lysozyme protein from Wolbachia prophage WORiA (ZP_00372884) was used as a query in a blastp search of the NCBI nonredundant protein database using Geneious Pro v5.5.6. All hits with E-values below 10−12 were collected and duplicate entries were removed. Sequences from field and laboratory samples were added to this collection and aligned with MUSCLE (Edgar, 2004), insertions and deletions were removed, and the eight most highly conserved residues from the MUSCLE alignment were mapped to a structure prediction of A. boonei lysozyme using PyMOL. Structure prediction was performed using the homology-based modeling tool Phyre2 (Kelley and Sternberg, 2009). For phylogenetic analyses, ProtTest (Abascal et al., 2005) was used to determine the best model of protein evolution based on the corrected Akaike information criterion (AICc). MrBayes (Ronquist et al., 2012) and PhyML (Guindon et al., 2010) were used to build a phylogenetic tree with Bayesian and maximum likelihood methods, respectively. For the global lysozyme phylogeny, the best model chosen by ProtTest (LG + I + G) was used to generate the maximum likelihood tree, while the third best model (WAG + I + G; ΔAICc: 74.82) was used to generate the Bayesian tree due to a lack of LG model availability in MrBayes. S. sanguinolenta and S. stauntoniana lysozymes were excluded from this analysis because frameshift mutations suggest the genes may be evolving in the absence of selection, while Aphidinae lysozymes were not included because of shorter sequences of the GH25 muramidase domain obtained through the use of degenerate primers that would have limited resolution of the tree.
In an iterative approach, each candidate example of HGT was used as a blastp query against the nr database and the top 15 (A. boonei, A. pisum, S. moellendorffii) or top 75 (A. oryzae) E-value hits were subjected to the same phylogenetic analysis as above. Evolutionary models used were A. boonei: WAG + I + G on a 148aa indel-free alignment, A. pisum: CpREV + I + G on a 190aa indel-free alignment, S. moellendorffii: WAG + I + G (ΔAICc: 44.28) on a 200aa indel-free alignment, A. oryzae: WAG + G (Bayesian, ΔAICc: 4.49) or LG + G (maximum likelihood) on a 186aa indel-free alignment. The fungal lysozyme was also phylogenetically analyzed on the DNA level using the top 25 E-value blastn hits to exon 2 of the A. oryzae lysozyme gene. jModelTest 2 (Darriba et al., 2012) was used to determine the best model of nucleic acid evolution (GTR + I + G, ΔAICc: 7.33) of a 282 bp indel-free alignment. The archaea HGT clade was also analyzed phylogenetically with a Bayesian tree of selected taxa using lysozyme protein sequences (WAG + I + G, 185aa) and compared to 16S rRNA (GTR + G, 1,156 bp) for the same strains obtained from SILVA (Quast et al., 2013). A schematic representation of putative HGT events was plotted on a Bayesian phylogeny based on the 16 s rRNA gene (GTR + G, 1,226 bp). Representative species used for this phylogeny were Magnetospirillum magneticum (Proteobacteria), Paenibacillus polymyxa (Firmicutes), Arthrospira maxima (Cyanobacteria), Crenarchaeota archaeon SCGC AAA471-B05, Chthoniobacter flavus (Verrucomicrobia), Thermotoga maritima (Thermotogae), Pedobacter saltans (Bacteroidetes), Streptomyces violaceusniger (Actinobacteria).
Statistical support for the HGT hypothesis was assessed with the Shimodaira–Hasegawa test (SH-test) (Shimodaira and Hasegawa, 1999) as implemented in RAxML v.8.0.20 (Stamatakis, 2014). An unresolved binary constraint tree was generated in MacClade v4.08, in which bacterial sequences are monophyletic, as are nonbacterial sequences, with all other topology unconstrained. This constraint tree was used to generate a maximum likelihood best tree with RAxML, using the same evolutionary models as above. The SH-test was then run comparing the maximum likelihood constrained tree to the unconstrained consensus Bayesian tree or to 100 bootstrap trees from the maximum likelihood analysis from PhyML. This procedure was repeated for a three-domain constraint tree consistent with the tree of life and with a constraint tree in which eukaryotic sequences were monophyletic and other sequences were unconstrained.
Lysozyme cloning and purification
A. boonei GH25 muramidase domain (ZP_04874596), P. polymyxa lysozyme (YP_003869492), and PhiBP lysozyme (CBA18122) were cloned and expressed with a 6x C-terminal histidine tag using an Expresso T7 Cloning and Expression System (Lucigen, Middleton, WI) according to the manufacturer's instructions. We also cloned the S. moellendorffii and A. oryzae GH25 muramidases, however recombinant 6× histidine-tagged proteins were insoluble when expressed in either E. coli or sf9 insect cells and attempts to solubilize them were unsuccessful. Sequence-confirmed expression plasmids and a control plasmid expressing cyan fluorescent protein (CFP) were transformed into HI-Control BL21 (DE3) E. coli cells. Cultures at an OD600 of ∼0.5 were induced with 1 mM IPTG for 6 hr, centrifuged, and frozen at −80°C until purification. Frozen pellets were resuspended in lysis buffer containing 10 mM Tris–HCl, pH 7.5, 300 mM NaCl, 0.5% Triton X-100, 0.3% sodium dodecyl sulfate, and 1 mM phenylmethylsulfonylfluoride and sonicated 5 times for 30 s with at least 1 min on ice between sonications. Samples were centrifuged and recombinant proteins were purified from supernatant using HisPur Ni-NTA chromatography cartridges (Thermo Scientific, Waltham, MA) according to manufacturer's instructions. Glycerol at a final concentration of 40% was added to enzymes in elution buffer for storage at −20°C for a maximum of 3 weeks before use in antibacterial assays. Purifications were analyzed with denaturing polyacrylamide gel electrophoresis and stained with GelCode Blue (Thermo Scientific).
