Cells of the human intestinal tract mapped across space and time.

The cellular landscape of the human intestinal tract is dynamic throughout life, developing in utero and changing in response to functional requirements and environmental exposures. Here, to comprehensively map cell lineages, we use single-cell RNA sequencing and antigen receptor analysis of almost half a million cells from up to 5 anatomical regions in the developing and up to 11 distinct anatomical regions in the healthy paediatric and adult human gut. This reveals the existence of transcriptionally distinct BEST4 epithelial cells throughout the human intestinal tract. Furthermore, we implicate IgG sensing as a function of intestinal tuft cells. We describe neural cell populations in the developing enteric nervous system, and predict cell-type-specific expression of genes associated with Hirschsprung's disease. Finally, using a systems approach, we identify key cell players that drive the formation of secondary lymphoid tissue in early human development. We show that these programs are adopted in inflammatory bowel disease to recruit and retain immune cells at the site of inflammation. This catalogue of intestinal cells will provide new insights into cellular programs in development, homeostasis and disease.

Somatic mutation landscapes at single-molecule resolution.

Somatic mutations drive the development of cancer and may contribute to ageing and other diseases1,2. Despite their importance, the difficulty of detecting mutations that are only present in single cells or small clones has limited our knowledge of somatic mutagenesis to a minority of tissues. Here, to overcome these limitations, we developed nanorate sequencing (NanoSeq), a duplex sequencing protocol with error rates of less than five errors per billion base pairs in single DNA molecules from cell populations. This rate is two orders of magnitude lower than typical somatic mutation loads, enabling the study of somatic mutations in any tissue independently of clonality. We used this single-molecule sensitivity to study somatic mutations in non-dividing cells across several tissues, comparing stem cells to differentiated cells and studying mutagenesis in the absence of cell division. Differentiated cells in blood and colon displayed remarkably similar mutation loads and signatures to their corresponding stem cells, despite mature blood cells having undergone considerably more divisions. We then characterized the mutational landscape of post-mitotic neurons and polyclonal smooth muscle, confirming that neurons accumulate somatic mutations at a constant rate throughout life without cell division, with similar rates to mitotically active tissues. Together, our results suggest that mutational processes that are independent of cell division are important contributors to somatic mutagenesis. We anticipate that the ability to reliably detect mutations in single DNA molecules could transform our understanding of somatic mutagenesis and enable non-invasive studies on large-scale cohorts.

Embryonic and early postnatal cranial bone volume and tissue mineral density values for C57BL/6J laboratory mice.

BACKGROUND: Laboratory mice are routinely used in craniofacial research based on the relatively close genetic relationship and conservation of developmental pathways between humans and mice. Since genetic perturbations and disease states may have localized effects, data from individual cranial bones are valuable for the interpretation of experimental assays. We employ high-resolution microcomputed tomography to characterize cranial bones of C57BL/6J mice at embryonic day (E) 15.5 and E17.5, day of birth (P0), and postnatal day 7 (P7) and provide estimates of individual bone volume and tissue mineral density (TMD). RESULTS: Average volume and TMD values are reported for individual bones. Significant differences in volume and TMD during embryonic ages likely reflect early mineralization of cranial neural crest-derived and intramembranously forming bones. Although bones of the face and vault had higher TMD values during embryonic ages, bones of the braincase floor had significantly higher TMD values by P7. CONCLUSIONS: These ontogenetic data on cranial bone volume and TMD serve as a reference standard for future studies using mice bred on a C57BL/6J genetic background. Our findings also highlight the importance of differentiating "control" data from mice that are presented as "unaffected" littermates, particularly when carrying a single copy of a cre-recombinase gene.

Foraging by an avian ecosystem engineer extensively modifies the litter and soil layer in forest ecosystems.

