Staphylococcus argenteus and S. schweitzeri, were recently proposed as novel species within S. aureus complex (SAC). S. argenteus has been reported in many countries and can threaten human health. S. schweitzeri has not been associated with human infections, but has been isolated from non-human primates. Questions regarding the evolution of pathogenicity of these two species will remain elusive until an exploratory evolutionary framework is established. We present genomic comparison analysis among members of SAC based on a pan-genome definition, which included 15 S. argenteus genomes (five newly sequenced), six S. schweitzeri genomes and 30 divergent S. aureus genomes. The three species had divergent core genomes and rare interspecific recombination was observed among the core genes. However, some subtypes of staphylococcal cassette chromosome mec (SCCmec) elements and prophages were present in different species. Of 111 tested virulence genes of S. aureus, 85 and 86 homologous genes were found in S. argenteus and S. schweitzeri, respectively. There was no difference in virulence gene content among the three species, but the sequence of most core virulence genes was divergent. Analysis of the agr locus and the genes in the capsular polysaccharides biosynthetic operon revealed that they both diverged before the speciation of SAC members. Furthermore, the widespread geographic distribution of S. argenteus, sequence type 2250, showed ambiguous biogeographical structure among geographically isolated populations, demonstrating an international spread of this pathogen. S. argenteus has spread among several countries, and invasive infections and persistent carriage may be not limited to currently reported regions. S. argenteus probably had undergone a recent host adaption and can cause human infections with a similar pathogenic potential.

In the process of developing a microplate-based growth assay, we discovered that our test organism, a native E. coli isolate, displayed very uniform doubling times (tau) only up to a certain threshold cell density. Below this cell concentration (100 CFU mL-1), the tau values were distributed unimodally (mutau = 18 +/- 0.71 min; n = 174). Inclusion of a small amount of ethyl acetate to the LB caused a collapse of the bimodal to a unimodal form. Comparable bimodal tau distribution results were also observed using E. coli cells diluted from mid-log phase cultures. Similar results were also obtained when using either an E. coli O157:H7 or a Citrobacter strain. When sterile-filtered LB supernatants, which formerly contained relatively low concentrations of bacteria(1,000-10,000 CFU mL-1), were employed as a diluent, there was an evident shift of the two populations towards each other but the bimodal effect was still apparent using either stationary or log phase cells. These data argue that there is a dependence of growth rate on starting cell density.

Escherichia coli serotype O157:H7 strain ATCC 43888 is a Shiga toxin-deficient human fecal isolate. Due to its reduced toxicity and its availability from a curated culture collection, the strain has been used extensively in applied research studies. Here, we report the Illumina-corrected PacBio whole-genome sequence of E. coli O157:H7 strain ATCC 43888.

The disruption of the bacterial cell wall plays an important part in achieving quantitative extraction of DNA from Eubacteria essential for accurate analyses of genetic material recovered from environmental samples. In this work we have tested a dozen commercial bacterial genomic DNA extraction methodologies on an average of 7.70 × 106 (±9.05%), 4.77 × 108 (±31.0%), and 5.93 × 108 (±4.69%) colony forming units (CFU) associated with 3 cultures (n = 3) each of Brochothrix thermosphacta (Bt; Gram-positive), Shigella sonnei (Ss; Gram-negative), and Escherichia coli O79 (Ec; Gram-negative). We have utilized real-time PCR (q PCR) quantification with two specific sets of primers associated with the 16S r RNA “gene” to determine the number of copies CFU-1 by comparing the unknown target DNA q PCR results with standards for each primer set. Based upon statistical analyses of our results, we determined that the Agencourt Genfind v2, High Pure PCR Template Prep Kit, and Omnilyse methods consistently provided the best yield of genomic DNA ranging from 141 to 934, 8 to 21, and 16 to 27 16S r DNA copies CFU-1 for Bt, Ss, and Ec. If one assumes 6–7 copies of the 16S r RNA gene per genome, between 1 and 3 genomes per actively dividing cell and ≥ 100 cells CFU-1 for Bt (found to be a reasonable assumption using an optical method expounded upon herein) or between 1 and 2 cells CFU-1 for either Ss or Ec, then the Omnilyse procedure provided nearly quantitative extraction of genomic DNA from these isolates (934 ± 19.9 copies CFU-1 for Bt; 20.8 ± 2.68 copies CFU-1 for Ss; 26.9 ± 3.39 copies CFU-1 for Ec). The Agencourt, High Pure, and Omnilyse technologies were subsequently assessed using 5 additional Gram-positive and 10 Gram-negative foodborne isolates (n = 3) using a set of “universal” 16S r DNA primers. Overall, the most notable DNA extraction method was found to be the Omnilyse procedure which is a “bead blender” technology involving high frequency agitation in the presence of zirconium silicate beads.

A method for coupling single-chain antibody fragments (scFvs) to immunomagnetic beads (IMBs) was developed and evaluated using scFvs specific for Listeria monocytogenes. A plasmid vector, pBAD380, was constructed that allowed the expression of histidine-tagged biotinylated scFvs in Escherichia coli. The gene encoding a scFv specific for L. monocytogenes was cloned into pBAD380 and the 6-histidine-tagged biotinylated anti-L. monocytogenes scFvs were coupled to streptavidin-coated IMBs. The ability of the anti-L. monocytogenes scFv-IMBs to capture L. monocytogenes and other Listeria species was evaluated in comparison to commercially available anti-Listeria IMBs. The anti-L. monocytogenes scFv-IMBs displayed higher efficiencies of capture (1.38-19.04%) for most strains of L. monocytogenes than were observed for the anti-Listeria IMBs (0.05-3.35%); also, the anti-L. monocytogenes scFv-IMBs exhibited improved specificity for L. monocytogenes as determined by cell capture efficiency in pure and mixed cultures.