Whole Genome and Core Genome Multilocus Sequence Typing and Single Nucleotide Polymorphism Analyses of Listeria monocytogenes Isolates Associated with an Outbreak Linked to Cheese, United States, 2013.

Epidemiological findings of a listeriosis outbreak in 2013 implicated Hispanic-style cheese produced by company A, and pulsed-field gel electrophoresis (PFGE) and whole genome sequencing (WGS) were performed on clinical isolates and representative isolates collected from company A cheese and environmental samples during the investigation. The results strengthened the evidence for cheese as the vehicle. Surveillance sampling and WGS 3 months later revealed that the equipment purchased by company B from company A yielded an environmental isolate highly similar to all outbreak isolates. The whole genome and core genome multilocus sequence typing and single nucleotide polymorphism (SNP) analyses results were compared to demonstrate the maximum discriminatory power obtained by using multiple analyses, which were needed to differentiate outbreak-associated isolates from a PFGE-indistinguishable isolate collected in a nonimplicated food source in 2012. This unrelated isolate differed from the outbreak isolates by only 7 to 14 SNPs, and as a result, the minimum spanning tree from the whole genome analyses and certain variant calling approach and phylogenetic algorithm for core genome-based analyses could not provide differentiation between unrelated isolates. Our data also suggest that SNP/allele counts should always be combined with WGS clustering analysis generated by phylogenetically meaningful algorithms on a sufficient number of isolates, and the SNP/allele threshold alone does not provide sufficient evidence to delineate an outbreak. The putative prophages were conserved across all the outbreak isolates. All outbreak isolates belonged to clonal complex 5 and serotype 1/2b and had an identical inlA sequence which did not have premature stop codons.IMPORTANCE In this outbreak, multiple analytical approaches were used for maximum discriminatory power. A PFGE-matched, epidemiologically unrelated isolate had high genetic similarity to the outbreak-associated isolates, with as few as 7 SNP differences. Therefore, the SNP/allele threshold should not be used as the only evidence to define the scope of an outbreak. It is critical that the SNP/allele counts be complemented by WGS clustering analysis generated by phylogenetically meaningful algorithms to distinguish outbreak-associated isolates from epidemiologically unrelated isolates. Careful selection of a variant calling approach and phylogenetic algorithm is critical for core-genome-based analyses. The whole-genome-based analyses were able to construct the highly resolved phylogeny needed to support the findings of the outbreak investigation. Ultimately, epidemiologic evidence and multiple WGS analyses should be combined to increase confidence levels during outbreak investigations.

Draft genome sequences of 25 Salmonella enterica serovar Agona strains isolated from poultry and associated food products harbouring multiple antibiotic resistance genes.

OBJECTIVES: Antimicrobial-resistant livestock-associated Salmonella enterica serovar Agona infection poses a significant public health threat worldwide. The present study aimed to identify antibiotic resistance genes in livestock-associated S. Agona strains isolated from chickens and associated food products (meat and eggs) in Pakistan via whole-genome sequencing. METHODS: The genomic DNAs of S. Agona strains (n=25) were sequenced using an Illumina MiSeq platform. The generated reads were trimmed and de novo assembled using CLC Genomics Workbench v.7. The draft genomes were annotated using the National Centre for Biotechnology Information (NCBI) Prokaryotic Genome Annotation Pipeline and were characterised by multilocus sequence typing (MLST). The antimicrobial-resistance genes (acquired and chromosomal mutations), extrachromosomal plasmids and Salmonella pathogenicity islands were predicted using ResFinder and CARD, PlasmidFinder and SPIFinder, respectively. RESULTS: The genome size of S. Agona ranges from 4.9 to 5.1 Mb with 52.1% GC contents. The strains belong to ST13 and harbour several antibiotic-resistance genes, including aac (6')-Iaa, aadA1, aadA2, bla OXA-10, qnrS1, cmlA, floR, tet(A), dfrA12 and point mutations in gyrB, gyrA, ParC conferring antibiotic resistance to fluoroquinolones. The strains also contain several plasmids and Salmonella pathogenicity islands. CONCLUSION: This study reports draft genomes of multidrug-resistant S. Agona from Pakistan isolated from chickens and associated food products. The data may help with understanding the antimicrobial resistance mechanisms and transmission dynamics of this serovar in poultry and associated food products and their possible transmission to humans.

Identification of the C4'-oxidized abasic site as the most abundant 2-deoxyribose lesion in radiation-damaged DNA using a novel HPLC-based approach.

A novel analytical high-performance liquid chromatography (HPLC)-based method of quantification of the yields of C4'-oxidized abasic sites, 1, in oxidatively damaged DNA has been elaborated. This new approach is based on efficient conversion of 1 into N-substituted 5-methylene-Δ(3)-pyrrolin-2-ones, 2, upon treatment of damaged DNA with primary amines in neutral or slightly acidic solutions with subsequent quantification of 2 by HPLC. The absolute and relative radiation-chemical yields of 1 in irradiated DNA solutions were re-evaluated using this method. The yields were compared with those of other 2-deoxyribose degradation products including 5-methylene-2(5H)-furanone, malondialdehyde, and furfural resulting from the C1', C4' and C5'-oxidations, respectively. The yield of free base release (FBR) determined in the same systems was employed as an internal measure of the total oxidative damage to the 2-deoxyribose moiety. Application of this technique identifies 1 as the most abundant sugar lesion in double-stranded (ds) DNA irradiated under air in solution (36% FBR). In single-stranded (ss) DNA this product is second by abundance (33% FBR) after 2-deoxyribonolactones (C1'-oxidation; 43% FBR). The production of nucleoside-5'-aldehydes (C5'-oxidation; 14% and 5% FBR in dsDNA and ssDNA, respectively) is in the third place. Taken together with the parallel reaction channel that converts C4'-radicals into malondialdehyde and 3'-phosphoglycolates, our results identify the C4'-oxidation as a prevalent pathway of oxidative damage to the sugar-phosphate backbone (50% or more of all 2-deoxyribose damages) in indirectly damaged DNA.

Caring for caregivers.

The need for managers to care for their employees has never been greater, because the need to retain good employees has never been greater. Pharmacists, nurse anesthetists, certified nurse aides, radiation oncology technicians, radiation technologists, and medical coders are in short supply in almost every part of the state, and more than 13 percent of nursing positions in the United States are open.