Diversity and antimicrobial resistance of Enterococcus from the Upper Oconee Watershed, Georgia.

AIM: It is well-known that enterococci are abundant in the environment; however, the role of surface water as a reservoir of antimicrobial-resistant enterococci remains largely undefined. In this study, surface water samples were collected over a 2-year period from the Upper Oconee watershed, Athens, GA to examine enterococci and their antimicrobial resistance. METHODS AND RESULTS: Approximately 97% (445/458) of the samples were positive for enterococci and a total of 637 enterococci were isolated. The predominant species were Enterococcus casseliflavus (33·6%) followed by Enterococcus faecalis (26·5%) and Enterococcus hirae (13·2%). Regardless of species, the highest levels of resistance were to lincomycin (88·5%) and tetracycline (13%); isolates also exhibited resistance to newer antimicrobials, daptomycin (8·9%) and tigecycline (6·4%). Multidrug resistance (resistance ≥3 antimicrobial classes) was observed to as many as five classes of antimicrobials. Resistant enterococci appeared to be randomly dispersed over the seasons rather than clustered by species or antimicrobial resistance. CONCLUSIONS: This study demonstrated that surface waters contain a large population of diverse species of antimicrobial-resistant enterococci, including resistance to new antimicrobials. SIGNIFICANCE AND IMPACT OF THE STUDY: These results may indicate the potential of human intestinal illness and/or colonization of the human gut with resistant enterococci as enterococci correlate with increased disease risk to humans during recreational exposure to water.

Welfare of anaesthesia trainees survey.

This study was designed to investigate levels of stress, anxiety or depression and to identify factors compounding or relieving stress in anaesthesia trainees within the Australian and New Zealand College of Anaesthetists (ANZCA) training scheme. An electronic survey was sent to 999 randomly selected trainees and 428 responses were received. In addition to demographics, psychological wellbeing was assessed using the Kessler Psychological Distress Scale (K10) and questions were asked about depression and anxiety, exacerbating factors, personal healthcare and strategies used to manage stress. The majority of respondents (73%) reported being satisfied or very satisfied with their job and training. However, 28% of respondents had K10 scores indicating high or very high distress levels. Eleven percent reported being currently on treatment for anxiety and/or depression. Twenty-two percent reported having experienced bullying and 14% sexism. Twenty-eight percent reported that they did not have a regular general practitioner (GP), and 41% reported having not visited their GP in the previous year. Forty-seven percent of trainees reported that they self-prescribe medications. Major stressors reported were exams, critical clinical events and fear of making errors. Two stressors previously not identified in similar studies were concern about job prospects in 71% of respondents and workplace-based assessments in 51%. This survey demonstrates significant psychological impairment and poor personal healthcare amongst many trainees. Education, careful continuing assessment of trainees' welfare and a review of current support and remedial measures may be required.

Anaesthesia on the Western Front-perspectives a century later.

This year, 2016, marks the centenary of some of the worst battles of World War I. It is timely to reflect on the experiences of those who lived through those events, and the significant effects on the development of anaesthesia that occurred as a result. War has been a catalyst that has accelerated the development of medicine and surgery throughout history. The challenges of the Western Front, with its massive numbers of injured, saw progress in the understanding and management of trauma and shock, and significant improvement in the provision of anaesthesia and the training of anaesthesia providers. The result was increased anaesthetic safety for the civilian population after the war and further development of anaesthesia and resuscitation as a specialised area of medicine.

The medical officer's journal: HMAS Sydney 1913-1922. An Australian Naval record of surgery and anaesthesia at the time of the outbreak of World War I.

The Medical Officer's Journal of HMAS Sydney was a record kept by the ship's Surgeon, Leonard Darby, of the conditions on the ship and the health, diseases, injuries and treatment of the sailor's under his care. Records of anaesthesia and surgery indicate chloroform was mostly administered as was ether occasionally. There was some use of intravenous and subcutaneous fluids for resuscitation. The journal also provides an eyewitness account of the Sydney-Emden battle on 9 November 1914 which occurred off the Cocos Keeling Islands and was a famous first victory for the young Australian Navy, making headlines around the world. The treatment of the many injured, mostly Germans, is described; with the two Australian surgeons and the surviving German surgeon working together.

Hepatic mitochondrial efficiency in lines of mice differing in feed intake.

