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Known SARS-CoV-2 variants of concern (VOCs) can be detected and differentiated using an RT-PCR-based genotyping approach, which offers quicker time to result, lower cost, higher flexibility, and use of the same laboratory instrumentation for detection of SARS-CoV-2 when compared with whole genome sequencing (WGS). In the current study, we demonstrate how we applied a genotyping approach for identification of all VOCs and that such technique can offer comparable performance to WGS for identification of known SARS-CoV-2 VOCs, including more recent strains, Omicron BA.1 and BA.2. [ABSTRACT FROM AUTHOR]

A large number of 186 Y-STR markers in nearly 2000 DNA-confirmed father-son pairs, covering an overall number of 352,999 meiotic transfers were analyzed to determine the key requirement for application of Y-chromosomal microsatellites (Y-STRs) which are widely used to infer population histories. The findings from the study might provide important insight into Y-chromosomal applications in forensic biology and identify future male individual from previous male lineage differentiation.

Activity of voltage-gated [K.sup.+] ([K.sub.v]) channels in pulmonary artery smooth muscle cells (PASMC) plays an important role in control of apoptosis and proliferation in addition to regulating membrane potential and pulmonary vascular tone. Bone morphogenetic proteins (BMPs) inhibit proliferation and induce apoptosis in normal human PASMC, whereas dysfunctional BMP signaling and downregulated [K.sub.v] channels are involved in pulmonary vascular medial hypertrophy associated with pulmonary hypertension. This study evaluated the effect of BMP-2 on [K.sub.v] channel function and expression in normal human PASMC. BMP-2 (100 nM for 18-24 h) significantly (>2-fold) upregulated mRNA expression of KCNA5, KCNA7, KCNA10, KCNC3, KCNC4, KCNF1, KCNG3, KCNS1, and KCNS3 but downregulated (at least 2-fold) KCNAB1, KCNA2, KCNG2, and KCNV2. The most dramatic change was the >10-fold downregulation of KCNG2 and KCNV2, two electrically silent [gamma]-subunits that form heterotetramers with functional [K.sub.v] channel [alpha]-subunits (e.g., KCNB1-2). Furthermore, the amplitude and current density of whole cell [K.sub.v] currents were significantly increased in PASMC treated with BMP-2. It has been demonstrated that [K.sub.+] currents generated by KCNB1 and KCNG1 (or KCNG2) or KCNB1 and KCNV2 heterotetramers are smaller than those generated by KCNB1 homotetramers, indicating that KCNG2 and KCNV2 (2 subunits that were markedly downregulated by BMP-2) are inhibitors of functional [K.sub.v] channels. These results suggest that BMP-2 divergently regulates mRNA expression of various [K.sub.v] channel [alpha]-, [beta]-, and [gamma]-subunits and significantly increases whole cell [K.sub.v] currents in human PASMC. Finally, we present evidence that attenuation of c-Myc expression by BMP-2 may be involved in BMP-2-mediated increase in [K.sub.v] channel activity and regulation of [K.sub.v] channel expression. The increased [K.sub.v] channel activity may be involved in the proapoptotic and/or antiproliferative effects of BMP-2 on PASMC. pulmonary arterial hypertension; patch clamp; membrane potential

In recent years, researchers have made great strides toward understanding the role of endogenous small RNAs (miRNAs) in gene regulation and harnessing the power of exogenous small RNAs (siRNAs and [...]

Abstract Background Rates of resistance to macrolide antibiotics in Streptococcus pneumoniae are rising around the world due to the spread of mobile genetic elements harboring mef(E) and erm(B) genes and post-vaccine clonal expansion of strains that carry them. Results Characterization of 592 clinical isolates collected in Arizona over a 10 year period shows 23.6% are macrolide resistant. The largest portion of the macrolide-resistant population, 52%, is dual mef(E)/erm(B)-positive. All dual-positive isolates are multidrug-resistant clonal lineages of Taiwan19F-14, mostly multilocus sequence type 320, carrying the recently described transposon Tn2010. The remainder of the macrolide resistant S. pneumoniae collection includes 31% mef(E)-positive, and 9% erm(B)-positive strains. Conclusions The dual-positive, multidrug-resistant S. pneumoniae clones have likely expanded by switching to non-vaccine serotypes after the heptavalent pneumococcal conjugate vaccine release, and their success limits therapy options. This upsurge could have a considerable clinical impact in Arizona.

