Novel linezolid resistance plasmids in Enterococcus from food animals in the USA.

Objectives: To sequence the genomes and determine the genetic mechanisms for linezolid resistance identified in three strains of Enterococcus isolated from cattle and swine caecal contents as part of the US National Antimicrobial Resistance Monitoring System (NARMS) surveillance programme. Methods: Broth microdilution was used for in vitro antimicrobial susceptibility testing to assess linezolid resistance. Resistance mechanisms and plasmid types were identified from data generated by WGS on Illumina® and PacBio® platforms. Conjugation experiments were performed to determine whether identified mechanisms were transmissible. Results: Linezolid resistance plasmids containing optrA were identified in two Enterococcus faecalis isolates and one Enterococcus faecium. The E. faecium isolate also carried the linezolid resistance gene cfr on the same plasmid as optrA. The linezolid resistance plasmids had various combinations of additional resistance genes conferring resistance to phenicols (fexA), aminoglycosides [spc and aph(3')-III] and macrolides [erm(A) and erm(B)]. One of the plasmids was confirmed to be transmissible by conjugation, resulting in linezolid resistance in the transconjugant. Conclusions: To the best of our knowledge, this is the first identification of linezolid resistance in the USA in bacteria isolated from food animals. The oxazolidinone class of antibiotics is not used in food animals in the USA, but the genes responsible for resistance were identified on plasmids with other resistance markers, indicating that there may be co-selection for these plasmids due to the use of different antimicrobials. The transmissibility of one of the plasmids demonstrated the potential for linezolid resistance to spread horizontally. Additional surveillance is necessary to determine whether similar plasmids are present in human strains of Enterococcus.

Establishing Genotypic Cutoff Values To Measure Antimicrobial Resistance in Salmonella.

Whole-genome sequencing (WGS) has transformed our understanding of antimicrobial resistance, helping us to better identify and track the genetic mechanisms underlying phenotypic resistance. Previous studies have demonstrated high correlations between phenotypic resistance and the presence of known resistance determinants. However, there has never been a large-scale assessment of how well resistance genotypes correspond to specific MICs. We performed antimicrobial susceptibility testing and WGS of 1,738 nontyphoidal Salmonella strains to correlate over 20,000 MICs with resistance determinants. Using these data, we established what we term genotypic cutoff values (GCVs) for 13 antimicrobials against Salmonella For the drugs we tested, we define a GCV as the highest MIC of isolates in a population devoid of known acquired resistance mechanisms. This definition of GCV is distinct from epidemiological cutoff values (ECVs or ECOFFs), which currently differentiate wild-type from non-wild-type strains based on MIC distributions alone without regard to genetic information. Due to the large number of isolates involved, we observed distinct MIC distributions for isolates with different resistance gene alleles, including for ciprofloxacin and tetracycline, suggesting the potential to predict MICs based on WGS data alone.

Whole-genome sequencing based characterization of antimicrobial resistance in Enterococcus.

Whole-genome sequencing (WGS) has transformed our understanding of antimicrobial resistance, yielding new insights into the genetics underlying resistance. To date, most studies using WGS to study antimicrobial resistance have focused on gram-negative bacteria in the family Enterobacteriaceae, such as Salmonella spp. and Escherichia coli, which have well-defined resistance mechanisms. In contrast, relatively few studies have been performed on gram-positive organisms. We sequenced 197 strains of Enterococcus from various animal and food sources, including 100 Enterococcus faecium and 97 E. faecalis. From analyzing acquired resistance genes and known resistance-associated mutations, we found that resistance genotypes correlated with resistance phenotypes in 96.5% of cases for the 11 drugs investigated. Some resistances, such as those to tigecycline and daptomycin, could not be investigated due to a lack of knowledge of mechanisms underlying these phenotypes. This study showed the utility of WGS for predicting antimicrobial resistance based on genotype alone.

The Role of Adverse Event Reporting in the FDA Response to a Multistate Outbreak of Liver Disease Associated with a Dietary Supplement.