Selection was practiced for high (MH) and low (ML) heat loss using direct calorimetry to create lines of mice differing in feed intake per unit average BW (FI/BW). A total of 25 generations of selection was practiced during maintenance of an unselected control (MC). As a percentage of MC, mice of MH and ML lines differed in heat loss and FI/BW by 56 and 34%, respectively, when selection ceased. The purpose of this study was to determine hepatic mitochondrial efficiency in mice at maintenance across the genetic lines. After measurement of feed intakeand BW over a 2-wk period, mice were euthanized, and their livers were extracted. Livers were homogenized and mitochondria were isolated in buffer for measurement of oxygen consumption and, hence, mitochondrial activity using a Clark-type oxygen electrode. Mitochondria of ML mice expressed greater (P < 0.03) respiratory control ratio by 22% than that for MH mice, revealing greater degree of uncoupling during electron transport in MH mice. Difference in FI/BW (P < 0.001) was large with MH mice consuming 34% more than ML mice. The respiratory control ratio differed between mice of MH and ML lines as expected; however, regression analysis revealed that it explained essentially no variation in feed intake per BW.

Differences in mitochondrial efficiency between lines of mice divergently selected for heat loss.

Divergent selection for heat loss was applied to lines of mice for 15 generations (G) in 3 replicates. Selection resumed at G42 and continued through G51 across all replicates. At the end of G51, differences in heat loss and feed intake per unit of BW were approximately 56 and 34%, respectively, between high heat loss (MH) and low heat loss (ML) lines, as a percentage of the control line (MC) mean. Rates of liver mitochondrial respiration states, degree of coupling, and mitochondrial efficiency were measured in G58 using a Clark-type oxygen electrode to investigate possible causes of underlying variation in maintenance requirements. Body composition, BW, liver weight, feed intake, and residual feed intake (RFI) were also measured or calculated. Results reported here represent data from 197 mature male mice from all replicates. There were no differences in BW (P = 0.91) between the selection lines. Selection had an effect on lean percentage (P = 0.02), with MH mice being leaner. Fat percentage differences between the selection lines tended toward significance (P = 0.13). Livers of MH mice were approximately 13% larger than livers of ML mice (P = 0.01). An effect of selection was observed (P < 0.01) in feed intake per unit BW, with MH mice consuming 29% more feed than ML mice in G58. Differences in state 2 and state 4 respiration rates were significant (P = 0.01), whereas state 3 rates approached significance (P = 0.06). Mitochondria of MH mice respired at a greater rate than mitochondria of ML mice in all states of respiration; ML mice had respiratory control ratios that were, on average, 8% greater than MH mice (P = 0.14). Although this difference only tended toward significance, we suspect a greater degree of coupling of mitochondrial processes exists in ML animals. Mice selected for reduced heat loss had ADP:oxygen ratios that were approximately 20% greater than MH mice (P = 0.03). Therefore, greater mitochondrial efficiency was expressed in the ML animals. Within a line-replicate, there was no correlation between ADP:O and feed intake per unit BW (P = 0.71). In addition, no correlation of ADP:O and RFI existed (P = 0.92). Although the selection lines differed in mitochondrial traits, including overall mitochondrial efficiency (ADP:oxygen), these differences were not a significant underlying cause of variation in feed intake per unit BW or in RFI estimates.

Renewed selection for heat loss in mice: direct responses and correlated responses in feed intake, body weight, litter size, and conception rate.

Divergent selection in mice was renewed in 3 independent replicates for high (MH) and low (ML) heat loss. An unselected control (MC) was maintained in all replicates. Heat loss was measured for individual male mice for 15 h, overnight in direct calorimeters. After 16 initial generations of selection followed by 26 generations of relaxed selection, divergent selection resumed for 9 generations. The realized selection applied was very close to the maximum possible selection according to the criteria and protocol. Selection differentials were greater for high than for low selection due to greater variation in the MH line. When corrected for SD, standardized selection differentials were similar for MH and ML selection. Unintended selection in MC was negligible. Realized heritability for divergence was 0.14 +/- 0.01, which was considerably less than that realized during the initial generations of selection (0.28 +/- 0.03). Realized heritabilities for MH selection (0.16 +/- 0.05) and for ML selection (0.07 +/- 0.06) were less, especially for ML selection, than were observed in the earlier generations. The difference in heat loss between MH and ML males was 55.7% of the MC mean at generation 51, compared with a difference of 53.6% in generation 15; this difference had decreased to 34.4% at the end of the relaxed selection (generation 42). For feed intake between 8 and 11 wk, MH and ML males differed by 34.0% of the MC mean by the end of the selection process. Body weight at 12 wk for MH and ML males was less than for MC males. Litter size response was positively related to the heat loss response. Conception rate was poorer in MH matings than in MC and ML matings.