Full-length A. boonei lysozyme and WORiA lysozyme were cloned into a pET-20b vector (EMD Millipore, Darmstadt, Germany) with a C-terminal 6× histidine tag and sequence-confirmed plasmids were transformed into BL21 (DE3) E. coli (EMD Millipore). Three colonies from each transformation were inoculated into LB media and grown to an OD600 of ∼0.5, induced for 4 hr with 1 mM IPTG and harvested for analysis on PAGE gels. Overnight cultures without induction were examined for bacterial death with a BacLight Live/Dead Stain (Life Technologies).
Antibacterial assays
Purified A. boonei GH25 muramidase, P. polymyxa lysozyme, PhiBP lysozyme, CFP, and commercially purchased CEWL (Sigma-Aldrich, St. Louis, MO) were diluted to 100 μg/ml in buffer EG (60% nickel column elution buffer, 40% glycerol) and filter sterilized. Bacteria to be tested were grown overnight in tryptic soy broth, split 1:10, and incubated to exponential growth before being diluted into each enzyme solution. Samples were incubated with shaking for 20 min at 37°C and then 5 μl was spotted onto tryptic soy agar and incubated overnight at 37°C. To evaluate whether antibacterial activity is dose-dependent, B. subtilis was incubated with A. boonei GH25 muramidase at 100 μg/ml, 75 μg/ml, 50 μg/ml, 25 μg/ml and 0 μg/ml and 100 μl was spread on tryptic soy agar plates. Replicates of 10 were performed for each concentration, plates were incubated overnight at 37°C, and colonies were counted the following morning. Bacterial strains used in these experiments are listed in Supplementary file 1.
A. boonei cultures
A. boonei and M. lauensis cultures were performed as previously described (Reysenbach et al. 2006) with the following modifications: yeast extract was added at 2.0 g/l, pH was adjusted to 4.8, and cultures were incubated at 65°C. For gene expression studies, 8.2 × 105 cells were inoculated into 5 ml cultures in 6 replicates each of monocultures and cocultures at 0.1:1, 1:1, and 1:0.1 ratios and 500 μl samples were collected after 4 and 12 hr of co-incubation and frozen for expression analysis. RNA was isolated from frozen samples using an RNeasy Mini Kit (Qiagen) and QIAshredder (Qiagen), DNA contamination was removed with a Turbo DNAfree Kit (Life Technologies), and reverse transcription was performed using a Superscript III first Strand Synthesis System (Life Technologies) along with no-reverse transcriptase controls. Quantitative PCR was performed with GoTaq qPCR Master Mix (Promega) using a CFX96 Real-Time System (Bio-Rad, Hercules, CA). Primers are listed in Supplementary file 1. For competition studies, 5 replicates of 5 ml cultures were inoculated as monocultures or 1:1 cocultures and 175 μl was collected every 4 hr for counting of relative species abundance with a hemocytometer. Relative fitness was calculated based on Malthusian parameters over the period of exponential growth as previously described (Lenski et al., 1991).
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We are grateful to members of the Bordenstein and Reysenbach labs for helpful discussions, and particularly to Christine Sislak for Mesoaciditoga lauensis cultures, Sarah Bordenstein for WO sequences, and Jessica Hardwicke for assistance with cell counting. We also thank Antonis Rokas, Jennifer Wisecleaver, Kristin Jernigan, and Elise Pfaltzgraff for technical assistance, and Julie Dunning Hotopp and Barton Slatko for helpful feedback on an earlier version of this manuscript, as well as Patrick Abbot for aphid samples and manuscript feedback, and Robert Brucker for support with figures.
Decision letter
eLife posts the editorial decision letter and author response on a selection of the published articles (subject to the approval of the authors). An edited version of the letter sent to the authors after peer review is shown, indicating the substantive concerns or comments; minor concerns are not usually shown. Reviewers have the opportunity to discuss the decision before the letter is sent (see review process). Similarly, the author response typically shows only responses to the major concerns raised by the reviewers.
Thank you for sending your work entitled "Antibacterial Gene Transfer Across the Tree of Life" for consideration at eLife. Your article has been favorably evaluated by Diethard Tautz (Senior editor), Wilfred van der Donk (Reviewing editor), and 3 reviewers (Peter Gogarten, Anthony Poole, and David Baltrus). The Reviewing editor and the reviewers discussed their comments before we reached this decision, and the Reviewing editor has assembled the following comments to help you prepare a revised submission.
The study provides clear evidence for the transfer of the GH25 muramidase between the three domains of life. Phylogenetic analyses establish that the eukaryotic and archaeal versions of the gene have bacterial neighbors and that the transfers occurred more recently than the splitting of the domains. Furthermore, the use of phenotypic tests as well as expression in E. coli establishes that the GH25 muramidase is functional across its range and not a pseudogene or other non-functional relic. The assessment of genomic context also supports that the GH25 muramidase gene has been transferred from bacteria to archaeal and eukaryote recipients.
