BackgroundMicrobiota that co-enrich during efforts to recover pathogens from foodborne outbreaks interfere with efficient detection and recovery. Here, dynamics of co-enriching microbiota during recovery of Listeria monocytogenes from naturally contaminated ice cream samples linked to an outbreak are described for three different initial enrichment formulations used by the Food and Drug Administration (FDA), the International Organization of Standardization (ISO), and the United States Department of Agriculture (USDA). Enrichment cultures were analyzed using DNA extraction and sequencing from samples taken every 4 h throughout 48 h of enrichment. Resphera Insight and CosmosID analysis tools were employed for high-resolution profiling of 16S rRNA amplicons and whole genome shotgun data, respectively.ResultsDuring enrichment, other bacterial taxa were identified, including Anoxybacillus, Geobacillus, Serratia, Pseudomonas, Erwinia, and Streptococcus spp. Surprisingly, incidence of L. monocytogenes was proportionally greater at hour 0 than when tested 4, 8, and 12 h later with all three enrichment schemes. The corresponding increase in Anoxybacillus and Geobacillus spp.indicated these taxa co-enriched in competition with L. monocytogenes during early enrichment hours. L. monocytogenes became dominant after 24 h in all three enrichments. DNA sequences obtained from shotgun metagenomic data of Listeria monocytogenes at 48 h were assembled to produce a consensus draft genome which appeared to have a similar tracking utility to pure culture isolates of L. monocytogenes.ConclusionsAll three methods performed equally well for enrichment of Listeria monocytogenes. The observation of potential competitive exclusion of L. mono by Anoxybacillus and Geobacillus in early enrichment hours provided novel information that may be used to further optimize enrichment formulations. Application of Resphera Insight for high-resolution analysis of 16S amplicon sequences accurately identified L. monocytogenes. Both shotgun and 16S rRNA data supported the presence of three slightly variable genomes of L. monocytogenes. Moreover, the draft assembly of a consensus genome of L. monocytogenes from shotgun metagenomic data demonstrated the potential utility of this approach to expedite trace-back of outbreak-associated strains, although further validation will be needed to confirm this utility.Electronic supplementary materialThe online version of this article (doi:10.1186/s12866-016-0894-1) contains supplementary material, which is available to authorized users.

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A rapid assay for eubacterial species identification is described using high-resolution melt analysis to characterize PCR products. Unique melt profiles generated from multiple hypervariable regions of the 16S rRNA gene for 100 clinically relevant bacterial pathogens, including category A and B biothreat agents and their surrogates, allowed highly specific species identification.Rapid and accurate diagnostic tools are critical for infectious disease surveillance and early diagnosis of disease (8,12). A simple platform which could deliver broad-based screening and specific pathogen identification would be invaluable for the timely recognition of emerging and biothreat (BT) outbreaks, as well as commonly encountered clinical infections (2,7,9,11,12).We previously reported a probe-based PCR assay, which utilizes conserved and variable 16S rRNA gene sequences for initial broad-based eubacterial detection and subsequent identification of specific bacterial agents (11). The assay demonstrated high analytical sensitivity but was limited by an inability to differentiate closely related pathogens due to decreased specificity of the TaqMan probe chemistry and high sequence homology within selected hypervariable regions of the 16S rRNA gene. Probe-based amplicon characterization accordingly limits testing to a finite number of anticipated pathogens. Alternative strategies for amplicon analysis, such as sequencing and mass spectrometry, allow broader-scale product characterization but are costly, time-consuming, and lacking in throughput (1, 6). High-resolution melt analysis (HRMA) offers a simple, low-cost, closed-tube approach to amplicon analysis with the capacity for single-nucleotide discrimination and easy integration with PCR analysis (10). We report a unique strategy for the rapid, highly specific identification of BTrelated and non-BT-related bacterial pathogens which couples eubacterial PCR with HRMA.

The rapidly growing supplement industry operates without a formal premarket approval process. Consumers rely on product labels to be accurate and true. Those products containing live microbials report both identity and viability on most product labels. This study used next-generation sequencing technology as an analytical tool in conjunction with classic culture methods to examine the validity of the labels on supplement products containing live microbials found in the United States marketplace. Our results show the importance of testing these products for identity, viability, and potential contaminants, as well as introduce a new culture-independent diagnostic approach for testing these products.