Ecosystem engineers physically modify their environment, thereby altering habitats for other organisms. Increasingly, "engineers" are recognized as an important focus for conservation and ecological restoration because their actions affect a range of ecosystem processes and thereby influence how ecosystems function. The Superb Lyrebird Menura novaehollandiae is proposed as an ecosystem engineer in forests of southeastern Australia due to the volume of soil and litter it turns over when foraging. We measured the seasonal and spatial patterns of foraging by Lyrebirds and the amount of soil displaced in forests in the Central Highlands, Victoria. We tested the effects of foraging on litter, soil nutrients and soil physical properties by using an experimental approach with three treatments: Lyrebird exclusion, Lyrebird exclusion with simulated foraging, and non-exclusion reference plots. Treatments were replicated in three forest types in each of three forest blocks. Lyrebirds foraged extensively in all forest types in all seasons. On average, Lyrebirds displaced 155.7 Mg/ha of litter and soil in a 12-month period. Greater displacement occurred where vegetation complexity (<50 cm height) was low. After two years of Lyrebird exclusion, soil compaction (top 7.5 cm) increased by 37% in exclusion plots compared with baseline measures, while in unfenced plots it decreased by 22%. Litter depth was almost three times greater in fenced than unfenced plots. Soil moisture, pH, and soil nutrients showed no difference between treatments. The enormous extent of litter and soil turned over by the Superb Lyrebird is unparalleled by any other vertebrate soil engineer in terrestrial ecosystems globally. The profound influence of such foraging activity on forest ecosystems is magnified by its year-round pattern and widespread distribution. The disturbance regime that Lyrebirds impose has implications for diverse ecosystem processes including decomposition and nutrient cycling, the composition of litter- and soil-dwelling invertebrate communities, the shaping of ground-layer vegetation patterns, and fire behavior and post-fire ecosystem recovery. Maintaining Lyrebird populations as a key facilitator of ecosystem function is now timely and critical as unprecedented wildfires in eastern Australia in summer 2019-2020 have severely burned ~12 million ha of forest, including ~30% of the geographic range of the Superb Lyrebird.

Copy number variation arising from gene conversion on the human Y chromosome.

We describe the variation in copy number of a ~ 10 kb region overlapping the long intergenic noncoding RNA (lincRNA) gene, TTTY22, within the IR3 inverted repeat on the short arm of the human Y chromosome, leading to individuals with 0-3 copies of this region in the general population. Variation of this CNV is common, with 266 individuals having 0 copies, 943 (including the reference sequence) having 1, 23 having 2 copies, and two having 3 copies, and was validated by breakpoint PCR, fibre-FISH, and 10× Genomics Chromium linked-read sequencing in subsets of 1234 individuals from the 1000 Genomes Project. Mapping the changes in copy number to the phylogeny of these Y chromosomes previously established by the Project identified at least 20 mutational events, and investigation of flanking paralogous sequence variants showed that the mutations involved flanking sequences in 18 of these, and could extend over > 30 kb of DNA. While either gene conversion or double crossover between misaligned sister chromatids could formally explain the 0-2 copy events, gene conversion is the more likely mechanism, and these events include the longest non-allelic gene conversion reported thus far. Chromosomes with three copies of this CNV have arisen just once in our data set via another mechanism: duplication of 420 kb that places the third copy 230 kb proximal to the existing proximal copy. Our results establish gene conversion as a previously under-appreciated mechanism of generating copy number changes in humans and reveal the exceptionally large size of the conversion events that can occur.

High-throughput and quantitative genome-wide messenger RNA sequencing for molecular phenotyping.

BACKGROUND: We present a genome-wide messenger RNA (mRNA) sequencing technique that converts small amounts of RNA from many samples into molecular phenotypes. It encompasses all steps from sample preparation to sequence analysis and is applicable to baseline profiling or perturbation measurements. RESULTS: Multiplex sequencing of transcript 3' ends identifies differential transcript abundance independent of gene annotation. We show that increasing biological replicate number while maintaining the total amount of sequencing identifies more differentially abundant transcripts. CONCLUSIONS: This method can be implemented on polyadenylated RNA from any organism with an annotated reference genome and in any laboratory with access to Illumina sequencing.