The potential antibacterial role of the GH25 muramidase gene in the archaeal recipient Aciduliprofundum boonei is addressed by showing that the product of the transferred gene exhibits antibiotic properties against expected species in purified form. Experimental work in a non-model system that lacks standard genetic tools is challenging, so these experiments along with the data in E. coli provide a good means of assessing function.
Finally, the authors test whether the gene can impact competitors. Again, without the capacity to undertake direct knockouts, the authors do an admirable job of testing this, by examining relative growth in both monocultures and cocultures with Mesoaciditoga lauensis, a bacterium known to cohabit the environments from which A. boonei has been isolated.
All reviewers agreed that this study is of high interest to the readership of eLife, but request you address the following points in a revised manuscript:
1) One question stems from the observation that not all field isolates appear to have lysozyme genes. If there are isolates that genuinely lack the GH25 muramidase genes, these would make a good control. It would be helpful if the authors could comment on whether they think the isolates definitely lack the GH25 muramidase gene, and whether they have tried the growth and coculture assays with any of these isolates.
2) The clarity of the manuscript would benefit greatly from the inclusion of a single phylogeny (recA-like genes?) across all domains of life with arrows actually showing putative transfer directions. The included phylogenies are great, but don't necessarily give an intuitive sense of how distant these transfers are. The inclusion of non-lysozyme containing taxa would really help tell the story to a wider audience.
3) The SH tests of phylogenies are significantly different and demonstrate that the constrained tree is a poorer fit than the unconstrained tree. However, this only demonstrates that at least one transfer has occurred while the study is about numerous independent transfers...it is possible to sequentially constrain the tree and perform independent SH tests for each potential transfer, which would make a stronger story.
4) In regards to the competition assays, the reviewers realize that the authors are dealing with intractable genetic systems, but were wondering if there might be cleaner ways of comparing growth between microbes, instead of independent vs. together. The current comparison is confounded because a variety of lysozyme independent reasons could be responsible for a shift in competitive outcome in co-culture (i.e. cross-feeding of archaea on bacterial waste products). Would it be possible to isolate lysozyme resistant bacteria and perform paired competition assays with sensitive and resistant versions? The reviewers felt that the story might be good enough as is without such experiments but wanted to share their thoughts with the authors. Regardless, the authors should point out the caveats of their approach in the paper.


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text:
Does Screening for Pain Correspond to High Quality Care for Veterans?
- First Online:
- Received:
- Revised:
- Accepted:
DOI: 10.1007/s11606-010-1301-5
- Cite this article as:
- Zubkoff, L., Lorenz, K.A., Lanto, A.B. et al. J GEN INTERN MED (2010) 25: 900. doi:10.1007/s11606-010-1301-5
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Routine numeric screening for pain is widely recommended, but its association with overall quality of pain care is unclear.
To assess adherence to measures of pain management quality and identify associated patient and provider factors.
A cross-sectional visit-based study.
One hundred and forty adult VA outpatient primary care clinic patients reporting a numeric rating scale (NRS) of moderate to severe pain (four or more on a zero to ten scale). Seventy-seven providers completed a baseline survey regarding general pain management attitudes and a post-visit survey regarding management of 112 participating patients.
We used chart review to determine adherence to four validated process quality indicators (QIs) including noting pain presence, pain character, and pain control, and intensifying pharmacological intervention. The average NRS was 6.7. Seventy-three percent of charts noted the presence of pain, 13.9% the character, 23.6% the degree of control, and 15.3% increased pain medication prescription. Charts were more likely to include documentation of pain presence if providers agreed that "patients want me to ask about pain" and "pain can have negative consequences on patient's functioning". Charts were more likely to document character of pain if providers agreed that "patients are able to rate their pain". Patients with musculoskeletal pain were less likely to have chart documentation of character of pain.
Despite routine pain screening in VA, providers seldom documented elements considered important to evaluation and treatment of pain. Improving pain care may require attention to all aspects of pain management, not just screening.
KEY WORDSpain quality assessment veterans
Pain is a common debilitating condition. Approximately 20% of patients and 25% to 50% of community-dwelling elders experience chronic pain.1,2Although many studies have identified under-treatment of pain,1, 2, 3 few have examined why providers vary in responding to patient-reported pain.3,4 Such information would help inform more successful approaches to pain management.
In 2003, the Department of Veterans Affairs (VA) implemented the "5th vital sign"—a rating also known as the numeric rating scale (NRS)3,4 to assure routine pain assessment and improve management.3,5, 6, 7, 8, 9 The NRS asks veteran patients to rate current pain from zero or "no pain" to ten or "worst possible pain".3,6,7
Moderate to severe pain, measured by a score on the NRS between four and ten, should typically trigger a comprehensive pain assessment and prompt intervention.5 However, one study at a single VA medical center, found that routine pain screening did not change provider medication management.3 That study occurred soon after implementing the 5th vital sign, using a pre-post design and did not examine the extent to which providers utilized the NRS for pain evaluation and management. This study builds on earlier work in developing an assessment across multiple sites with established NRS procedures and examines multiple processes of care, from assessment of pain to treatment.
We examined indicators of four key processes of pain evaluation and management in a regional cohort of facilities where routine pain screening was implemented during the last 10 years. We estimated adherence to these indicators and examined patient and provider factors associated with these processes of care.
