Detection and Identification of Salmonella enterica, Escherichia coli, and Shigella spp. via PCR-ESI-MS: Isolate Testing and Analysis of Food Samples

An assay to identify the common food-borne pathogens Salmonella, Escherichia coli, Shigella, and Listeria monocytogenes was developed in collaboration with Ibis Biosciences (a division of Abbott Molecular) for the Plex-ID biosensor system, a platform that uses electrospray ionization mass spectroscopy (ESI-MS) to detect the base composition of short PCR amplicons. The new food-borne pathogen (FBP) plate has been experimentally designed using four gene segments for a total of eight amplicon targets. Initial work built a DNA base count database that contains more than 140 Salmonella enterica, 139 E. coli, 11 Shigella, and 36 Listeria patterns and 18 other Enterobacteriaceae organisms. This assay was tested to determine the scope of the assay\u27s ability to detect and differentiate the enteric pathogens and to improve the reference database associated with the assay. More than 800 bacterial isolates of S. enterica, E. coli, and Shigella species were analyzed. Overall, 100% of S. enterica, 99% of E. coli, and 73% of Shigella spp. were detected using this assay. The assay was also able to identify 30% of the S. enterica serovars to the serovar level. To further characterize the assay, spiked food matrices and food samples collected during regulatory field work were also studied. While analysis of preenrichment media was inconsistent, identification of S. enterica from selective enrichment media resulted in serovar-level identifications for 8 of 10 regulatory samples. The results of this study suggest that this high-throughput method may be useful in clinical and regulatory laboratories testing for these pathogens

Identification and Characterization of Class 1 Integron Resistance Gene Cassettes among Salmonella Strains Isolated from Imported Seafood ▿

A total of 210 Salmonella isolates, representing 64 different serovars, were isolated from imported seafood samples, and 55/210 isolates were found to be resistant to at least one antibiotic. Class 1 integrons from three multidrug-resistant Salmonella enterica strains (Salmonella enterica serovars Newport [strain 62], Typhimurium var. Copenhagen [strain 629], and Lansing [strain 803], originating from Hong Kong, the Philippines, and Taiwan, respectively) were characterized. Southern hybridization of plasmids isolated from these strains, using a class 1 integron probe, showed that trimethoprim-sulfamethoxazole and streptomycin resistance genes were located on a megaplasmid in strain 629. Our study indicates that imported seafood could be a reservoir for Salmonella isolates resistant to multiple antibiotics

Dual-layered nanogel-coated hollow lipid/polypeptide conjugate assemblies for potential pH-triggered intracellular drug release.

To achieve effective intracellular anticancer drug delivery, the polymeric vesicles supplemented with the pH-responsive outlayered gels as a delivery system of doxorubicin (DOX) were developed from self-assembly of the lipid/polypeptide adduct, distearin grafted poly(γ-glutamic acid) (poly(γ-GA)), followed by sequential deposition of chitosan and poly(γ-GA-co-γ-glutamyl oxysuccinimide)-g-monomethoxy poly(ethylene glycol) in combination with in situ covalent cross-linking on assembly surfaces. The resultant gel-caged polymeric vesicles (GCPVs) showed superior performance in regulating drug release in response to the external pH change. Under typical physiological conditions (pH 7.4 and 37 °C) at which the γ-GA/DOX ionic pairings remained mostly undisturbed, the dense outlayered gels of GCPVs significantly reduced the premature leakage of the uncomplexed payload. With the environmental pH being reduced from pH 7.4 to 4.7, the drug liberation was appreciably promoted by the massive disruption of the ionic γ-GA/DOX complexes along with the significant swelling of nanogel layers upon the increased protonation of chitosan chain segments. After being internalized by HeLa cells via endocytosis, GCPVs exhibited cytotoxic effect comparable to free DOX achieved by rapidly releasing the payload in intracellular acidic endosomes and lysosomes. This strongly implies the great promise of such unique GCPVs as an intracellular drug delivery carrier for potential anticancer treatment

Recipes, compositions and average molecular weights of the derived polypeptide adducts.

a<p>Determined by ¹H-NMR in DMSO-<i>d₆</i> at 20°C.</p>b<p>Obtained by theoretical calculation as follows: poly(γ-GA) M_w 15600 (g/mol) + number of conjugated distearin moieties × 625 (M_w of distearin)</p>c<p>Obtained by theoretical calculation as follows: poly(γ-GA) M_w 15600 (g/mol) + number of conjugated Osu moieties ×114 (M_w of OSu) + number of mPEG grafts ×5000 (M_w of mPEG).</p

Angle-dependent DLS/SLS data and TEM images of lipid/polypeptide conjugate vesicles (a, b) and DOX-loaded GCPVs (c, d).

<p>Angle-dependent DLS/SLS data and TEM images of lipid/polypeptide conjugate vesicles (a, b) and DOX-loaded GCPVs (c, d).</p

pH-dependent characteristics of DOX-loaded GCPVs and DOX-loaded chitosan/ poly(γ-GA)-deposited polymeric vesicles.

<p>(a) DLS colloidal particle size distribution profiles of DOX-loaded GCPVs in aqueous media of various pH. (b) Zeta potentials of DOX-loaded GCPVs and DOX-loaded chitosan/ poly(γ-GA)-deposited polymeric vesicles in different pH aqueous media.</p

¹H-NMR spectra of (a) poly(γ-GA-co-γ-DSGA) and (b) poly(γ-GA-co-γ-GAOSu)-g-mPEG in DMSO-<i>d₆</i> at 25°C.

<p>¹H-NMR spectra of (a) poly(γ-GA-co-γ-DSGA) and (b) poly(γ-GA-co-γ-GAOSu)-g-mPEG in DMSO-<i>d₆</i> at 25°C.</p

Flow cytometric histogram profiles of HeLa cells incubated with free DOX, DOX-loaded vesicles and DOX-loaded GCPV.

<p>DOX fluorescence intensity of HeLa cells incubated with free DOX (red), DOX-loaded vesicles (green) and DOX-loaded GCPV (blue) at 37°C for 1 and 2 h, respectively. Untreated cells (black) were used as a control.</p