Genomic comparison of Escherichia coli O104:H4 isolates from 2009 and 2011 reveals plasmid, and prophage heterogeneity, including shiga toxin encoding phage stx2.

In May of 2011, an enteroaggregative Escherichia coli O104:H4 strain that had acquired a Shiga toxin 2-converting phage caused a large outbreak of bloody diarrhea in Europe which was notable for its high prevalence of hemolytic uremic syndrome cases. Several studies have described the genomic inventory and phylogenies of strains associated with the outbreak and a collection of historical E. coli O104:H4 isolates using draft genome assemblies. We present the complete, closed genome sequences of an isolate from the 2011 outbreak (2011C-3493) and two isolates from cases of bloody diarrhea that occurred in the Republic of Georgia in 2009 (2009EL-2050 and 2009EL-2071). Comparative genome analysis indicates that, while the Georgian strains are the nearest neighbors to the 2011 outbreak isolates sequenced to date, structural and nucleotide-level differences are evident in the Stx2 phage genomes, the mer/tet antibiotic resistance island, and in the prophage and plasmid profiles of the strains, including a previously undescribed plasmid with homology to the pMT virulence plasmid of Yersinia pestis. In addition, multiphenotype analysis showed that 2009EL-2071 possessed higher resistance to polymyxin and membrane-disrupting agents. Finally, we show evidence by electron microscopy of the presence of a common phage morphotype among the European and Georgian strains and a second phage morphotype among the Georgian strains. The presence of at least two stx2 phage genotypes in host genetic backgrounds that may derive from a recent common ancestor of the 2011 outbreak isolates indicates that the emergence of stx2 phage-containing E. coli O104:H4 strains probably occurred more than once, or that the current outbreak isolates may be the result of a recent transfer of a new stx2 phage element into a pre-existing stx2-positive genetic background.

Comprehensive biothreat cluster identification by PCR/electrospray-ionization mass spectrometry.

Technology for comprehensive identification of biothreats in environmental and clinical specimens is needed to protect citizens in the case of a biological attack. This is a challenge because there are dozens of bacterial and viral species that might be used in a biological attack and many have closely related near-neighbor organisms that are harmless. The biothreat agent, along with its near neighbors, can be thought of as a biothreat cluster or a biocluster for short. The ability to comprehensively detect the important biothreat clusters with resolution sufficient to distinguish the near neighbors with an extremely low false positive rate is required. A technological solution to this problem can be achieved by coupling biothreat group-specific PCR with electrospray ionization mass spectrometry (PCR/ESI-MS). The biothreat assay described here detects ten bacterial and four viral biothreat clusters on the NIAID priority pathogen and HHS/USDA select agent lists. Detection of each of the biothreat clusters was validated by analysis of a broad collection of biothreat organisms and near neighbors prepared by spiking biothreat nucleic acids into nucleic acids extracted from filtered environmental air. Analytical experiments were carried out to determine breadth of coverage, limits of detection, linearity, sensitivity, and specificity. Further, the assay breadth was demonstrated by testing a diverse collection of organisms from each biothreat cluster. The biothreat assay as configured was able to detect all the target organism clusters and did not misidentify any of the near-neighbor organisms as threats. Coupling biothreat cluster-specific PCR to electrospray ionization mass spectrometry simultaneously provides the breadth of coverage, discrimination of near neighbors, and an extremely low false positive rate due to the requirement that an amplicon with a precise base composition of a biothreat agent be detected by mass spectrometry.

Rapid identification of vector-borne flaviviruses by mass spectrometry.

Flaviviruses are a highly diverse group of RNA viruses classified within the genus Flavivirus, family Flaviviridae. Most flaviviruses are arthropod-borne, requiring a mosquito or tick vector. Several flaviviruses are highly pathogenic to humans; however, their high genetic diversity and immunological relatedness makes them extremely challenging to diagnose. In this study, we developed and evaluated a broad-range Flavivirus assay designed to detect both tick- and mosquito-borne flaviviruses by using RT-PCR/electrospray ionization mass spectrometry (RT-PCR/ESI-MS) on the Ibis T5000 platform. The assay was evaluated with a panel of 13 different flaviviruses. All samples were correctly identified to the species level. To determine the limit of detection for the mosquito-borne primer sets, serial dilutions of RNA from West Nile virus (WNV) were assayed and could be detected down to an equivalent viral titer of 0.2 plaque-forming units/mL. Analysis of flaviviruses in their natural biological background included testing Aedes aegypti mosquitoes that were laboratory-infected with dengue-1 virus. The assay accurately identified the virus within infected mosquitoes, and we determined the average viral genome per mosquito to be 2.0 x 10(6). Using human blood, serum, and urine spiked with WNV and mouse blood and brain tissues from Karshi virus-infected mice, we showed that these clinical matrices did not inhibit the detection of these viruses. Finally, we used the assay to test field-collected Ixodes scapularis ticks collected from sites in New York and Connecticut. We found 16/322 (5% infection rate) ticks positive for deer tick virus, a subtype of Powassan virus. In summary, we developed a single high-throughput Flavivirus assay that could detect multiple tick- and mosquito-borne flaviviruses and thus provides a new analytical tool for their medical diagnosis and epidemiological surveillance.