Subjects and Setting
The Helping Veterans Experience Less Pain (HelpVets) study is a VA-funded cross-sectional cohort study designed to evaluate adherence to basic pain care management. We conducted the study from March 2006 to March 2007 in primary care, oncology, women's health and cardiology outpatient clinics at two VA hospitals and six associated clinics in the VA Greater Los Angeles Healthcare System.
Participants: Patients
We approached patients in the waiting areas of the outpatient study clinics; patients meeting inclusion criteria were invited to participate. Eligible patients were seen by participating providers, had vital signs taken during their visit, passed a brief cognitive screening test, spoke English, had intact hearing, had not previously participated and agreed to have their medical records reviewed. To assure an adequate number of subjects with possible pain-related conditions, we included all patients who reported fair or poor health, and every other patient who reported good (or better) health. In total, 6,138 patients were approached. Of those approached, 862 refused screening; 4,337 were ineligible (2,265 had not yet had vital signs taken, 942 had not visited a provider, 310 visited non-participating clinics, 171 exhibited cognitive problems, 103 visited non-participating providers, 61 previously participated, 49 were hearing impaired). Of the remaining 939 eligible patients, 650 completed the patient baseline survey yielding a 69.2% response rate. Further details of the methods are elsewhere.6 The present analysis includes the 140 patients reporting moderate to severe pain (an NRS of four or higher).3,5,7, 8, 9
Patient Baseline Survey
The baseline survey asked patients about the degree to which pain was discussed during the nurse and provider visits, current treatment regimens and general quality of life issues.
Participants: Providers
Provider Baseline Survey
We asked all providers (n = 260) in relevant clinics to complete a baseline survey about attitudes, beliefs, and practices prior to patient interviews. Of those approached, 209 (74%) completed surveys. This study includes 77 providers (45 staff physicians, nine fellows, five house staff, 14 nurse practitioners and four physician assistants) who cared for 140 patients in the analytic sample. Of the 77 providers, 57% had one patient, 17% had two patients, 14% had three patients, 10% had four patients and 1% had five patients.
Provider Post-visit Survey
After the patient visit, providers were asked to complete a post-visit survey that assessed provider's perception of, and discussion about patient's pain as well as whether they initiated or intensified a pharmacological intervention. If applicable, we queried about reasons for not changing treatment. Sixty-five of the 77 providers completed post-visit surveys for the 140 patients in the initial analytic sample, yielding valid post-visit provider data for 112 patients.
Additional Data Sources
Medical Record Review
Data elements for all four indicators were abstracted from clinic notes in the VA's electronic medical record (Computerized Patient Record System or CPRS) by one, experienced abstractor. To assure accuracy, the abstractor reviewed questionable records with physician investigators.
Pharmacy Data
We obtained pharmacy data from the regional data warehouse to supplement medical record estimates of pain regimen alteration. We identified pain medications dispensed within 90 days before and 90 days after each study visit. Of the 747 total prescriptions dispensed, 282 prescriptions were 24 unique agents used to treat pain including: acetaminophen, non-steroidal anti-inflammatory drugs (NSAIDs), anti-seizure medicines used for analgesia, opioids and topical analgesics. A Ph.D. data abstractor characterized each prescription for each patient based on days of supply, dose, instructions and frequency of dispensing during the study. Increases in any of these factors resulted in an "increase" rating, while a decrease in any factors received a "decrease" rating. Prescriptions that remained consistent received a "no change" rating. After reviewing all medicines, each patient received an overall rating of "increase", "decrease", or "no change". A physician over-read 10% of all characteristics and to resolve any clinical difficulties that the abstractor faced. Of note, the available pharmacy data did not include non-VA pharmaceutical use.
Conceptual Framework
The Theory of Planned Behavior by Azjen and Fishbein explores the relationship between beliefs, attitudes, intentions and subsequent behavior.10 It postulates that a person's attitude toward performing a behavior influences intent as do beliefs about the behavior by persons(s) who are important to the individual.11, 12, 13 This theoretical model helps explain how provider beliefs, attitudes, and intentions may be important in understanding feelings about the NRS and responses to a moderate to severe pain rating. Provider attitudes toward a behavior (shaped by beliefs), subjective norms (determined by social standards), and provider perceived behavioral control about documenting and treating pain were all key constructs in evaluating determinants of pain management. However, it was infeasible to collect some variables in the framework and they were omitted from this analysis.
Measures: Dependent Variables (Quality Indicators)
The four quality indicators (QIs) evaluating pain assessment and management were adapted from the Assessing Care of Vulnerable Elders (ACOVE)14, 15, 16, 17, 18 and Quality Assessment (QA) Tools measurement sets.19,20 Specifically, the four stem from the following recommendations for patients with moderate to severe pain on the NRS: 1. evaluate the presence or absence of pain; 2. evaluate the character of the pain; 3. note the degree of pain control; and 4. initiate or intensify pain treatment.
We assessed the degree to which providers adhered to each individual quality indicator, leaving interpretation deliberately liberal to meet the intent of the indicators. For example, any documentation of pain (or absence of pain) in the progress note met the first quality indicator. The character of pain included many terms such as type (e.g., throbbing, intermittent), location, and/or mitigating or worsening factors. General statements about improvement or stability of pain symptoms satisfied evaluation of pain control. Pharmacy prescriptions were carefully examined for initiation or intensification of pain management; we also assessed progress notes for advice on current prescriptions or over-the-counter medication(s).