SASI-Seq: sample assurance Spike-Ins, and highly differentiating 384 barcoding for Illumina sequencing.

BACKGROUND: A minor but significant fraction of samples subjected to next-generation sequencing methods are either mixed-up or cross-contaminated. These events can lead to false or inconclusive results. We have therefore developed SASI-Seq; a process whereby a set of uniquely barcoded DNA fragments are added to samples destined for sequencing. From the final sequencing data, one can verify that all the reads derive from the original sample(s) and not from contaminants or other samples. RESULTS: By adding a mixture of three uniquely barcoded amplicons, of different sizes spanning the range of insert sizes one would normally use for Illumina sequencing, at a spike-in level of approximately 0.1%, we demonstrate that these fragments remain intimately associated with the sample. They can be detected following even the tightest size selection regimes or exome enrichment and can report the occurrence of sample mix-ups and cross-contamination.As a consequence of this work, we have designed a set of 384 eleven-base Illumina barcode sequences that are at least 5 changes apart from each other, allowing for single-error correction and very low levels of barcode misallocation due to sequencing error. CONCLUSION: SASI-Seq is a simple, inexpensive and flexible tool that enables sample assurance, allows deconvolution of sample mix-ups and reports levels of cross-contamination between samples throughout NGS workflows.

Comparative activities of telavancin combined with nafcillin, imipenem, and gentamicin against Staphylococcus aureus.

Beta-lactams enhance the killing activity of vancomycin. Due to structural and mechanistic similarities between vancomycin and telavancin, we investigated the activity of telavancin combined with nafcillin and imipenem compared to the known synergistic combination of telavancin and gentamicin. Thirty strains of Staphylococcus aureus, 10 methicillin-susceptible S. aureus (MSSA), 10 methicillin-resistant S. aureus (MRSA), and 10 heterogeneously vancomycin-intermediate S. aureus (hVISA), were tested for synergy by time-kill methodology. Six strains (2 each of MSSA, MRSA, and hVISA) were further evaluated in an in vitro pharmacokinetic/pharmacodynamic (PK/PD) model with simulated regimens of 10 mg/kg of body weight of telavancin once daily alone and combined with 2 g nafcillin every 4 h, 500 mg imipenem every 6 h, or 5 mg/kg gentamicin once daily over 72 h. In the synergy test, 67% of strains displayed synergy with the combination of telavancin and gentamicin, 70% with telavancin and nafcillin, and 63% with telavancin and imipenem. In the PK/PD model, the activities of all three combinations against MRSA and hVISA were superior to all individual drugs alone (P ≤ 0.002) and were similar to each other (P ≥ 0.187). The activities of all three combinations against MSSA were generally similar to each other except for one strain where the combination of telavancin and imipenem was superior to all other regimens (P ≤ 0.011). The activity of the combination of telavancin and beta-lactam agents was similar to that of telavancin and gentamicin against S. aureus, including resistant strains. Because beta-lactam combinations are less likely to be nephrotoxic than telavancin plus gentamicin, these beta-lactam combinations may have clinical utility.

Clinical Outcomes in Patients with Heterogeneous Vancomycin-Intermediate Staphylococcus aureus Bloodstream Infection.