Abstract Background Divergence of bacterial populations into distinct subpopulations is often the result of ecological isolation. While some studies have suggested the existence of Salmonella enterica subsp. enterica subclades, evidence for these subdivisions has been ambiguous. Here we used a comparative genomics approach to define the population structure of Salmonella enterica subsp. enterica, and identify clade-specific genes that may be the result of ecological specialization. Results Multi-locus sequence analysis (MLSA) and single nucleotide polymorphisms (SNPs) data for 16 newly sequenced and 30 publicly available genomes showed an unambiguous subdivision of S. enterica subsp. enterica into at least two subpopulations, which we refer to as clade A and clade B. Clade B strains contain several clade-specific genes or operons, including a β-glucuronidase operon, a S-fimbrial operon, and cell surface related genes, which strongly suggests niche specialization of this subpopulation. An additional set of 123 isolates was assigned to clades A and B by using qPCR assays targeting subpopulation-specific SNPs and genes of interest. Among 98 serovars examined, approximately 20% belonged to clade B. All clade B isolates contained two pathogenicity related genomic islands, SPI-18 and a cytolethal distending toxin islet; a combination of these two islands was previously thought to be exclusive to serovars Typhi and Paratyphi A. Presence of β-glucuronidase in clade B isolates specifically suggests an adaptation of this clade to the vertebrate gastrointestinal environment. Conclusions S. enterica subsp. enterica consists of at least two subpopulations that differ specifically in genes involved in host and tissue tropism, utilization of host specific carbon and nitrogen sources and are therefore likely to differ in ecology and transmission characteristics.

Abstract Background The bacterial genus Listeria contains pathogenic and non-pathogenic species, including the pathogens L. monocytogenes and L. ivanovii, both of which carry homologous virulence gene clusters such as the prfA cluster and clusters of internalin genes. Initial evidence for multiple deletions of the prfA cluster during the evolution of Listeria indicates that this genus provides an interesting model for studying the evolution of virulence and also presents practical challenges with regard to definition of pathogenic strains. Results To better understand genome evolution and evolution of virulence characteristics in Listeria, we used a next generation sequencing approach to generate draft genomes for seven strains representing Listeria species or clades for which genome sequences were not available. Comparative analyses of these draft genomes and six publicly available genomes, which together represent the main Listeria species, showed evidence for (i) a pangenome with 2,032 core and 2,918 accessory genes identified to date, (ii) a critical role of gene loss events in transition of Listeria species from facultative pathogen to saprotroph, even though a consistent pattern of gene loss seemed to be absent, and a number of isolates representing non-pathogenic species still carried some virulence associated genes, and (iii) divergence of modern pathogenic and non-pathogenic Listeria species and strains, most likely circa 47 million years ago, from a pathogenic common ancestor that contained key virulence genes. Conclusions Genome evolution in Listeria involved limited gene loss and acquisition as supported by (i) a relatively high coverage of the predicted pan-genome by the observed pan-genome, (ii) conserved genome size (between 2.8 and 3.2 Mb), and (iii) a highly syntenic genome. Limited gene loss in Listeria did include loss of virulence associated genes, likely associated with multiple transitions to a saprotrophic lifestyle. The genus Listeria thus provides an example of a group of bacteria that appears to evolve through a loss of virulence rather than acquisition of virulence characteristics. While Listeria includes a number of species-like clades, many of these putative species include clades or strains with atypical virulence associated characteristics. This information will allow for the development of genetic and genomic criteria for pathogenic strains, including development of assays that specifically detect pathogenic Listeria strains.

Staphylococcus aureus is an important clinical pathogen worldwide and understanding this organism's phylogeny and, in particular, the role of recombination, is important both to understand the overall spread of virulent lineages and to characterize outbreaks. To further elucidate the phylogeny of S. aureus, 35 diverse strains were sequenced using whole genome sequencing. In addition, 29 publicly available whole genome sequences were included to create a single nucleotide polymorphism (SNP)-based phylogenetic tree encompassing 11 distinct lineages. All strains of a particular sequence type fell into the same clade with clear groupings of the major clonal complexes of CC8, CC5, CC30, CC45 and CC1. Using a novel analysis method, we plotted the homoplasy density and SNP density across the whole genome and found evidence of recombination throughout the entire chromosome, but when we examined individual clonal lineages we found very little recombination. However, when we analyzed three branches of multiple lineages, we saw intermediate and differing levels of recombination between them. These data demonstrate that in S. aureus, recombination occurs across major lineages that subsequently expand in a clonal manner. Estimated mutation rates for the CC8 and CC5 lineages were different from each other. While the CC8 lineage rate was similar to previous studies, the CC5 lineage was 100-fold greater. Fifty known virulence genes were screened in all genomes in silico to determine their distribution across major clades. Thirty-three genes were present variably across clades, most of which were not constrained by ancestry, indicating horizontal gene transfer or gene loss. [ABSTRACT FROM AUTHOR]