Measures: Independent Variables and Covariates
Selection of independent variables representing provider and patient attitudes and demographics was guided by: 1. Literature review1,3,13,21, 22, 23, 24; 2. Conceptual framework10, 11, 12, 13; and 3. Experts in palliative care and research.19,20,25, 26, 27, 28, 29, 30
Baseline Provider Variables
Baseline patient variables
Post-visit provider variables
The post-visit analytic variables included dichotomous questions (yes or no responses) that asked whether during the visit the provider: (1) noticed the nurse's NRS; (2) thought the patient was in pain; (3) talked about the symptom of pain; (4) had reasons for not discussing pain, as applicable (four different reasons); and (5) reasons for not initiating or intensifying treatment (five options).
Institutional Review Board (IRB) approvals were obtained by the VA Greater Los Angeles Healthcare System and the VA Long Beach Healthcare System.
We performed weighted bivariate logistic regressions to measure associations of the provider baseline variables with the four quality indicators. All associations with p-value <0.10 were included in multivariate analyses. We employed weighted logistic regressions fitting nested models by sequentially adding blocks of variables in StataSE 10.0. In each model, we first entered the block representing the provider attitude variables because we felt those variables were, conceptually, the most important for predicting the quality indicators. We then added a second block containing variables for two diagnoses (substance abuse and musculoskeletal pain), as these were previously identified as potential barriers in responding to pain. Patient and provider demographic variables (patient race and age, provider race) were added into the model last. The nested models were performed as a sensitivity analysis to confirm our model. We report the "full" model containing all variables.
In addition, we examined whether there were differences in provider reporting and medical record documentation by evaluating the percent of patients who received initiation or intensification of treatment (based on chart review) and compared that information to responses on the provider post-visit survey.
All analyses were weighted for patient survey non-response based on age, race, general health, cognition score, and adjusted for over-sampling of persons in fair to poor health. No adjustment for clustering by provider was needed given minimal variation in the number of patients per provider, with 74% of providers having one or two patients.
Provider Attitudes About Patients with Paina
% Providers whom strongly agree with:
Percent (%)
Patients are able to rate their pain intensity
0–10 ratings by nurses accurately reflect patients painb
Patients want me to ask about their pain
Pain medicines should be given when pain is severe
Pain has negative consequences on patient functioning
Pain rating is an important part of vital signs
Patients are more satisfied when pain concerns are addressed
Frequencies of Quality Indicators from Chart Review
When patient reports moderate to severe pain, was... (N = 140)
Yes (%)
The presence of pain noted?
102 (72.8)
The character of the pain noted?
19 (13.9)
The degree of pain control?
33 (23.6)
Treatment intensified or initiated?
21 (15.3)
Nested Logistic Regressions to Predict Quality of Care Indicators
N = 120c
Presence of Pain
Character of Painb
Degree of Pain Control
Initiate/ Intensify Treatment
Independent variables
"% Outpatients with pain"
"Patients are able to rate their own pain intensity"
"0–10 pain ratings by nurses accurately reflect patients pain intensity"
"Patients want me to ask about their pain problems"
"Pain medication should be given when pain is severe"
"Pain can have negative consequences for a patient's functioning"
"When reviewing patients' vital signs, I pay attention to 0–10 pain score"
"Patients more satisfied when address pain concerns"
Substance abuse
Musculoskeletal pain
Patient race (white vs. others)
Patient age
Provider race (white vs. others)
Presence of Pain Noted
Providers were more likely to document the presence of pain if they believed that: "patients are able to rate their own pain intensity" (OR = 2.02, p = 0.01), "patients want him/her to ask about pain" (OR = 3.49, p = 0.01) or if "pain medications should be given when pain is severe" (OR = 3.17, p = 0.01). Providers were also more likely to document pain presence if they perceived a "pain rating as an important vital sign" (OR = 1.82, p = 0.01) and "patients were more satisfied when they address pain concerns" (OR = 2.41, p = 0.02), but less likely to document pain for patients with substance abuse history (OR = .399, p = 0.02). In multivariate analysis (Table 3), two provider attitudes were significantly associated with the notation of pain presence: "patients want me to ask about pain" (OR = 4.20, p = 0.04) and "pain has negative consequences on patient functioning" (OR = 7.10, p = 0.01).
Character of Pain Noted
In bivariate analysis, documentation of pain character was found less often for patients who had a diagnosis of musculoskeletal pain (OR = 0.25, p = 0.01). In multivariate analysis, "patients are able to rate own pain intensity" was associated with better performance of pain character being noted (OR = 2.48, p = 0.05); those with musculoskeletal pain were less likely to have the character of pain documented (OR = 0.22, p = 0.05). The model was significant (χ2 = 29.12, p = 0.01).
Degree of Pain Control Noted
In bivariate analyses, the following were associated with noting degree of pain control: provider's perception that patients could "accurately rate their pain intensity" (OR = 2.03, p = 0.01), the need for providing "pain medications when pain is severe" (OR = 2.83, p = 0.01), pain having "negative consequences on patients' functioning" (OR = 3.75, p = 0.01), and paying "attention to 0–10 pain scores when reviewing patient vital signs" (OR = 1.82, p = 0.04). Patient age was inversely associated with documenting pain control (OR = 0.98, p = 0.10). These relationships did not persist in multivariate analyses.