The prevalence of heterogeneous vancomycin-intermediate Staphylococcus aureus (hVISA) infections varies in the literature, a problem complicated by the lack of routine screening procedures; however, limited data suggest that hVISA has been associated with persistent bloodstream infections (BSI) and vancomycin failure, yet these studies have been confounded by design issues. We conducted this study to compare the characteristics of patients with BSI caused by hVISA with those with vancomycin-susceptible Staphylococcus aureus (VSSA) treated with vancomycin. This retrospective, multicenter matched (1:1) cohort study compared the clinical characteristics and outcomes of hVISA and VSSA. Patients with hVISA methicillin-resistant Staphylococcus aureus (MRSA) BSI from 2004 to 2012 were matched to VSSA-MRSA BSI patients. The primary outcome was failure of vancomycin treatment, defined as a composite of persistent bacteremia (≥7 days), persistent signs and symptoms, change of MRSA antibiotic, recurrent BSI, or MRSA-related mortality. We identified 122 matched cases. The overall vancomycin failure rate was 57% (82% hVISA versus 33% VSSA; P < 0.001). The individual components of failure in hVISA versus VSSA were persistent bacteremia, 59% versus 21% (P < 0.001); change in MRSA therapy, 54% versus 25% (P = 0.001); MRSA-related mortality, 21% versus 10% (P = 0.081); and recurrence of BSI, 26% versus 2% (P < 0.001). Using logistic regression analysis and adjusting for covariates, hVISA (adjusted odds ratio [aOR], 11.1; 95% confidence interval [CI], 4.3 to 28.7) and intensive care unit (ICU) admission (aOR, 4.5; 95% CI, 1.8 to 11.6) were still independently associated with vancomycin failure. Relative to VSSA BSI, patients with hVISA were more likely to experience failure of vancomycin treatment, including persistent bacteremia and recurrence. Our results indicate that hVISA was responsible for considerable morbidity.

Evaluation of the combination of daptomycin and nafcillin against vancomycin-intermediate Staphylococcus aureus.

OBJECTIVES: Continued selective pressure from glycopeptide use has led to non-susceptible strains of Staphylococcus aureus, including vancomycin-intermediate S. aureus (VISA). Though relatively uncommon, VISA presents a particularly difficult clinical challenge when it arises. Pertinent to this investigation is the correlation between vancomycin intermediacy and daptomycin non-susceptibility. The aim of this study was to evaluate the potential for synergy between daptomycin and nafcillin against VISA. METHODS: Twenty VISA strains were evaluated for daptomycin and nafcillin MICs by broth microdilution in duplicate. Potential for synergy was assessed by time-kill at 0.5× MIC in triplicate. Four strains displaying synergy in time-kill analysis were analysed in an in vitro pharmacokinetic (PK)/pharmacodynamic (PD) model in duplicate over 72 h. RESULTS: In time-kill experiments, 55% of strains (11/20) displayed synergy with the combination. In the PK/PD model, no differences between daptomycin-alone and combination regimens were observed for the strain with the lowest daptomycin MIC (0.5 mg/L). For the strain with a daptomycin MIC of 1 mg/L, 6 mg/kg daptomycin+nafcillin was superior to 6 mg/kg daptomycin alone (P=0.002) and 10 mg/kg daptomycin+nafcillin was superior to all other regimens (P ≤ 0.004). When the daptomycin MIC increased to 2 mg/L, 10 mg/kg daptomycin+nafcillin was superior to 6 mg/kg daptomycin+nafcillin, which was superior to both 6 and 10 mg/kg daptomycin alone (P ≤ 0.019). CONCLUSIONS: Daptomycin and nafcillin in combination significantly improved antibacterial activity against VISA. This effect was more pronounced as the daptomycin susceptibility of the strain declined.

Improvements to services at the European Nucleotide Archive.

The European Nucleotide Archive (ENA; http://www.ebi.ac.uk/ena) is Europe's primary nucleotide sequence archival resource, safeguarding open nucleotide data access, engaging in worldwide collaborative data exchange and integrating with the scientific publication process. ENA has made significant contributions to the collaborative nucleotide archival arena as an active proponent of extending the traditional collaboration to cover capillary and next-generation sequencing information. We have continued to co-develop data and metadata representation formats with our collaborators for both data exchange and public data dissemination. In addition to the DDBJ/EMBL/GenBank feature table format, we share metadata formats for capillary and next-generation sequencing traces and are using and contributing to the NCBI SRA Toolkit for the long-term storage of the next-generation sequence traces. During the course of 2009, ENA has significantly improved sequence submission, search and access functionalities provided at EMBL-EBI. In this article, we briefly describe the content and scope of our archive and introduce major improvements to our services.

Differential effects of ecosystem engineering by the superb lyrebird Menura novaehollandiae and herbivory by large mammals on floristic regeneration and structure in wet eucalypt forests.