OBJECTIVE: Liver disease is a potential complication from using dietary supplements. This study investigated an outbreak of non-viral liver disease associated with the use of OxyELITE Pro(TM), a dietary supplement used for weight loss and/or muscle building. METHODS: Illness details were ascertained from MedWatch reports submitted to the U.S. Food and Drug Administration (FDA) describing consumers who ingested OxyELITE Pro alone or in combination with other dietary supplements. FDA's Forensic Chemistry Center analyzed samples of OxyELITE Pro. RESULTS: From February 2012 to February 2014, FDA received 114 reports of adverse events of all kinds involving consumers who ingested OxyELITE Pro. The onset of illness for the first report was December 2010 and for the last report was January 2014. Thirty-three states, two foreign nations, and Puerto Rico submitted reports. Fifty-five of the reports (48%) described liver disease in the absence of viral infection, gallbladder disease, autoimmune disease, or other known causes of liver damage. A total of 33 (60%) of these patients were hospitalized, and three underwent liver transplantation. In early 2013, OxyELITE Pro products entered the market with a formulation distinct from products sold previously. The new formulation replaced 1,3-dimethylamylamine with aegeline. However, the manufacturer failed to submit to FDA a required "new dietary ingredient" notice for the use of aegeline in OxyELITE Pro products. Laboratory analysis identified no drugs, poisons, pharmaceuticals, toxic metals, usnic acid, N-Nitroso-fenfluramine, pyrrolizidine alkaloids, aristocholic acid, or phenethylamines in the products. CONCLUSIONS: Vigilant surveillance is required for adverse events linked to the use of dietary supplements.

Live cell analysis of G protein beta5 complex formation, function, and targeting.

The G protein beta(5) subunit differs from other beta subunits in having divergent sequence and subcellular localization patterns. Although beta(5)gamma(2) modulates effectors, beta(5) associates with R7 family regulators of G protein signaling (RGS) proteins when purified from tissues. To investigate beta(5) complex formation in vivo, we used multicolor bimolecular fluorescence complementation in human embryonic kidney 293 cells to compare the abilities of 7 gamma subunits and RGS7 to compete for interaction with beta(5). Among the gamma subunits, beta(5) interacted preferentially with gamma(2), followed by gamma(7), and efficacy of phospholipase C-beta2 activation correlated with amount of beta(5)gamma complex formation. beta(5) also slightly preferred gamma(2) over RGS7. In the presence of coexpressed R7 family binding protein (R7BP), beta(5) interacted similarly with gamma(2) and RGS7. Moreover, gamma(2) interacted preferentially with beta(1) rather than beta(5). These results suggest that multiple coexpressed proteins influence beta(5) complex formation. Fluorescent beta(5)gamma(2) labeled discrete intracellular structures including the endoplasmic reticulum and Golgi apparatus, whereas beta(5)RGS7 stained the cytoplasm diffusely. Coexpression of alpha(o) targeted both beta(5) complexes to the plasma membrane, and alpha(q) also targeted beta(5)gamma(2) to the plasma membrane. The constitutively activated alpha(o) mutant, alpha(o)R179C, produced greater targeting of beta(5)RGS7 and less of beta(5)gamma(2) than did alpha(o). These results suggest that alpha(o) may cycle between interactions with beta(5)gamma(2) or other betagamma complexes when inactive, and beta(5)RGS7 when active. Moreover, the ability of beta(5)gamma(2) to be targeted to the plasma membrane by alpha subunits suggests that functional beta(5)gamma(2) complexes can form in intact cells and mediate signaling by G protein-coupled receptors.

Analysis of G protein betagamma dimer formation in live cells using multicolor bimolecular fluorescence complementation demonstrates preferences of beta1 for particular gamma subunits.

The specificity of G protein betagamma signaling demonstrated by in vivo knockouts is greater than expected based on in vitro assays of betagamma function. In this study, we investigated the basis for this discrepancy by comparing the abilities of seven beta1gamma complexes containing gamma1, gamma2, gamma5, gamma7, gamma10, gamma11, or gamma12 to interact with alphas and of these gamma subunits to compete for interaction with beta1 in live human embryonic kidney (HEK) 293 cells. betagamma complexes were imaged using bimolecular fluorescence complementation, in which fluorescence is produced by two nonfluorescent fragments (N and C) of cyan fluorescent protein (CFP) or yellow fluorescent protein (YFP) when brought together by proteins fused to each fragment. Plasma membrane targeting of alphas-CFP varied inversely with its expression level, and the abilities of YFP-N-beta1YFP-C-gamma complexes to increase this targeting varied by 2-fold or less. However, there were larger differences in the abilities of the CFP-N-gamma subunits to compete for association with CFP-C-beta1. When the intensities of coexpressed CFP-C-beta1CFP-N-gamma (cyan) and CFP-C-beta1YFP-N-gamma2 (yellow) complexes were compared under conditions in which CFP-C-beta1 was limiting, the CFP-N-gamma subunits exhibited a 4.5-fold range in their abilities to compete with YFP-N-gamma2 for association with CFP-C-beta1. CFP-N-gamma12 and CFP-N-gamma1 were the strongest and weakest competitors, respectively. Taken together with previous demonstrations of a role for betagamma in the specificity of receptor signaling, these results suggest that differences in the association preferences of coexpressed beta and gamma subunits for each other can determine which complexes predominate and participate in signaling pathways in intact cells.