Correlated responses in maternal performance following divergent selection for heat loss in mice.

Divergent selection in mice was applied in 3 independent replicates for high (maintenance high; MH) and low (maintenance low; ML) heat loss for 16 generations. An unselected control (maintenance control; MC) was also maintained in all replicates. Selection ceased for 26 generations; heat-loss measurement and selection resumed at generation 42. Lactation performance, dam weight, dam feed intake, and efficiency of production of pup weight were recorded or calculated for MH and ML dams in all 3 replicates at generation 46 or 47 with the objective of determining whether selection for heat loss has created correlated responses in maternal performance. One-half of the dams reared their own litters, and one-half reared cross-fostered (across lines) litters. Between 10 and 12 litters were used from each replicate-line-rearing class. Litter size was recorded, and litters were standardized to 8 pups within 24 h of birth. For cross fostering, MH litters were matched to ML litters born within 24 h of each other, and MH-ML litter pairs were cross-fostered at 3 d of age. A weigh-suckle-weigh protocol was used to obtain milk production estimates over a 2-h suckling period at 6, 9, 12, and 15 d. Dam (plus litter) feed intake was also recorded at these times and was calculated as the disappearance of feed over 3-d intervals. Dams of the MH selection tended (P < 0.11) to have greater litter size than those of the ML selection; litter size of MC dams was intermediate. Line of dam affected milk production (P = 0.04) and dam feed intake (P < 0.03) as MH dams produced more milk and consumed more feed than ML dams. Average milk production for the 2-h measurement period was 1.70 +/- 0.07 and 1.41 +/- 0.07 g, and average 3-d feed consumption was 50.8 +/- 1.2 and 45.2 +/- 1.2 g for MH and ML dams, respectively. Cross-fostering had no effect (P > 0.86) on milk production. Line of dam tended to affect 21-d litter weight (P = 0.15) with litters reared by MH dams weighing more than those reared by ML dams, but there was no difference (P > 0.86) in 21-d dam weights. Efficiency of producing litter weight (litter 15-d weight: dam plus litter feed intake from d 6 to 15) was greater (0.49 vs. 0.46, SE = 0.009; P = 0.03) for ML than for MH dams. Selection for reduced heat loss (lower maintenance feed intake in the ML line) resulted in reduced milk production and feed intake in dams and greater efficiency of litter weight production.

Differential effects of calmodulin and protein kinase C antagonists on bone resorption and acid transport activity.

Tamoxifen inhibits bone resorption by disrupting calmodulin-dependent processes. Since tamoxifen inhibits protein kinase C in other cells, we compared the effects of tamoxifen and the PKC inhibitor, bis indolylmaleimide II (bIM), on bone resorption and acid transport activity in isolated membrane vesicles. Bis indolylmaleimide inhibited bone resorption 50% with an IC50 approximately 3 microM, as well as acid transport activity in a concentration -dependent manner with an IC50 of approximately 0.4 IM. The IC50 of bIM for inhibiting acid transport activity was similar to that of calmodulin antagonists. The potassium ionophore, valinomycin, failed to restore bIM or tamoxifen-dependent inhibition of acid transport, suggesting that bIM and tamoxifen both inhibit H(+)-ATPase activity. Half maximal inhibitory concentrations of tamoxifen and bIM were not additive in acid transport assays, suggesting different sites of action. Furthermore, exogenous calmodulin blocked tamoxifen, but not bIM, -dependent inhibition of acid transport. We also compared the effects of tamoxifen and bIM on phosphorylation of proteins in isolated membrane fractions as determined by 32P incorporation and autoradiography. Tamoxifen had no effect on protein phosphorylation in contrast to bIM, which inhibited phosphorylation of eight proteins with different apparent kinetics. The data suggest that, while tamoxifen and bIM both affect H(+)-ATPase activity, the mechanisms of action are different.

Regulation of gonadotropin releasing hormone release by neuropeptide Y at the median eminence during the preovulatory period in ewes.