Background Within the last decade, Salmonella enterica subsp. enterica serovar Cerro (S. Cerro) has become one of the most common serovars isolated from cattle and dairy farm environments in the northeastern US. The fact that this serovar is commonly isolated from subclinically infected cattle and is rarely associated with human disease, despite its frequent isolation from cattle, has led to the hypothesis that this emerging serovar may be characterized by reduced virulence. We applied comparative and population genomic approaches to (i) characterize the evolution of this recently emerged serovar and to (ii) gain a better understanding of genomic features that could explain some of the unique epidemiological features associated with this serovar. Results In addition to generating a de novo draft genome for one Salmonella Cerro strain, we also generated whole genome sequence data for 26 additional S. Cerro isolates, including 16 from cattle operations in New York (NY) state, 2 from human clinical cases from NY in 2008, and 8 from diverse animal sources (7 from Washington state and 1 from Florida). All isolates sequenced in this study represent sequence type ST367. Population genomic analysis showed that isolates from the NY cattle operations form a well-supported clade within S. Cerro ST367 (designated here "NY bovine clade"), distinct from isolates from Washington state, Florida and the human clinical cases. A molecular clock analysis indicates that the most recent common ancestor of the NY bovine clade dates back to 1998, supporting the recent emergence of this clone. Comparative genomic analyses revealed several relevant genomic features of S. Cerro ST367, that may be responsible for reduced virulence of S. Cerro, including an insertion creating a premature stop codon in sopA. In addition, patterns of gene deletion in S. Cerro ST367 further support adaptation of this clone to a unique ecological or host related niche. Conclusions Our results indicate that the increase in prevalence of S. Cerro ST367 is caused by a highly clonal subpopulation and that S. Cerro ST367 is characterized by unique genomic deletions that may indicate adaptation to specific ecological niches and possibly reduced virulence in some hosts. [ABSTRACT FROM AUTHOR]

We propose that haplotyped loci with high heterozygosity can be useful in human identification, especially within families, if recombination is very low among the sites. Three or more SNPs extending over small molecular intervals (<10 KB) can be identified in the human genome to define miniature haplotypes with moderate levels of linkage disequilibrium. Properly selected, these mini-haplotypes (or minihaps) consist of multiple haplotype lineages (alleles) that have evolved from the ancestral human haplotype but show no evidence of recurring recombination, allowing each distinct haplotype to be equated with an allele, all copies of which are essentially identical by descent. Historic recombinants, representing rare events that have drifted to common frequencies over many generations, can be identified in some cases, they do not equate to frequently recurring recombination. We have identified examples in our data collected on various projects and present eight such mini-haplotypes comprised of informative SNPs. We also discuss the ideal characteristics and advantages of minihaps for human familial identification and ancestry inference, and compare them to other types of forensic markers in use and/or that have been proposed. We expect that it is possible to carry out a systematic search and identify a useful panel of mini-haplotypes, with even better properties than the examples presented here. [ABSTRACT FROM AUTHOR]

A letter to the editor is presented related to application of next generationsequencing in clinical research.

The genetic diversity represented by >2,500 different Salmonella serovars provides a yet largely uncharacterized reservoir of mobile elements that can contribute to the frequent emergence of new pathogenic strains of this important zoonotic pathogen. Currently, our understanding of Salmonella mobile elements is skewed by the fact that most studies have focused on highly virulent or common serovars. To gain a more global picture of mobile elements in Salmonella, we used prediction algorithms to screen for mobile elements in 16 sequenced Salmonella genomes representing serovars for which no prior genome scale mobile element data were available. From these results, selected mobile elements underwent further analyses in the form of validation studies, comparative analyses, and PCR-based population screens. Through this analysis we identified a novel plasmid that has two cointegrated replicons (IncI1-IncFIB); this plasmid type was found in four genomes representing different Salmonella serovars and contained a virulence gene array that had not been previously identified. A Salmonella Montevideo isolate contained an IncHI and an IncN2 plasmid, which both encoded antimicrobial resistance genes. We also identified two novel genomic islands (SGI2 and SGI3), and 42 prophages with mosaic architecture, seven of them harboring known virulence genes. Finally, we identified a novel integrative conjugative element (ICE) encoding a type IVb pilus operon in three non-typhoidal Salmonella serovars. Our analyses not only identified a considerable number of mobile elements that have not been previously reported in Salmonella, but also found evidence that these elements facilitate transfer of genes that were previously thought to be limited in their distribution among Salmonella serovars. The abundance of mobile elements encoding pathogenic properties may facilitate the emergence of strains with novel combinations of pathogenic traits. [ABSTRACT FROM AUTHOR]