Pain Treatment Initiated or Intensified
Performance on this indicator was not significantly associated with any of the provider attitudes or other variables in either bivariate or multivariate analyses.
Post-visit Survey
Providers completed at least part of the post visit survey following 112 (80%) of patient visits. Providers reported they noticed the NRS during 67% of visits and in 64% of visits reported the patient was in pain.
While medical record and pharmacy data review found documentation of treatment intensification in just 15% of visits, providers self-reported initiating or intensifying treatment at 48% of the visits. Thus, agreement between the two sources was poor (kappa = 0.076, 95%CI = −0.1, 0.25).
Providers gave various reasons for not initiating or intensifying treatment for 40 such patients. The most common reason was that "the patient preferred not to change his/her current approach" (56%) followed by "everything has been done" (34.7%), "patient not experiencing pain" (26.0%), "patient misusing pain medication" (8.5%) and "patients not responding to previous treatments" (5.4%).
We found that among patients who reported moderate to severe pain at an appointment, providers often noted the presence of pain but did not clinically evaluate it further or modify pharmacologic treatment. Provider attitudes were weak predictors of adherence to quality indicators, although some provider attitudes toward the NRS were correlated with QI adherence.
Similar to prior studies, 3,6,25 our results question whether the 5th vital sign has substantively improved pain assessment and management. More notably, Mularski et al.3 reported that NRS implementation did not change visit-associated pain documentation or treatment intensification. Our results extend previous work in three ways. First, our indicators were closely adapted from ACOVE14, 15, 16, 17, 18 and were subjected to a rigorous development process. As such, the quality indicators allowed us to more broadly measure processes of care (i.e., notation, character and severity of pain). Second, Mularski et al.3 conducted their study between 1999 and 2001, soon after the institution of the NRS when providers were perhaps not familiar with it. Our results, a decade later, suggest the screening tool still has had limited impact on pain management. Third, our work extends Mularski's findings from a single VA site to multiple sites.
Why the 5th vital sign has not produced more substantive improvements is unclear. One possibility is the NRS does not accurately represent pain levels or that providers do not perceive it as accurate, and hence do not act based on the rating scale. Indeed, 12% of providers disagreed or strongly disagreed that "patients are able to rate their own pain intensity on the 0–10 scale" (Table 1), suggesting concern with the tool. Another possibility is that providers do not approach pain evaluation uniformly. Chronic pain patients are vexing for many providers22, 23, 24, 25, 26 and the subjective nature of pain likely adds to variance in addressing it. While rating pain numerically may create a standard measurement, it may not yield a standardized approach to its subsequent evaluation and management. Further research is needed on variations in pain management but, in the meantime, providers and patients might benefit from clinical decision support tools (e.g., clinical reminders) within CPRS.
Measuring pain management poses unique methodological challenges. Chart review may not be optimal (for example, pharmacy records may not represent all provider actions), though it remains the current gold standard. However, our study looked for new or adjusted prescriptions within 90 days of a visit to evaluate for initiation or intensification. Even so, our results identified a divergence between providers' self-described actions and pharmacy records. This could point to inaccurate information reflected in the pharmacy records, although this is unlikely within the VA. However, our pharmacy data did not include non-VA prescriptions and this may have importance since some patients, particularly those who have an $8 per month co-payment for each prescription, purchase medications privately. Several non-pharmaceutical intensification strategies were reported by patients to be useful (e.g., physical therapy, ice). Also, at times, initiation or intensification may not have been the most appropriate clinical action. When intensification did not occur at the visit, providers most commonly reported that patient preference was not to change treatment, "everything had been done", or the patient was not experiencing pain. Furthermore, visit based quality indicators may not be accurate proxies for measuring the quality of pain care at a single visit. Future research could consider longitudinal information to better understand factors influencing pain management. In addition, qualitative interview of providers may help to understand adherence to and predictors of the quality indicators. We examined only VA outpatients, limiting generalizability to other populations and healthcare systems. However, the VA is a leader in chronic care quality measurement2,3,17,20,23,24 and our study sites implemented routine pain screening a decade ago suggesting our findings represent a 'best case scenario.'
Our study provides greater understanding of how the 5th vital sign and providers' attitudes toward pain influence the quality of pain care. We found that simply implementing the NRS does not necessarily improve subsequent pain evaluation and management, as measured by four standardized quality indicators. Future research should examine what additional provider factors are associated with meeting the quality indicators and consider assessing non-pharmacologic interventions. Focused research could include bolstering the 5th vital sign beyond categorizing pain at one point in time; for example, additional screening question(s)31, 32, 33 might address whether a patient's current regimen provides sufficient pain control. Whether adding such screening questions would improve pain management is unknown, but it appears that using the NRS scale alone does not guarantee high quality pain management.
Funded by the VA HSR&D 11R #03-150 and Associated Health Professions Postdoctoral Training in Health Services TPP 65-007. Dr. Zubkoff was a Health Services Fellow at the Greater Los Angeles Healthcare Veterans Affairs during the time of this study and has since relocated to the New England Healthcare Engineering Partnership at the White River Junction Veterans Affairs.
Conflict of Interest
None disclosed.
Open Access
This article is distributed under the terms of the Creative Commons Attribution Noncommercial License which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author(s) and source are credited.


text:
Donald, that is a most remarkable arilbred! I take it that Autumn Echo is a
reblooming TB. Possibly you have a
reblooming variegated AB. I think
it's prettier than most variegated TBs. Please take good care of it. If it will grow decently, introduce it.