Diagnosis of the etiologic agent of respiratory viral infection relies traditionally on culture or antigen detection. This pilot was conducted evaluation comparing performance characteristics of the RT-PCR and Electrospray Ionization Mass Spectrometry (RT-PCR/ESI-MS) platform to conventional virological methods for identifying multiple clinically relevant respiratory viruses in nasopharyngeal aspirates. The RT-PCR/ESI-MS respiratory virus surveillance kit was designed to detect respiratory syncytial virus, influenza A and B, parainfluenza types 1-4, adenoviridae types A-F, coronaviridae, human bocavirus, and human metapneumovirus. Patients (N=192) attending an emergency department during the 2007-8 respiratory season consented, and “excess” frozen archived nasopharyngeal aspirates were analysed; 46 were positive by conventional virology and 69 by RT-PCR/ESI-MS, among which there were six samples with multiple viral pathogens detected. The sensitivity and specificity of the assay were 89.1% and 80.3%, respectively. Additional viruses that were not identified by conventional virology assays were detected (4 human bocaviruses and 7 coronaviruses). Samples in which the RT-PCR/ESI-MS results disagreed with conventional virology were sent for analysis by a third method using a commercial RT-PCR-based assay, which can identify viruses not detectable by conventional virologic procedures. Time to first result of RT-PCR/ESI-MS was 8 hours. RT-PCR/ESI-MS demonstrated capacity to detect respiratory viruses identifiable and unidentifiable by conventional methods rapidly.

Septic arthritis (SA) is a rheumatologic emergency associated with significant morbidity and mortality. Delayed or inadequate treatment of SA can lead to irreversible joint destruction and disability. Current methods of diagnosing SA rely on synovial fluid analysis and culture which are known to be imprecise and time-consuming. We report a novel adaptation of a probe-based real-time PCR assay targeting the 16S rRNA gene for early and accurate diagnosis of bacterial SA. The assay algorithm consists of initial broad-range eubacterial detection, followed by Gram typing and species characterization of the pathogen. The platform demonstrated a high analytical sensitivity with a limit of detection of 10 1 CFU/ml with a panel of SA-related organisms. Gram typing and pathogen-specific probes correctly identified their respective targets in a mock test panel of 36 common clinically relevant pathogens. One hundred twenty-one clinical synovial fluid samples from patients presenting with suspected acute SA were tested. The sensitivity and specificity of the assay were 95% and 97%, respectively, versus synovial fluid culture results. Gram-typing probes correctly identified 100% of eubacterial positive samples as to gram-positive or gram-negative status, and pathogen-specific probes correctly identified the etiologic agent in 16/20 eubacterial positive samples. The total assay time from sample collection to result is 3 h. We have demonstrated that a real-time broad-based PCR assay has high analytical and clinical performance with an improved time to detection versus culture for SA. This assay may be a useful diagnostic adjunct for clinicians, particularly those practicing in the acute care setting where rapid pathogen detection and identification would assist in disposition and treatment decisions.Septic arthritis (SA) is a rheumatologic emergency associated with significant morbidity and mortality (6, 9). Delayed or inadequate treatment of SA can lead to irreversible joint destruction with subsequent disability. Accordingly, prompt diagnosis and early initiation of therapy are critical in improving the outcome (7).The diagnosis of SA in the acute care setting is challenging because of the relatively poor sensitivity and specificity of clinical examination findings and lack of a rapid reliable diagnostic assay. Further, overreliance on conventional laboratory tests for synovial fluid analysis is hindered by the relatively poor performance characteristics of these methods (11,12,16). In particular, the sensitivity of Gram staining has been reported in the range of 29% to 50% (3, 4), and the sensitivity of culture may be only 82% (9). Lack of a rapid and accurate diagnostic tool results in acute care clinicians often choosing the conservative approach of hospital admission and empirical broadspectrum antibiotics for patients with suspected SA. The benefits of this management strategy may be offset, however, by added costs and potential iatrogenic complications associated with unnecessary treatment and hospitalizations, as well ...

US forests are plagued by chronic deer feeding on native plants and the invasion of exotic plants. One possible synergy of these issues is that white-tailed deer preferentially consume native plants and ignore exotic plants. This issue is difficult to test due to the inability to closely measure deer feeding. We used a DNA barcoding protocol to examine the plant DNA found in deer feces at a Virginia site. We identified most plant species and found exotics, as well as native plants, in the feces examined. Deer apparently avoid some exotic plants but are not the sole reason for the abundance of most exotics.

Echinacea is a common botanical used in dietary supplements, primarily to treat upper respiratory tract infections and to support immune function. There are currently thought to be nine species in the genus Echinacea. Due to very low molecular divergence among sister species, traditional DNA barcoding has not been successful for differentiation of Echinacea species. Here, we present the use of full chloroplast genomes to distinguish between all 9 reported species. Total DNA was extracted from specimens stored at the National Museum of Natural History, Smithsonian Institution, which had been collected from the wild with species identification documented by experts in the field. We used Next Generation Sequencing (NGS) and CLC Genomics Workbench to assemble complete chloroplast genomes for all nine species. Full chloroplasts unambiguously differentiated all nine species, compared with the very few single nucleotide polymorphisms (SNPs) available with core DNA barcoding markers. SNPs for any two Echinacea chloroplast genomes ranged from 181 to 910, and provided robust data for unambiguous species delimitation. Implications for DNA-based species identification assays derived from chloroplast genome sequences are discussed in light of product safety, adulteration and quality issues.