Reducing the risk of Salmonella contamination in pet food is critical for both companion animals and humans, and its importance is reflected by the substantial increase in the demand for pathogen testing. Accurate and rapid detection of foodborne pathogens improves food safety, protects the public health, and benefits food producers by assuring product quality while facilitating product release in a timely manner. Traditional culture-based methods for Salmonella screening are laborious and can take 5 to 7 days to obtain definitive results. In this study, we developed two methods for the detection of low levels of Salmonella in pet food using real-time PCR: (i) detection of Salmonella in 25 g of dried pet food in less than 14 h with an automated magnetic bead-based nucleic acid extraction method and (ii) detection of Salmonella in 375 g of composite dry pet food matrix in less than 24 h with a manual centrifugation-based nucleic acid preparation method. Both methods included a preclarification step using a novel protocol that removes food matrix-associated debris and PCR inhibitors and improves the sensitivity of detection. Validation studies revealed no significant differences between the two real-time PCR methods and the standard U.S. Food and Drug Administration Bacteriological Analytical Manual (chapter 5) culture confirmation method. [ABSTRACT FROM AUTHOR]

Three species--Vibrio cholerae, Vibrio parahaemolyticus, and Vibrio vulnificus--account for the majority of vibrio infections in humans. Rapid and accurate identification of Vibrio species has been problematic because phenotypic characteristics are variable within species. Additionally, biochemical identification and confirmation require 2 or more days to complete. Rapid and sensitive molecular techniques for the detection of vibrio pathogens would be useful for the surveillance and management of outbreaks. To facilitate the identification of human-pathogenic species, we designed and validated a highly sensitive, specific, and robust multiplex real-time PCR assay to identify V. cholerae, V. parahaemolyticus, and V. vulnificus using a four-dye configuration in a convenient lyophilized format. Multiple Vibrio strains were sequenced to verify candidate target TaqMan sites. Several individual assays within the multiplex contain multiple primers or probes to ensure detection of polymorphic variants. V. cholerae, V. parahaemolyticus, and V. vulnificus were detected either individually or in mixtures at ≤30 genomic copies. V. cholerae was specifically detected in the presence or absence of Vibrio mimicus. The Vibrio multiplex assay showed 100% specificity to all targets analyzed and no detection of nearest neighbor strains. Each assay exhibited 100% ± 10% efficiency. Multiplex real-time PCR can simplify pathogen detection and reduce costs per test since three species can be analyzed in a single reaction tube. Rapid and accurate detection of pathogenic vibrios in shellfish or seawater samples will improve the microbiological safety of seafood for consumers. [ABSTRACT FROM AUTHOR]

The HID EVOlution—qPCR/STR Setup System enables automation of DNA quantitative real-time polymerase chain reaction (PCR) setup, normalization of DNA sample, and PCR setup for short tandem repeat (STR) analysis. The HID EVOlution System tracks sample and reagent information and facilitates data transfer of DNA quantification, normalization, and PCR setup for STR analysis steps, eliminating the need for manual processing and repetitive data entry. Instruments for the automated system include a Tecan Freedom EVO 150 robot for liquid handling, the 7500 Real-Time PCR System for DNA quantification, the GeneAmp PCR System 9700 for STR amplification, and the 3130xl Genetic Analyzer for the detection of amplified STR fragments. Validation studies including reproducibility, accuracy, correlation, and contamination studies were performed. Results demonstrated clean liquid-handling capabilities and maintenance of sample integrity. Variation in average allele peak height obtained using automated protocol was similar to that obtained using the manual protocol. [Copyright &y& Elsevier]

Background: In the event of biocrimes or infectious disease outbreaks, high-resolution genetic characterization for identifying the agent and attributing it to a specific source can be crucial for an effective response. Until recently, in-depth genetic characterization required expensive and time-consuming Sanger sequencing of a few strains, followed by genotyping of a small number of marker loci in a panel of isolates at or by gel-based approaches such as pulsed field gel electrophoresis, which by necessity ignores most of the genome. Next-generation, massively parallel sequencing (MPS) technology (specifically the Applied Biosystems sequencing by oligonucleotide ligation and detection (SOLiD™) system) is a powerful investigative tool for rapid, cost-effective and parallel microbial whole-genome characterization. Results: To demonstrate the utility of MPS for whole-genome typing of monomorphic pathogens, four Bacillus anthracis and four Yersinia pestis strains were sequenced in parallel. Reads were aligned to complete reference genomes, and genomic variations were identified. Resequencing of the B. anthracis Ames ancestor strain detected no false-positive single-nucleotide polymorphisms (SNPs), and mapping of reads to the Sterne strain correctly identified 98% of the 133 SNPs that are not clustered or associated with repeats. Three geographically distinct B. anthracis strains from the A branch lineage were found to have between 352 and 471 SNPs each, relative to the Ames genome, and one strain harbored a genomic amplification. Sequencing of four Y. pestis strains from the Orientalis lineage identified between 20 and 54 SNPs per strain relative to the CO92 genome, with the single Bolivian isolate having approximately twice as many SNPs as the three more closely related North American strains. Coverage plotting also revealed a common deletion in two strains and an amplification in the Bolivian strain that appear to be due to insertion element-mediated recombination events. Most private SNPs (that is, a, variant found in only one strain in this set) selected for validation by Sanger sequencing were confirmed, although rare falsepositive SNPs were associated with variable nucleotide tandem repeats. Conclusions: The high-throughput, multiplexing capability, and accuracy of this system make it suitable for rapid whole-genome typing of microbial pathogens during a forensic or epidemiological investigation. By interrogating nearly every base of the genome, rare polymorphisms can be reliably discovered, thus facilitating high-resolution strain tracking and strengthening forensic attribution. [ABSTRACT FROM AUTHOR]