Identification of pathogenic Vibrio species by multilocus PCR-electrospray ionization mass spectrometry and its application to aquatic environments of the former soviet republic of Georgia.

The Ibis T5000 is a novel diagnostic platform that couples PCR and mass spectrometry. In this study, we developed an assay that can identify all known pathogenic Vibrio species and field-tested it using natural water samples from both freshwater lakes and the Georgian coastal zone of the Black Sea. Of the 278 total water samples screened, 9 different Vibrio species were detected, 114 (41%) samples were positive for V. cholerae, and 5 (0.8%) samples were positive for the cholera toxin A gene (ctxA). All ctxA-positive samples were from two freshwater lakes, and no ctxA-positive samples from any of the Black Sea sites were detected.

Application of the Ibis-T5000 pan-Orthopoxvirus assay to quantitatively detect monkeypox viral loads in clinical specimens from macaques experimentally infected with aerosolized monkeypox virus.

Monkeypox virus (MPXV), a member of the family Poxviridae and genus Orthopoxvirus, causes a smallpox-like disease in humans. A previously described pan-Orthopoxvirus assay, based on a broad-range polymerase chain reaction (PCR) coupled with electrospray ionization mass spectrometry (PCR/ESI-MS), was evaluated for its ability to detect MPXV from spiked human and aerosol-infected cynomolgous macaque (Macaca fascicularis) samples. Detection of MPXV DNA from macaque tissue, blood, and spiked human blood by the PCR/ESI-MS pan-Orthopoxvirus assay was comparable, albeit at slightly higher levels, to the current gold standard method of real-time PCR with the pan-Orthopoxvirus assay and had a limit of detection of 200 plaque-forming units. Furthermore, the platform was able to distinguish MPXV and vaccinia viruses that were spiked into macaque blood samples at various concentrations. This platform provides a new tool for the diagnosis and monitoring of orthopoxviral loads during vaccine or antiviral studies, but also could provide rapid identification during natural outbreaks or bioterrorism attacks.

Usefulness of multilocus polymerase chain reaction followed by electrospray ionization mass spectrometry to identify a diverse panel of bacterial isolates.

Polymerase chain reaction electrospray ionization mass spectrometry (PCR/ESI-MS) was tested for its ability to accurately identify a blinded panel of 156 diverse bacterial isolates, mostly human and/or animal pathogens. Here, 142/156 (91%) isolates were correctly identified to the genus level and 115/156 (74%) were correctly identified to the species level. Only 9% were misidentified. This study shows that multilocus PCR/ESI-MS has the potential to be a useful technique for identifying a broad range of bacteria.

Assay for and replication of Karshi (mammalian tick-borne flavivirus group) virus in mice.

Little is known about the replication of Karshi virus, a member of the mammalian tick-borne flavivirus group, in its rodent hosts. Therefore, we developed a quantitative real-time RT-PCR assay and measured the amount of viral RNA in selected tissues of infected Swiss Webster mice. Two-day-old mice were highly susceptible, with 100% fatality 9 to 12 days after infection, whereas infection in 9-day-old mice was less virulent, with death occurring only rarely. In nearly all cases, mice inoculated when 2 days old contained similar numbers of viral genome equivalents from blood and liver samples from any given mouse, with titers declining after day 7. In contrast, the amount of viral RNA in the brain began to rise rapidly 4 days after exposure, peaked at about 6 days after virus exposure (titer of > 10(13) genome equivalents/g), and remained at that level until euthanasia or death. Viral profiles were similar in needle-inoculated or tick-exposed mice.

Molecular identification of the biowarfare simulant Serratia marcescens from a 50-year-old munition buried at Fort Detrick, Maryland.

Serratia marcescens are Gram-negative bacteria that were often used by the U.S. military and others to track movement of bacteria in the environment. As part of ongoing construction at Fort Detrick, Maryland, what appeared to be a small bomblet was found buried in the ground at the site of an old test grid. A sample of a clear, straw-colored liquid was aseptically removed from the plastic reservoir; the results of routine cultures on standard bacteriological media were negative. DNA was extracted from the sample and found to be 99% identical to S. marcescens. These results demonstrate the ability to identify the contents of a biological munition that had been buried for approximately 50 years.