The median eminence (ME) of the hypothalamus is known to be an important brain site where hypophysiotropic release might be regulated by excitatory and inhibitory signals impinging on their neuronal terminals. Since a role for neuropeptide Y (NPY) on preovulatory luteinizing hormone (LH) release has been suggested, we hypothesized that NPY might act at the ME to control preovulatory gonadotropin-releasing hormone (GnRH) release and thus the onset of the preovulatory surge of LH. To examine this possibility, we used the ewe as an animal model to determine: (a) immunocytochemical distribution of GnRH and NPY in the ewe ME; (b) changes in in vivo release of NPY and GnRH using ME push-pull cannula (PPC) perfusate samples, as well as in plasma LH, during the luteal, follicular and preovulatory phases of a synchronized estrous cycle, and (c) effects of ME perfusion of NPY or a Y1-NPY antagonist, or an NPY antiserum on in vivo release of ME-GnRH and plasma LH during a synchronized follicular phase. Immunolocalization reveals a dense plexus of beaded GnRH-containing neurites in the arcuate nucleus and in its vicinity, the pituitary stalk and the palisade. In contrast, a dense plexus of NPY-containing neurites occurs in the internal layer, with occasional fibers found in the intermediate and lateral external zone of the ME. In the area between the lateral internal and lateral external layers, both NPY and GnRH-containing processes were found, thus providing opportunities for synaptic and/or paracrine interactions between NPY- and GnRH-containing neurons. Hormonal analysis indicated that a synchronized preovulatory surge of LH is elicited within a 2-hour window by the sequential implantation and removal of silastic-encased estradiol (E2) or progesterone (P4) implants. In this paradigm, there was a parallel increase in ME release of both NPY and GnRH preceding the synchronized LH surge. The onset of this synchronized LH surge was advanced by ME perfusion of exogenous NPY and was both delayed and blunted by ME perfusion with the NPY antagonist (both were perfused through the PPC probe for 2 h, starting 2-3 h before the expected onset of the LH surge). In addition, NPY perfusion in the ME increases, while perfusion of the Y1-NPY antagonist or of the NPY antiserum decreases ME-PPC GnRH content and plasma levels of LH in early follicular ewes. Finally, perfusion of NPY antiserum during an ongoing LH surge disrupted LH release. These results suggest that interactions between NPY and GnRH neurons are important in controlling the timing, magnitude and maintenance of the preovulatory LH surge.

Osteoclasts secrete the chemotactic cytokine mim-1.

Osteoclasts are terminally differentiated, multinucleated cells of monocytic origin. In this study, we report that osteoclasts secrete a 35 kD protein and that phorbol myristate acetate treatment stimulates secretion dramatically. Peptide digests of the protein were analyzed by mass spectroscopy. The protein was identified as myb induced myeloid protein-1 precursor (mim-1 protein). Mim-1 is expressed specifically by hematopoietic cells and has no known function. It is homologous with the neutrophil chemokine, chondromodulin II, which stimulates proliferation of osteoblasts and chondrocytes. Western analysis showed that osteoclasts secrete mim-1 into culture media. Immunofluorescence studies demonstrated a cytoplasmic and perinuclear distribution of mim-1 in both avian osteoclasts and human osteoclast-like cells. Expression and secretion of a chemokine-like protein by osteoclasts suggests a novel signaling pathway in the bone microenvironment that may be involved in coordinating bone remodeling.

Cellular uptake and in situ binding of a peptide agonist for calmodulin.

We have used the method of inverted hydropathy to develop peptides that interact with EF hands of calmodulin (CaM). Previously we have shown these peptides specifically interact with their desired target in a productive manner, in that they activated CaM in the absence of Ca(2+). Therefore, we sought to determine whether these peptides would enter cells, remain intact, and interact with CaM in the interior of the cell. Using several techniques we have demonstrated cellular uptake, stability, and an intracellular interaction with CaM with fluorescein-labeled and radiolabeled peptides in Jurkat T cells. The results suggest that these peptides may be useful in the study and the manipulation of Ca(2+)-mediated pathways in cells.

Calmodulin and HIV type 1: interactions with Gag and Gag products.

The level of calmodulin increases in cells expressing HIV-1 envelope glycoprotein. Although a calmodulin increase is bound to alter many cellular metabolic and signaling pathways, the benefits to the virus of these alterations must be indirect. However, the possibility exists that increased cellular calmodulin benefits the virus by directly associating with nonenvelope viral proteins. We have, therefore, investigated whether calmodulin can interact with HIV structural proteins Gag, p17, and p24. Calmodulin binds Gag and p17 but not p24 in (125)I-labeled calmodulin overlays of SDS-polyacrylamide gels. Removal of calcium by addition of EGTA eliminates this binding. A computer algorithm for predicting helical regions that should bind calmodulin predicts that there are two calmodulin-binding regions near the N terminus of p17. Intrinsic tryptophan fluorimetry shows that two peptides, each of which includes one of the predicted regions, bind calmodulin: p17(11-25) binds calmodulin with a 2-to-1 stoichiometry and dissociation constant of approximately 10(-9) M(2), and p17(31-46) also binds calmodulin with a dissociation constant of about 10(-9) M. These binding sites are nearly contiguous, forming an extended calmodulin-binding domain p17(11-46). In H-9 cells, Gag and calmodulin colocalize within the resolution of confocal light microscopy.