The Y-chromosomal short tandem repeat (Y-STR) polymorphisms included in the AmpF lSTR® Yfiler® polymerase chain reaction amplification kit have become widely used for forensic and evolutionary applications where a reliable knowledge on mutation properties is necessary for correct data interpretation. Therefore, we investigated the 17 Yfiler Y-STRs in 1,730–1,764 DNA-confirmed father–son pairs per locus and found 84 sequence-confirmed mutations among the 29,792 meiotic transfers covered. Of the 84 mutations, 83 (98.8%) were single-repeat changes and one (1.2%) was a double-repeat change (ratio, 1:0.01), as well as 43 (51.2%) were repeat gains and 41 (48.8%) repeat losses (ratio, 1:0.95). Medians from Bayesian estimation of locus-specific mutation rates ranged from 0.0003 for DYS448 to 0.0074 for DYS458, with a median rate across all 17 Y-STRs of 0.0025. The mean age (at the time of son’s birth) of fathers with mutations was with 34.40 (±11.63) years higher than that of fathers without ones at 30.32 (±10.22) years, a difference that is highly statistically significant ( p < 0.001). A Poisson-based modeling revealed that the Y-STR mutation rate increased with increasing father’s age on a statistically significant level ( α = 0.0294, 2.5% quantile = 0.0001). From combining our data with those previously published, considering all together 135,212 meiotic events and 331 mutations, we conclude for the Yfiler Y-STRs that (1) none had a mutation rate of >1%, 12 had mutation rates of >0.1% and four of <0.1%, (2) single-repeat changes were strongly favored over multiple-repeat ones for all loci but 1 and (3) considerable variation existed among loci in the ratio of repeat gains versus losses. Our finding of three Y-STR mutations in one father–son pair (and two pairs with two mutations each) has consequences for determining the threshold of allelic differences to conclude exclusion constellations in future applications of Y-STRs in paternity testing and pedigree analyses. [ABSTRACT FROM AUTHOR]

The HID EVOlution–Extraction System (Tecan Group Ltd., Mannedorf, Switzerland) was developed to automate DNA extraction from biological samples using the PrepFiler Automated Forensic DNA Extraction Kit (Applied Biosystems, Foster City, CA). The system consists of a Tecan Freedom EVO 150 robot (Tecan Group Ltd., Mannedorf, Switzerland), a graphical user interface designed for use with Freedom EVOware software v 2.1 SP1 (Tecan Group Ltd., Mannedorf, Switerland) as well as instrument hardware and plastic to support the PrepFiler reagents and protocol. The DNA quality and quantity obtained were comparable to that observed with the corresponding manual extraction protocol. Purified DNA was free of inhibitors and ready for downstream applications, such as real-time quantitative PCR and PCR for short tandem repeat (STR) analysis. The DNA quantity and quality obtained were consistent as demonstrated by the quantification and STR results. Our studies indicate that the HID EVOlution–Extraction System can easily be adopted in forensic laboratories to alleviate some of the bottlenecks of sample preparation in forensic laboratories. [Copyright &y& Elsevier]

The Quantifiler® Duo DNA Quantification kit enables simultaneous quantification of human DNA and human male DNA as well as detection of inhibitors of PCR in a single real-time PCR well. Pooled human male genomic DNA is used to generate standard curves for both human (ribonuclease P RNA component H1) and human male (sex determining region Y) specific targets. A shift in the cycle threshold ( CT) values for the internal positive control monitors the presence of PCR inhibitors in a sample. The assay is human specific and exhibits a high dynamic range from 0.023 to 50 ng/μL. In addition, the multiplex assay can detect as little as 25 pg/μL of human male DNA in the presence of a 1000-fold excess of human female DNA. The multiplex assay provides assessment of the DNA extract and guidance for the selection of the appropriate AmpFℓSTR® Amplification Kit to obtain interpretable short tandem repeat profiles. [ABSTRACT FROM AUTHOR]