Global surveillance of emerging Influenza virus genotypes by mass spectrometry.

BACKGROUND: Effective influenza surveillance requires new methods capable of rapid and inexpensive genomic analysis of evolving viral species for pandemic preparedness, to understand the evolution of circulating viral species, and for vaccine strain selection. We have developed one such approach based on previously described broad-range reverse transcription PCR/electrospray ionization mass spectrometry (RT-PCR/ESI-MS) technology. METHODS AND PRINCIPAL FINDINGS: Analysis of base compositions of RT-PCR amplicons from influenza core gene segments (PB1, PB2, PA, M, NS, NP) are used to provide sub-species identification and infer influenza virus H and N subtypes. Using this approach, we detected and correctly identified 92 mammalian and avian influenza isolates, representing 30 different H and N types, including 29 avian H5N1 isolates. Further, direct analysis of 656 human clinical respiratory specimens collected over a seven-year period (1999-2006) showed correct identification of the viral species and subtypes with >97% sensitivity and specificity. Base composition derived clusters inferred from this analysis showed 100% concordance to previously established clades. Ongoing surveillance of samples from the recent influenza virus seasons (2005-2006) showed evidence for emergence and establishment of new genotypes of circulating H3N2 strains worldwide. Mixed viral quasispecies were found in approximately 1% of these recent samples providing a view into viral evolution. CONCLUSION/SIGNIFICANCE: Thus, rapid RT-PCR/ESI-MS analysis can be used to simultaneously identify all species of influenza viruses with clade-level resolution, identify mixed viral populations and monitor global spread and emergence of novel viral genotypes. This high-throughput method promises to become an integral component of influenza surveillance.

Detection of biological threat agents by real-time PCR: comparison of assay performance on the R.A.P.I.D., the LightCycler, and the Smart Cycler platforms.

BACKGROUND: Rapid detection of biological threat agents is critical for timely therapeutic administration. Fluorogenic PCR provides a rapid, sensitive, and specific tool for molecular identification of these agents. We compared the performance of assays for 7 biological threat agents on the Idaho Technology, Inc. R.A.P.I.D., the Roche LightCycler, and the Cepheid Smart Cycler. METHODS: Real-time PCR primers and dual-labeled fluorogenic probes were designed to detect Bacillus anthracis, Brucella species, Clostridium botulinum, Coxiella burnetii, Francisella tularensis, Staphylococcus aureus, and Yersinia pestis. DNA amplification assays were optimized by use of Idaho Technology buffers and deoxynucleotide triphosphates supplemented with Invitrogen Platinum Taq DNA polymerase, and were subsequently tested for sensitivity and specificity on the R.A.P.I.D., the LightCycler, and the Smart Cycler. RESULTS: Limit of detection experiments indicated that assay performance was comparable among the platforms tested. Exclusivity and inclusivity testing with a general bacterial nucleic acid cross-reactivity panel containing 60 DNAs and agent-specific panels containing nearest neighbors for the organisms of interest indicated that all assays were specific for their intended targets. CONCLUSION: With minor supplementation, such as the addition of Smart Cycler Additive Reagent to the Idaho Technology buffers, assays for DNA templates from biological threat agents demonstrated similar performance, sensitivity, and specificity on all 3 platforms.

Isolation of naturally occurring aluminium ligands using immobilized metal affinity chromatography for analysis by ESI-MS.

Aluminium (iii) is one of the most abundant metal ions found in soil. Typically, Al(+3) is bound to minerals, but its bioavailability and toxicity toward vascular plants increases with increasing soil acidity. Ectomycorrhizal fungi, which live symbiotically on the roots of numerous woody plants, often confer Al(+3) resistance to host plants by reducing metal availability to the plant by unknown mechanisms. A potential mechanism of detoxification is binding of the Al(+3) by organic compounds that are exuded by the fungi into the surrounding soil and solution. A novel method has been developed to purify and characterize Al(+3) binding ligands from Pisolithus tinctorius exudate solutions using Al(+3) immobilized metal affinity chromatography (IMAC), reversed phase chromatography, and mass spectrometry. Fungal exudates produced by P. tinctorius exhibit a strong binding capacity for Al(+3), allowing their selective enrichment and collection using this IMAC method. Elution of the ligands requires the use of high pH. RP-HPLC separation and elemental analysis of the IMAC elutent indicates that the Al(+3) and the exudate ligands both elute from the column but are not bound in a complex. Thus, reversed phase HPLC at pH 10 is used for separation of the ligands and Al(+3) prior to MS analysis. The strongest binding IMAC fraction is analyzed by electrospray ionization mass spectrometry in positive and negative ion modes. This report provides new methods for the direct purification and analysis of naturally occurring ligands that bind hard metal ions.