Ecosystem engineers that modify the soil and ground-layer properties exert a strong influence on vegetation communities in ecosystems worldwide. Understanding the interactions between animal engineers and vegetation is challenging when in the presence of large herbivores, as many vegetation communities are simultaneously affected by both engineering and herbivory. The superb lyrebird Menura novaehollandiae, an ecosystem engineer in wet forests of south-eastern Australia, extensively modifies litter and soil on the forest floor. The aim of this study was to disentangle the impacts of engineering by lyrebirds and herbivory by large mammals on the composition and structure of ground-layer vegetation. We carried out a 2-year, manipulative exclusion experiment in the Central Highlands of Victoria, Australia. We compared three treatments: fenced plots with simulated lyrebird foraging; fenced plots excluding herbivores and lyrebirds; and open controls. This design allowed assessment of the relative impacts of engineering and herbivory on germination rates, seedling density, vegetation cover and structure, and community composition. Engineering by lyrebirds enhanced the germination of seeds in the litter layer. After 2 years, more than double the number of germinants were present in "engineered" than "non-engineered" plots. Engineering did not affect the density of seedlings, but herbivory had strong detrimental effects. Herbivory also reduced the floristic richness and structural complexity (<0.5 m) of forest vegetation, including the cover of herbs. Neither process altered the floristic composition of the vegetation within the 2-year study period. Ecosystem engineering by lyrebirds and herbivory by large mammals both influence the structure of forest-floor vegetation. The twofold increase in seeds stimulated to germinate by engineering may contribute to the evolutionary adaptation of plants by allowing greater phenotypic expression and selection than would otherwise occur. Over long timescales, engineering and herbivory likely combine to maintain a more-open forest floor conducive to ongoing ecosystem engineering by lyrebirds.

Petabyte-scale innovations at the European Nucleotide Archive.

Dramatic increases in the throughput of nucleotide sequencing machines, and the promise of ever greater performance, have thrust bioinformatics into the era of petabyte-scale data sets. Sequence repositories, which provide the feed for these data sets into the worldwide computational infrastructure, are challenged by the impact of these data volumes. The European Nucleotide Archive (ENA; http://www.ebi.ac.uk/embl), comprising the EMBL Nucleotide Sequence Database and the Ensembl Trace Archive, has identified challenges in the storage, movement, analysis, interpretation and visualization of petabyte-scale data sets. We present here our new repository for next generation sequence data, a brief summary of contents of the ENA and provide details of major developments to submission pipelines, high-throughput rule-based validation infrastructure and data integration approaches.

Heterogeneous vancomycin resistance in Staphylococcus aureus: a review of epidemiology, diagnosis, and clinical significance.

OBJECTIVE: To provide an overview of heterogeneously glycopeptide intermediate Staphylococcus aureus, its epidemiology, methods of diagnosis, and clinical relevance. DATA SOURCES: Literature was retrieved in December 2009 through Academic Search Premier, Alt-Health Watch, MEDLINE, OVID, and PubMed, using the search terms heterogeneous glycopeptide-intermediate Staphylococcus aureus, hGISA, hVISA, vancomycin-intermediate Staphylococcus aureus, GISA, and VISA (from 2003 to December 2009). Additional references were obtained through review of publication citations. STUDY SELECTION AND DATA EXTRACTION: All articles retrieved through the literature search and reference review were evaluated for inclusion in this review. DATA SYNTHESIS: Heterogeneously glycopeptide-intermediate S. aureus (hGISA) has been shown to significantly complicate treatment for patients with bacteremia and often escapes detection in clinical laboratories. Population analysis profile area under the curve ratio (PAP-AUC) is considered to be the gold standard of hGISA detection; however, it is labor-intensive, expensive, and not feasible in a clinical setting. The Etest macromethod is a reasonable alternative to PAP, yielding high sensitivity and specificity, and has potential for routine use in clinical laboratories. Due to these difficulties in detection, an exact prevalence is difficult to obtain though, based on available data, it appears that approximately 5-15% of isolates display this phenotype. hGISA is associated with prolonged bacteremia, high bacterial load infections, prolonged antibiotic therapy, prolonged hospitalization, treatment failure, and potential for increased mortality. CONCLUSIONS: Overall, hGISA is associated with a poor clinical course in patients with bacteremia. Information is still lacking regarding the actual prevalence of hGISA as well as the extent of its clinical impact. A uniform method of diagnosis must be established in order to better account for all cases of hGISA. More studies must be conducted to assess clinical outcomes of hGISA.