Activity of voltage-gated K+ (Kv) channels in pulmonary artery smooth muscle cells (PASMC) plays an important role in control of apoptosis and proliferation in addition to regulating membrane potential and pulmonary vascular tone. Bone morphogenetic proteins (BMPs) inhibit proliferation and induce apoptosis in normal human PASMC, whereas dysfunctional BMP signaling and downregulated Kv channels are involved in pulmonary vascular medial hypertrophy associated with pulmonary hypertension. This study evaluated the effect of BMP-2 on Kv channel function and expression in normal human PASMC. BMP-2 (100 nM for 18–24 h) significantly (>2-fold) upregulated mRNA expression of KCNA5, KCNA7, KCNA10, KCNC3, KCNC4, KCNF1, KCNG3, KCNS1, and KCNS3 but downregulated (at least 2-fold) KCNAB1, KCNA2, KCNG2, and KCNV2. The most dramatic change was the >10-fold downregulation of KCNG2 and KCNV2, two electrically silent γ-subunits that form heterotetramers with functional Kv channel α-subunits (e.g., KCNB1-2). Furthermore, the amplitude and current density of whole cell Kv currents were significantly increased in PASMC treated with BMP-2. It has been demonstrated that K+ currents generated by KCNB1 and KCNG1 (or KCNG2) or KCNB1 and KCNV2 heterotetramers are smaller than those generated by KCNB1 homotetramers, indicating that KCNG2 and KCNV2 (2 subunits that were markedly downregulated by BMP-2) are inhibitors of functional Kv channels. These results suggest that BMP-2 divergently regulates mRNA expression of various Kv channel α-, β-, and γ-subunits and significantly increases whole cell Kv currents in human PASMC. Finally, we present evidence that attenuation of c-Myc expression by BMP-2 may be involved in BMP-2-mediated increase in Kv channel activity and regulation of Kv channel expression. The increased Kv channel activity may be involved in the proapoptotic and/or antiproliferative effects of BMP-2 on PASMC. [ABSTRACT FROM AUTHOR]

The article presents a study which validated the applied biosystems MicroSEQ real-time polymerase chain reaction method to detect Escherichia coli (E. coli) O157:H7 in several good matrixes. The test method was compared to the U.S. Department of Agriculture's "Microbiology Laboratory Guidebook" 5.04 reference method for detecting E. coli in ground beef and beef trim and to the ISO 16654 reference method for detecting E. coli in spinach, orange juice and apple juice.

In this study, we report a whole-genome single nucleotide polymorphism (SNP)-based evolutionary approach to study the epidemiology of a multistate outbreak of Salmonella enterica subsp. enterica serovar Montevideo. This outbreak included 272 cases that occurred in 44 states between July 2009 and April 2010. A case-control study linked the consumption of salami made with contaminated black and red pepper to the outbreak. We sequenced, on the SOLiD System, 47 isolates with XbaI PFGE pattern JIXX01.0011, a common pulsed-field gel electrophoresis (PFGE) pattern associated with isolates from the outbreak. These isolates represented 20 isolates collected from human sources during the period of the outbreak and 27 control isolates collected from human, food, animal, and environmental sources before the outbreak. Based on 253 high-confidence SNPs, we were able to reconstruct a tip-dated molecular clock phylogeny of the isolates and to assign four human isolates to the actual outbreak. We developed an SNP typing assay to rapidly discriminate between outbreak-related cases and non-outbreak-related cases and tested this assay on an extended panel of 112 isolates. These results suggest that only a very small percentage of the human isolates with the outbreak PFGE pattern and obtained during the outbreak period could be attributed to the actual pepper-related outbreak (20%), while the majority (80%) of the putative cases represented background cases. This study demonstrates that next-generation-based SNP typing provides the resolution and accuracy needed for outbreak investigations of food-borne pathogens that cannot be distinguished by currently used subtyping methods. [ABSTRACT FROM AUTHOR]

A complete system for real-time PCR detection of Listeria species was validated in five food matrixes and five environmental surfaces, namely, hot dogs, roast beef, lox (smoked salmon), pasteurized whole cow's milk, dry infant formula, stainless steel, plastic cutting board, ceramic tile, rubber sheets, and sealed concrete. The system consists of the MicroSEQ Listeria spp. Detection Kit, two sample preparation kits (PrepSEQ Nucleic Acid Extraction Kit and PrepSEQ Rapid Spin Sample Preparation Kit), the Applied Biosystems 7500 Fast Real-Time PCR instrument, and the RapidFinderTM Express v1.1 Software for data analysis. The test method was compared to the ISO 11290-1 reference method using an unpaired study design. The MicroSEQ Listeria spp. Detection Kit and the ISO 11290-1 reference method showed equivalent detection based on Chi-square analysis for all matrixes except hot dogs. For hot dogs, the MicroSEQ method detected more positives than the reference method for the low- and high-level inoculations, with all of the presumptive positives confirmed by the reference method. An independent validation study confirmed these findings on lox and stainless steel surface. The MicroSEQ kit detected all 50 Listeria strains tested and none of the 31 nontarget bacteria strains.