Francelle Edwards Glendale, AZ Zone 9
-----Original Message-----
From: Donald Eaves
Sent: Saturday, April 10, 2004 11:04 AM
To: firstname.lastname@example.com
Subject: [iris-photos] variegated
AB sdlg
Early last fall I posted some photos of the TB X AB
seedlings that have
turned up with variegated foliage. They had
only recently been removed from
the pots. I didn't expect any of those late
line-outs to bloom, but quite a
few are going to bloom or try. Quite a few
bloomouts, too. But perhaps the
correct term for the foliage would be 'broken
color' instead of variegated.
This bloom looks like something from the
Kasperaks. The stalk is only about
six inches high. It really should have
waited 'til next year to bloom. Not
even a clue about the ID of the pollen
parent. AUTUMN ECHO just was in a
pathway toward other things and I would stop and
daub whatever I was using
at the time. It got lots of varied daubing,
but I have no idea which ones
resulted in the pods.
Donald Eaves


text:
This is the full research report for 220.127.116.11, which is an IP address.
Dynamic Broadband Services
Telefonia Dialog S.A. - Dialog Telecom
Netia Telekom S.A. Contact Role
Poleczki 13
02-822 Warszawa, Poland
Telephone: 4822352 0000
Fax: 4822352 2213
Netia Telekom S.A. Contact Role
Poleczki 13
02-822 Warszawa, Poland
Telephone: 4822352 0000
Fax: 4822352 2213
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text:
Seventeen - Beauty And The Senior 2 (DVDRip/1.46 GB)
14-03-2016, 22:53
What man doesn't dream of a horny young woman waking him up with a blowjob? It is exactly what Anastasia and Leda love to do! Of course their "victims" wake up, and what happens next?! Meanwhile Luciana has a very special way to thank the painter for his services, and Eva climbs on top of a ping pong table to offer her tight little behind to Hugo. Something he obviously can't refuse!
● Released: 2016
● Studio: Seventeen
● Starring: Anastasia, Eva, Luciana, Leda, Hugo
● Genre: Teens, Young, Older Men, Oral, Outdoor, Cumshot, Anal
● Quality: DVDRip
● Video: Xvid 704x400 25fps 1760kbps
● Duration: 01:47:39
● Size: 1.46 GB
Download: Seventeen - Beauty And The Senior 2 (DVDRip/1.46 GB)


text:
ED Magedson – Founder
Ripoff Report
Frontier Pharmacy - Cp2pay.comInternet U.S.A.
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Fix it the right way.
Corporate Advocacy Program™
This report was posted on Ripoff Report on 11/16/2007 11:44 AM and is a permanent record located here: http://www.ripoffreport.com/reports/frontier-pharmacy-cp2paycom/internet/frontier-pharmacy-cp2paycom-cp2paycom-frontier-pharmacy-charges-account-monthly-with-n-285178. The posting time indicated is Arizona local time. Arizona does not observe daylight savings so the post time may be Mountain or Pacific depending on the time of year.
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You are currently using an unsupported web browser.
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Airport Motel is located in Gurgaon, Haryana. Its rooms offer a spectacular view of lush green lawns, have elegantly appointed furnishings, individual climate control, color television, a well stocked mini-bar, attached bath and all standard amenities.
The motel is an ideal venue...
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text:
- Home
- Jewelry
- Jewelry Organizers & Storage
- Bead Storage & Jewelry Making Organizer Supplies
- Acrylic Bead Container-Clear
Acrylic Bead Container-Clear
Product ID: 2025-251
9 1/2x6 3/8x1 1/8 inch acrylic bead container 1 piece per pkg.
Note: Storage items are often shown with the craft supplies they are intended to organize. Unless otherwise noted above, these supplies are not included in your purchase.
Color: Clear
Material: Other
Product Type: Organizers
Shape: Other
Our Price: $6.47
1 piece per package.


text:
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This step is elective but will greatly increase your chances of your profile being viewed by others. Let people know a bit about yourself, your interests and upload some good photos of yourself.
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Announcement All jewelry handmade, by me, Jen at PoleStar Jewelry®
All jewelry handmade, by me, Jen at PoleStar Jewelry®
- All 527
- Birthstone of the Month 20
- Sterling Silver Earrings 167
- Sterling Silver Necklace 256
- 14K Gold Filled Earrings 25
- 14K Gold Filled Necklace 28
- 14K Gold Jewelry 3
- Sterling Silver Bracelet 3
- Gold Bracelet 1
- Brass and Copper Jewelry 10
- Sale and Clearance 14
deiseal on May 19, 2017
Necklace shipped promptly and arrived in a cute little box; item is exactly as pictured and is a lovely statement piece. Very pleased with purchase and service. Will buy from seller again.
kcolleen on May 15, 2017
Very pretty earrings - just as pictured and super fast shipping! Thanks!
LJ Barat on May 5, 2017
The chain is nowhere near as substantial as in the picture. Very pricey for what you get.
Shop Story
I thank you for visiting my shop and taking time to read a little more about me.
My name is Jen Cowgill and I make, photograph, market and do everything involved in running PoleStar Jewelry® .
Providing quality jewelry that makes each day a little more enjoyable for you is my goal in each piece I create. I also take the presentation of the jewelry seriously and strive to represent the jewelry how you would see it looking at it very closely with your eye.
