Multi-site Analysis Reveals Widespread Antibiotic Resistance in the Marine Pathogen <Emphasis Type="Italic">Vibrio vulnificus</Emphasis>

Vibrio vulnificus is a serious opportunistic human pathogen commonly found in subtropical coastal waters, and is the leading cause of seafood-borne mortality in the USA. This taxon does not sustain prolonged presence in clinical or agricultural settings, where it would undergo human-induced selection for antibiotic resistance. Therefore, few studies have verified the effectiveness of commonly prescribed antibiotics in V. vulnificus treatment. Here we screened 151 coastal isolates and 10 primary septicaemia isolates against 26 antimicrobial agents representing diverse modes of action. The frequency of multiple resistances to antibiotics from all sources was unexpectedly high, particularly during summer months, and a substantial proportion of isolates (17.3%) were resistant to eight or more antimicrobial agents. Numerous isolates demonstrated resistance to antibiotics routinely prescribed for V. vulnificus infections, such as doxycycline, tetracycline, aminoglycosides and cephalosporins. These resistances were detected at similar frequencies in virulent and non-virulent strains (PCR-based virulence typing) and were present in septicaemia isolates, underlying the public health implications of our findings. Among environmental isolates, there were no consistent differences in the frequency of resistance between pristine and anthropogenically impacted estuaries, suggesting natural rather than human-derived sources of resistance traits. This report is the first to demonstrate prevalent antibiotic resistance in a human pathogen with no clinical reservoirs, implying the importance of environmental studies in understanding the spread, evolution and public health relevance of antibiotic resistance factors. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Noninvasive determination of perfused artery dimensions ex vivo using a pressure-diameter relationship

Based on the decisive effects of the hemodynamic and mechanical environments on the development and remodeling of arteries in vivo, several groups have cultured tissue-engineered vessels and excised vessels in various mechanically active perfusion systems. To facilitate the interpretation and design of such studies, accurate estimates of the applied forces and resulting stresses are required, which in turn require an accurate estimate of vessel dimensions. The measured pressure drop along the length of the vessel could be used to calculate the average inner diameter, but practical considerations, including the modest accuracy of many pressure transducers, limit this approach. Using nine porcine arteries harvested from pigs weighing between 25 and 100 kg, we show that when real-time measurements of the pressure drop and the outer diameter during a vasoactive event are fit to a theoretical model, offset errors in the pressure measurement can be compensated for and estimates of vessel wall transverse area with an average error of 4.1% (not exceeding 8.3%) are achieved.     

Mechanical properties of native and ex vivo remodeled porcine saphenous veins

When grafted into an arterial environment in vivo, veins remodel in response to the new mechanical environment, thereby changing their mechanical properties and potentially impacting their patency as bypass grafts. Porcine saphenous veins were subjected for one week to four different ex vivo hemodynamic environments in which pressure and shear stress were varied independently, as well as an environment that mimicked that of an arterial bypass graft. After one week of ex vivo culture, the mechanical properties of intact saphenous veins were evaluated to relate specific aspects of the mechanical environment to vein remodeling and corresponding changes in mechanics. The compliance of all cultured veins tended to be less than that of fresh veins; however, this trend was more due to changes in medial and luminal areas than changes in the intrinsic properties of the vein wall. A combination of medial hypertrophy and eutrophic remodeling leads to significantly smaller (p<0.05) wall stresses measured in all cultured veins except those subjected to bypass graft conditions relative to stresses measured in fresh veins at corresponding pressures. Our results suggest that the mechanical environment effects changes in vessel size, as well as the nature of the remodeling, which contribute to altering vein mechanical properties.     

Nutritional Intervention Restores Muscle but Not Kidney Phenotypes in Adult Calcineurin Aα Null Mice

Mice lacking the α isoform of the catalytic subunit of calcineurin (CnAα) were first reported in 1996 and have been an important model to understand the role of calcineurin in the brain, immune system, bones, muscle, and kidney. Research using the mice has been limited, however, by failure to thrive and early lethality of most null pups. Work in our laboratory led to the rescue of CnAα−/− mice by supplemental feeding to compensate for a defect in salivary enzyme secretion. The data revealed that, without intervention, knockout mice suffer from severe caloric restriction. Since nutritional deprivation is known to significantly alter development, it is imperative that previous conclusions based on CnAα−/− mice are revisited to determine which aspects of the phenotype were attributable to caloric restriction versus a direct role for CnAα. In this study, we find that defects in renal development and function persist in adult CnAα−/− mice including a significant decrease in glomerular filtration rate and an increase in blood urea nitrogen levels. These data indicate that impaired renal development we previously reported was not due to caloric restriction but rather a specific role for CnAα in renal development and function. In contrast, we find that rather than being hypoglycemic, rescued mice are mildly hyperglycemic and insulin resistant. Examination of muscle fiber types shows that previously reported reductions in type I muscle fibers are no longer evident in rescued null mice. Rather, loss of CnAα likely alters insulin response due to a reduction in insulin receptor substrate-2 (IRS2) expression and signaling in muscle. This study illustrates the importance of re-examining the phenotypes of CnAα−/− mice and the advances that are now possible with the use of adult, rescued knockout animals.     

Amino acids stimulate cholecystokinin release through the Ca2+-sensing receptor

Cholecystokinin (CCK) is produced by discrete endocrine cells in the proximal small intestine and is released following the ingestion of food. CCK is the primary hormone responsible for gallbladder contraction and has potent effects on pancreatic secretion, gastric emptying, and satiety. In addition to fats, digested proteins and aromatic amino acids are major stimulants of CCK release. However, the cellular mechanism by which amino acids affect CCK secretion is unknown. The Ca(2+)-sensing receptor (CaSR) that was originally identified on parathyroid cells is not only sensitive to extracellular Ca(2+) but is activated by extracellular aromatic amino acids. It has been postulated that this receptor may be involved in gastrointestinal hormone secretion. Using transgenic mice expressing a CCK promoter driven/enhanced green fluorescent protein (GFP) transgene, we have been able to identify and purify viable intestinal CCK cells. Intestinal mucosal CCK cells were enriched >200-fold by fluorescence-activated cell sorting. These cells were then used for real-time PCR identification of CaSR. Immunohistochemical staining with an antibody specific for CaSR confirmed colocalization of CaSR to CCK cells. In isolated CCK cells loaded with a Ca(2+)-sensitive dye, the amino acids phenylalanine and tryptophan, but not nonaromatic amino acids, caused an increase in intracellular Ca(2+) ([Ca(2+)](i)). The increase in [Ca(2+)](i) was blocked by the CaSR inhibitor Calhex 231. Phenylalanine and tryptophan stimulated CCK release from intestinal CCK cells, and this stimulation was also blocked by CaSR inhibition. Electrophysiological recordings from isolated CCK-GFP cells revealed these cells to possess a predominant outwardly rectifying potassium current. Administration of phenylalanine inhibited basal K(+) channel activity and caused CCK cell depolarization, consistent with changes necessary for hormone secretion. These findings indicate that amino acids have a direct effect on CCK cells to stimulate CCK release by activating CaSR and suggest that CaSR is the physiological mechanism through which amino acids regulate CCK secretion.