Three mechanistically different sample extraction methodologies, namely, silica spin columns, phenol-chloroform, and an automated magnetic capture of polymer-complexed DNA (via an Automate Express instrument), were compared for their abilities to purify nucleic acids from blood culture fluids for use in TaqMan assays for detection of Staphylococcus aureus. The extracts from silica columns required 100- to 1000-fold dilutions to sufficiently reduce the powerful PCR inhibitory effects of the anticoagulant sodium polyanetholsulfonate, a common additive in blood culture media. In contrast, samples extracted by either phenol-chloroform or the Automate Express instrument required little or no dilution, respectively, allowing for an approximate 100-fold improvement in assay sensitivity. Analysis of 60 blood culture bottles indicated that these latter two methodologies could be used to detect lower numbers of pathogens and that a growing S. aureus culture could be detected 2 hours earlier than when using silica columns. Of the three tested methodologies, the Automate Express instrument had the shortest time to result, requiring only approximately 80 minutes to process 12 samples. These findings highlight the importance of considering the mechanism when selecting a DNA extraction methodology, given that certain PCR inhibitors act in a similar fashion to DNA in certain chemical environments, resulting in copurification, whereas other methodologies use different chemistries that have advantages during the DNA purification of certain types of samples., (Copyright © 2012 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.)

Background: Species of Cronobacter are widespread in the environment and are occasional food-borne pathogens associated with serious neonatal diseases, including bacteraemia, meningitis, and necrotising enterocolitis. The genus is composed of seven species: C. sakazakii, C. malonaticus, C. turicensis, C. dublinensis, C. muytjensii, C. universalis, and C. condimenti. Clinical cases are associated with three species, C. malonaticus, C. turicensis and, in particular, with C. sakazakii multilocus sequence type 4. Thus, it is plausible that virulence determinants have evolved in certain lineages., Methodology/principal Findings: We generated high quality sequence drafts for eleven Cronobacter genomes representing the seven Cronobacter species, including an ST4 strain of C. sakazakii. Comparative analysis of these genomes together with the two publicly available genomes revealed Cronobacter has over 6,000 genes in one or more strains and over 2,000 genes shared by all Cronobacter. Considerable variation in the presence of traits such as type six secretion systems, metal resistance (tellurite, copper and silver), and adhesins were found. C. sakazakii is unique in the Cronobacter genus in encoding genes enabling the utilization of exogenous sialic acid which may have clinical significance. The C. sakazakii ST4 strain 701 contained additional genes as compared to other C. sakazakii but none of them were known specific virulence-related genes., Conclusions/significance: Genome comparison revealed that pair-wise DNA sequence identity varies between 89 and 97% in the seven Cronobacter species, and also suggested various degrees of divergence. Sets of universal core genes and accessory genes unique to each strain were identified. These gene sequences can be used for designing genus/species specific detection assays. Genes encoding adhesins, T6SS, and metal resistance genes as well as prophages are found in only subsets of genomes and have contributed considerably to the variation of genomic content. Differences in gene content likely contribute to differences in the clinical and environmental distribution of species and sequence types.

The genetic diversity represented by >2,500 different Salmonella serovars provides a yet largely uncharacterized reservoir of mobile elements that can contribute to the frequent emergence of new pathogenic strains of this important zoonotic pathogen. Currently, our understanding of Salmonella mobile elements is skewed by the fact that most studies have focused on highly virulent or common serovars. To gain a more global picture of mobile elements in Salmonella, we used prediction algorithms to screen for mobile elements in 16 sequenced Salmonella genomes representing serovars for which no prior genome scale mobile element data were available. From these results, selected mobile elements underwent further analyses in the form of validation studies, comparative analyses, and PCR-based population screens. Through this analysis we identified a novel plasmid that has two cointegrated replicons (IncI1-IncFIB); this plasmid type was found in four genomes representing different Salmonella serovars and contained a virulence gene array that had not been previously identified. A Salmonella Montevideo isolate contained an IncHI and an IncN2 plasmid, which both encoded antimicrobial resistance genes. We also identified two novel genomic islands (SGI2 and SGI3), and 42 prophages with mosaic architecture, seven of them harboring known virulence genes. Finally, we identified a novel integrative conjugative element (ICE) encoding a type IVb pilus operon in three non-typhoidal Salmonella serovars. Our analyses not only identified a considerable number of mobile elements that have not been previously reported in Salmonella, but also found evidence that these elements facilitate transfer of genes that were previously thought to be limited in their distribution among Salmonella serovars. The abundance of mobile elements encoding pathogenic properties may facilitate the emergence of strains with novel combinations of pathogenic traits.