When you convo me or connect with me through social media, you will be talking to me. I am proud to run a business from home and be an example of a working mother to my two sons, who are in elementary school.
Whether it is through a transaction with no communication other than my thank you and shipping notice to you, or a friendly chat through email; I want you to feel that the jewelry you own or gift will last for years and know that if you do not feel that way, I will remedy it. I am here to make you feel happy with your purchase.
The happiness that comes with shopping on with me is not only knowing you have a quality product, but that you can also receive the support of the maker that you cannot get in todays world of big box mass produced merchandise.
I am proud to be a part of the handmade ethos, and honored you are shopping with me.
Thanks for stopping by and feel free to contact me any time.
Shop members
Jennifer Cowgill
Owner, Maker, Designer, Packaging
My name is Jen and I love making and wearing handmade jewelry. I am honored to have the opportunity to share that with you.
Shop policies
Payment options
- Accepts Etsy gift cards
Returns & exchanges
- Custom or personalized orders
Privacy policy
- To communicate with you about your order
- To fulfill your order
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text:
6 Part 1
Matt looked at Anna. He looked at her brave, but frightened, face. He looked at the blood on her clothes and knew that she had proved herself. Still, he wished he did not have to be the one to tell her this.
"Do you really want me to tell you, Anna?" he said. "I can understand it if you don't. I can understand if you just want to live a normal life, without this knowledge. It's a heavy burden to bear, but if you feel that you are ready…" His words were solemn, reflecting the expression on his pale, beautiful face.
Anna squared her shoulders. She had no idea what they were about to tell her, but she wanted to face it bravely.
"I am ready," she said. Her words echoed around the room, which was silent despite the amount of people in it. Kathryn and Callie looked frightened, for they too had no idea what Anna was about to find out.
"So young …" said Matt, and smiled sadly.
"I'm only a year younger than you, so don't start that!" exclaimed Anna, furiously. "Don't patronize me, Matt!"
"I may only be a year older in human terms, but do you know how long I have truly lived?" he said sincerely. There was silence.
"Just … just tell me," Anna said at last, choking slightly.
"Anna, you have fairy blood."
Anna looked at him. She looked directly into his golden-green eyes, and in that moment she knew that he was telling her the truth. He was telling her the truth, in the way that no one ever had.
"Thank you," she said, and the tears that had been welling up in her eyes finally fell, released from their bonds. Tears that she had been bottling up since she was a small child, afraid to let them go for what they might bring. Afraid, because even then she had known that she was different. As they fell, they sparkled in the light that streamed through the window, where the moon still shone, even though the dawn was breaking over the edge of the horizon like an egg dropped from a great height. They seemed to glow silver, with an almost magical appeal, and Anna knew this was just a confirmation of what she had been told.
"You are one who needs to know truth, fairy child," said the Telcontar who had spoken before. Anna wondered vaguely whether he was some sort of second-in-command. "You are a being based on truth and justice, and you will not rest until it is achieved. Unlike many of your disreputable race, you have a heart of gold, and you will carry out your duty, even if it kills you." There was a long silence; a feeling almost of ceremony in the room.
"Thank you, Anna O'Sullivan, for you truly are a fairy child. We hope that you can bring honour back to your disgraced race, for surely if anyone can it is you."
The Telcontar bowed once. They turned and left the room in silence. Even Matt did not look back. Slowly, Kathryn and Callie followed them. Anna left the room, unable to bear sitting there alone, for with knowledge had come much suffering.


text:
Time again for another march toward t-shirt doom! Discuss this week's life-or-death struggle for sales supremacy as chronicled on the Reckoning page all week long.
Here's how the Reckoning week runs now: every Tuesday at midnight, we retire the previous week's lowest-selling shirts and launch
this here discussion thread. Tuesday around noon we'll post the all-new, all-early Reckoning Recon. On Friday, we'll juice demand and
prime the pump with a little weekend reminder post. Finally, on the following Monday, the Day of Reckoning post sounds the last call
for the Reckoned shirts. All of those posts will be linked below. Monday night (or Tuesday morning), it starts all over again. The
Reckoning, she is a hungry beast...
May the odds be ever in your favor!
I love shirts!!!!!!!!!
I don't get it.
Trust Me! gets reckoned after 80 weeks in the Top Ten—a week and a half after the TMNT movie is released??
I just don't get it...
EwingKlipspringer wrote:I don't get it.
Trust Me! gets reckoned after 80 weeks in the Top Ten—a week and a half after the TMNT movie is released??
I just don't get it...
Maybe the reckoning just wasn't big enough for two patrickspens TMNT shirts?
Volunteer Moderator
EwingKlipspringer wrote:... I just don't get it...
It's been hovering around the line for a while now, so it was just a matter of time, and one good week of sales can't make up for 2-1/2 slow weeks.
Good Bye TRUST ME...you had a great and well deserved run! An end of an era...and it looks like Starcholgy is in danger also...I think it is because of these quickie flash shirts that sell great for a week or so, and don't show the lasting power, but still take a spot in the top 20....BINGE was getting a little low but had a spurt it looks like...
View All →


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Product code: 17563
Despatched in 3-5 days.
Assorted in car warning sign with 2 slogans.
E.g. ""Precoius Princess in Car"", ""Mummy's angel"".
Affixes to the inside of the window with a suction cup provided.
2 signs per pack.
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