Priorities for nucleotide trace, sequence and annotation data capture at the Ensembl Trace Archive and the EMBL Nucleotide Sequence Database.

The Ensembl Trace Archive (http://trace.ensembl.org/) and the EMBL Nucleotide Sequence Database (http://www.ebi.ac.uk/embl/), known together as the European Nucleotide Archive, continue to see growth in data volume and diversity. Selected major developments of 2007 are presented briefly, along with data submission and retrieval information. In the face of increasing requirements for nucleotide trace, sequence and annotation data archiving, data capture priority decisions have been taken at the European Nucleotide Archive. Priorities are discussed in terms of how reliably information can be captured, the long-term benefits of its capture and the ease with which it can be captured.

Heart transplantation techniques after hybrid single-ventricle palliation.

The Norwood procedure for hypoplastic left heart syndrome (HLHS) aims to provide an unobstructed systemic outflow tract, unrestrictive inter-atrial communication, controlled source of pulmonary blood flow, and reliable source of coronary blood flow. The hybrid palliative strategy of pulmonary artery banding and ductal stenting has emerged as an alternative treatment for neonates with HLHS. Neonates who have undergone a hybrid Norwood but are not candidates for the three-stage single-ventricle pathway may need heart transplantation. Patients who have undergone hybrid Norwood or those with visceral heterotaxy who have undergone ductal stenting and bilateral PA bands represent a technically challenging group of patients for heart transplantation, but it appears to be a favorable approach and we describe our experience with three patients who underwent heart transplant after a hybrid Norwood procedure.

Single-cell atlas of the first intra-mammalian developmental stage of the human parasite Schistosoma mansoni.

Over 250 million people suffer from schistosomiasis, a tropical disease caused by parasitic flatworms known as schistosomes. Humans become infected by free-swimming, water-borne larvae, which penetrate the skin. The earliest intra-mammalian stage, called the schistosomulum, undergoes a series of developmental transitions. These changes are critical for the parasite to adapt to its new environment as it navigates through host tissues to reach its niche, where it will grow to reproductive maturity. Unravelling the mechanisms that drive intra-mammalian development requires knowledge of the spatial organisation and transcriptional dynamics of different cell types that comprise the schistomulum body. To fill these important knowledge gaps, we perform single-cell RNA sequencing on two-day old schistosomula of Schistosoma mansoni. We identify likely gene expression profiles for muscle, nervous system, tegument, oesophageal gland, parenchymal/primordial gut cells, and stem cells. In addition, we validate cell markers for all these clusters by in situ hybridisation in schistosomula and adult parasites. Taken together, this study provides a comprehensive cell-type atlas for the early intra-mammalian stage of this devastating metazoan parasite.

Activity of telavancin against Staphylococcus aureus strains with various vancomycin susceptibilities in an in vitro pharmacokinetic/pharmacodynamic model with simulated endocardial vegetations.