In this study, we report a whole-genome single nucleotide polymorphism (SNP)-based evolutionary approach to study the epidemiology of a multistate outbreak of Salmonella enterica subsp. enterica serovar Montevideo. This outbreak included 272 cases that occurred in 44 states between July 2009 and April 2010. A case-control study linked the consumption of salami made with contaminated black and red pepper to the outbreak. We sequenced, on the SOLiD System, 47 isolates with XbaI PFGE pattern JIXX01.0011, a common pulsed-field gel electrophoresis (PFGE) pattern associated with isolates from the outbreak. These isolates represented 20 isolates collected from human sources during the period of the outbreak and 27 control isolates collected from human, food, animal, and environmental sources before the outbreak. Based on 253 high-confidence SNPs, we were able to reconstruct a tip-dated molecular clock phylogeny of the isolates and to assign four human isolates to the actual outbreak. We developed an SNP typing assay to rapidly discriminate between outbreak-related cases and non-outbreak-related cases and tested this assay on an extended panel of 112 isolates. These results suggest that only a very small percentage of the human isolates with the outbreak PFGE pattern and obtained during the outbreak period could be attributed to the actual pepper-related outbreak (20%), while the majority (80%) of the putative cases represented background cases. This study demonstrates that next-generation-based SNP typing provides the resolution and accuracy needed for outbreak investigations of food-borne pathogens that cannot be distinguished by currently used subtyping methods.

Increasingly, more food companies are relying on molecular methods, such as PCR, for pathogen detection due to their improved simplicity, sensitivity, and rapid time to results. This report describes the validation of a new Real-Time PCR method to detect Listeria monocytogenes in nine different food matrixes. The complete system consists of the MicroSEQ L. monocytogenes Detection Kit, sample preparation, the Applied Biosystems 7500 Fast Real-Time PCR instrument, and RapidFinder Express software. Two sample preparation methods were validated: the PrepSEQ Nucleic Acid extraction kit and the PrepSEQ Rapid Spin sample preparation kit. The test method was compared to the ISO 11290-1 reference method using an unpaired-study design to detect L. monocytogenes in roast beef, cured bacon, lox (smoked salmon), lettuce, whole cow's milk, dry infant formula, ice cream, salad dressing, and mayonnaise. The MicroSEQ L. monocytogenes Detection Kit and the ISO 11290-1 reference method showed equivalent detection based on Chi-square analysis for all food matrixes when the samples were prepared using either of the two sample preparation methods. An independent validation confirmed these findings on smoked salmon and whole cow's milk. The MicroSEQ kit detected all 50 L. monocytogenes strains tested, and none of the 30 nontargeted bacteria strains.

Background: Divergence of bacterial populations into distinct subpopulations is often the result of ecological isolation. While some studies have suggested the existence of Salmonella enterica subsp. enterica subclades, evidence for these subdivisions has been ambiguous. Here we used a comparative genomics approach to define the population structure of Salmonella enterica subsp. enterica, and identify clade-specific genes that may be the result of ecological specialization., Results: Multi-locus sequence analysis (MLSA) and single nucleotide polymorphisms (SNPs) data for 16 newly sequenced and 30 publicly available genomes showed an unambiguous subdivision of S. enterica subsp. enterica into at least two subpopulations, which we refer to as clade A and clade B. Clade B strains contain several clade-specific genes or operons, including a β-glucuronidase operon, a S-fimbrial operon, and cell surface related genes, which strongly suggests niche specialization of this subpopulation. An additional set of 123 isolates was assigned to clades A and B by using qPCR assays targeting subpopulation-specific SNPs and genes of interest. Among 98 serovars examined, approximately 20% belonged to clade B. All clade B isolates contained two pathogenicity related genomic islands, SPI-18 and a cytolethal distending toxin islet; a combination of these two islands was previously thought to be exclusive to serovars Typhi and Paratyphi A. Presence of β-glucuronidase in clade B isolates specifically suggests an adaptation of this clade to the vertebrate gastrointestinal environment., Conclusions: S. enterica subsp. enterica consists of at least two subpopulations that differ specifically in genes involved in host and tissue tropism, utilization of host specific carbon and nitrogen sources and are therefore likely to differ in ecology and transmission characteristics.