Live cell imaging of Gs and the beta2-adrenergic receptor demonstrates that both alphas and beta1gamma7 internalize upon stimulation and exhibit similar trafficking patterns that differ from that of the beta2-adrenergic receptor.

To visualize and investigate the regulation of the localization patterns of Gs and an associated receptor during cell signaling, we produced functional fluorescent fusion proteins and imaged them in HEK-293 cells. alphas-CFP, with cyan fluorescent protein (CFP) inserted into an internal loop of alphas, localized to the plasma membrane and exhibited similar receptor-mediated activity to that of alphas. Functional fluorescent beta1gamma7 dimers were produced by fusing an amino-terminal yellow fluorescent protein (YFP) fragment to beta1 (YFP-N-beta1) and a carboxyl-terminal YFP fragment to gamma7 (YFP-C-gamma7). When expressed together, YFP-N-beta1 and YFP-C-gamma7 produced fluorescent signals in the plasma membrane that were not seen when the subunits were expressed separately. Isoproterenol stimulation of cells co-expressing alphas-CFP, YFP-N-beta1/YFP-C-gamma7, and the beta2-adrenergic receptor (beta2AR) resulted in internalization of both fluorescent signals from the plasma membrane. Initially, alphas-CFP and YFP-N-beta1/YFP-C-gamma7 stained the cytoplasm diffusely, and subsequently they co-localized on vesicles that exhibited minimal overlap with beta2AR-labeled vesicles. Moreover, internalization of beta2AR-GFP, but not alphas-CFP or YFP-N-beta1/YFP-C-gamma7, was inhibited by a fluorescent dominant negative dynamin 1 mutant, Dyn1(K44A)-mRFP, indicating that the Gs subunits and beta2AR utilize different internalization mechanisms. Subsequent trafficking of the Gs subunits and beta2AR also differed in that vesicles labeled with the Gs subunits exhibited less overlap with RhoB-labeled endosomes and greater overlap with Rab11-labeled endosomes. Because Rab11 regulates traffic through recycling endosomes, co-localization of alphas and beta1gamma7 on these endosomes may indicate a means of recycling specific alphasbetagamma combinations to the plasma membrane.

Visualization of G protein betagamma dimers using bimolecular fluorescence complementation demonstrates roles for both beta and gamma in subcellular targeting.

To investigate the role of subcellular localization in regulating the specificity of G protein betagamma signaling, we have applied the strategy of bimolecular fluorescence complementation (BiFC) to visualize betagamma dimers in vivo. We fused an amino-terminal yellow fluorescent protein fragment to beta and a carboxyl-terminal yellow fluorescent protein fragment to gamma. When expressed together, these two proteins produced a fluorescent signal in human embryonic kidney 293 cells that was not obtained with either subunit alone. Fluorescence was dependent on betagamma assembly in that it was not obtained using beta2 and gamma1, which do not form a functional dimer. In addition to assembly, BiFC betagamma complexes were functional as demonstrated by more specific plasma membrane labeling than was obtained with individually tagged fluorescent beta and gamma subunits and by their abilities to potentiate activation of adenylyl cyclase by alpha(s) in COS-7 cells. To investigate isoform-dependent targeting specificity, the localization patterns of dimers formed by pair-wise combinations of three different beta subunits with three different gamma subunits were compared. BiFC betagamma complexes containing either beta1 or beta2 localized to the plasma membrane, whereas those containing beta5 accumulated in the cytosol or on intracellular membranes. These results indicate that the beta subunit can direct trafficking of the gamma subunit. Taken together with previous observations, these results show that the G protein alpha, beta, and gamma subunits all play roles in targeting each other. This method of specifically visualizing betagamma dimers will have many applications in sorting out roles for particular betagamma complexes in a wide variety of cell types.