We investigated the activity of telavancin, a novel lipoglycopeptide, alone and combined with gentamicin or rifampin (rifampicin) against strains of Staphylococcus aureus with various vancomycin susceptibilities. Strains tested included methicillin (meticillin)-resistant S. aureus (MRSA) 494, methicillin-sensitive S. aureus (MSSA) 1199, heteroresistant glycopeptide-intermediate S. aureus (hGISA) 1629, which was confirmed by a population analysis profile, and glycopeptide-intermediate S. aureus (GISA) NJ 992. Regimens of 10 mg/kg telavancin daily and 1 g vancomycin every 12 h were investigated alone and combined with 5 mg/kg gentamicin daily or 300 mg rifampin every 8 h in an in vitro model with simulated endocardial vegetations over 96 h. Telavancin demonstrated significantly greater killing than did vancomycin (P < 0.01) for all isolates except MRSA 494 (P = 0.07). Telavancin absolute reductions, in log(10) CFU/g, at 96 h were 2.8 +/- 0.5 for MRSA 494, 2.8 +/- 0.3 for MSSA 1199, 4.2 +/- 0.2 for hGISA 1629, and 4.1 +/- 0.3 for GISA NJ 992. Combinations of telavancin with gentamicin significantly enhanced killing compared to telavancin alone against all isolates (P < 0.001) except MRSA 494 (P = 0.176). This enhancement was most evident against hGISA 1629, where killing to the level of detection (2 log(10) CFU/g) was achieved at 48 h (P < 0.001). The addition of rifampin to telavancin resulted in significant (P < 0.001) enhancement of killing against only MSSA 1199. No changes in telavancin susceptibilities were observed. These results suggest that telavancin may have therapeutic potential, especially against strains with reduced susceptibility to vancomycin. Combination therapy, particularly with gentamicin, may improve bacterial killing against certain strains.

Impact of inoculum size and heterogeneous vancomycin-intermediate Staphylococcus aureus (hVISA) on vancomycin activity and emergence of VISA in an in vitro pharmacodynamic model.

The activity of vancomycin against heterogeneous vancomycin-intermediate Staphylococcus aureus (hVISA) and non-hVISA isolates, using an in vitro pharmacodynamic model, was reduced in the presence of a high inoculum amount (10(8) CFU/ml). A high bacterial load of >10(5) CFU/ml persisted for all strains with doses up to 5 g every 12 h against high inoculum amounts. No change in the vancomycin MIC was detected in any isolate at a moderate inoculum amount (10(6) CFU/ml), and bactericidal activity occurred only against the non-hVISA isolate (time to 99% kill, 7.5 h; P = 0.001).

Evaluation of vancomycin and daptomycin against methicillin-resistant Staphylococcus aureus and heterogeneously vancomycin-intermediate S. aureus in an in vitro pharmacokinetic/pharmacodynamic model with simulated endocardial vegetations.

OBJECTIVES: Glycopeptides have historically been the drugs of choice for the treatment of infections caused by methicillin-resistant Staphylococcus aureus (MRSA). However, the continued selective pressure has led to the emergence of non-susceptible strains including heterogeneously vancomycin-intermediate S. aureus (hVISA). Infections with hVISA have been associated with poor outcomes including vancomycin treatment failures. The objective of this study was to evaluate vancomycin and daptomycin against vancomycin-susceptible MRSA and hVISA in a pharmacokinetic/pharmacodynamic (PK/PD) model with simulated endocardial vegetations. METHODS: Six clinical isolates obtained from patients at the Detroit Medical Center were used: MRSA 494, MRSA 67, hVISA R1720, hVISS R2295, hVISA R3640 and hVISA R1629. All heteroresistant strains were confirmed by a population analysis profile ratio, with Mu3 as a control strain. Vancomycin regimens of 1 g every 12 h and 2 g every 12 h and daptomycin regimens of 6, 10 and 12 mg/kg daily were utilized in a PK/PD model over 72 h. RESULTS: Against MRSA isolates, vancomycin displayed minimal activity and minimal-to-no activity against hVISA. In general, the use of high dose vancomycin over standard dose vancomycin did not improve activity except against one of six isolates (MRSA 494). Daptomycin was bactericidal against both MRSA and hVISA isolates, although the rate of kill was slower against hVISA. CONCLUSIONS: Overall, daptomycin achieved rapid and effective kill against both MRSA and hVISA while vancomycin displayed slow and minimal kill against MRSA and minimal-to-no activity against hVISA, regardless of high dose exposure.