Familial Wilms' tumor with neural elements: characterization by histology, immunohistochemistry, and genetic analysis.

Wilms' tumor (WT) is the most common renal malignancy of children. While most occur sporadically, a small percentage are familial or occur as part of a developmental syndrome. Classic WTs exhibit a triphasic histologic pattern composed of blastema, epithelium, and stroma. Occasionally, heterologous elements may also be observed. In this study we investigated a series of four WTs that occurred within a single familial aggregate and contained focal areas of neural differentiation. The tumors were evaluated histologically for the presence of neural elements and immunohistochemically for expression of neural-related markers. Genetic linkage analysis was performed on 3 of the 4 WTs. In addition to the classic triphasic histology, the WTs contained tumor rosettes (4/4), ganglion cells (2/4), foci of ganglioneuromatous differentiation (2/4), and anaplasia (1/4). Staining for chromogranin, S-100, synaptophysin, vimentin, and neuron-specific enolase was positive in all 4 tumors within the areas of neural differentiation whereas staining for CD99 (013) and glial fibrillary acidic protein was negative. Linkage analysis studies suggest that the familial predisposition gene segregating in this family is at 19q13.4. To our knowledge, this is the first reported series of WTs with neural differentiation that occurred within a single family aggregate. Genetic linkage analysis of this family is consistent with linkage to the FWT2 WT predisposition gene at 19q13.4. We propose that these tumors may represent a unique manifestation of tumor susceptibility in this family.

Thoracoscopic localization of nonpalpable rib tumors for excisional biopsy.

Nonpalpable tumors of the rib can be difficult to localize accurately at the time of excisional biopsy. Furthermore, the ability of current imaging techniques to assess pleural or pulmonary involvement is not reliable. An intraoperative localization method using single port thoracoscopy is discussed which allows optimal placement of a biopsy incision and provides an accurate assessment of any tumor invasion into the adjacent pleura or lung.

A prospective trial of telepathology for intraoperative consultation (frozen sections).

Telepathology is a maturing technology that, for a variety of reasons, has not been widely deployed. In addition, clinical validation is relatively modest compared with accepted telemedicine applications such as teleradiology. A prototype telepathology system (Tele-Path(sm)) featuring high-resolution images selected from a remote microscope site has been developed at the University of Alabama at Birmingham (UAB). To validate the diagnostic efficacy of the system, a prospective study was undertaken of parallel diagnoses by conventional microscopy and telepathology with a remotely operated microscope. Slides from 99 intraoperative consultations from 29 tissue/ organ sites in the University of Alabama Hospitals by 9 academic pathologists were used in the study. Each microscopic and telepathology diagnosis was compared with the final diagnosis rendered by a referee pathologist. Diagnoses were classified as correct, false positive, or false negative or classification error. Of the 99 frozen sections evaluated, 3 cases were deferred. Of the remaining 96 cases, 2 received incorrect diagnoses in both the microscopic and telepathology arms of the study. Three errors occurred only in the telepathology arm. There was 1 false-positive diagnosis, 1 false-negative diagnosis, and 1 classification error. Statistical analysis indicated no significant difference between telepathology and conventional microscopy. Qualitative data indicated that the pathologists were generally satisfied with the performance of the system. Telepathology using this system paradigm is sufficiently accurate for real time utilization in a complex surgical environment. Telepathology therefore may be an effective model to support the surgical services of hospitals lacking full-time pathology coverage, resulting in full-time access to anatomic pathology services.

Functional differences of the PDS gene product are associated with phenotypic variation in patients with Pendred syndrome and non-syndromic hearing loss (DFNB4).

The PDS gene encodes a transmembrane protein, known as pendrin, which functions as a transporter of iodide and chloride. Mutations in this gene are responsible for Pendred syndrome and autosomal recessive non-syndromic hearing loss at the DFNB4 locus on chromosome 7q31. A screen of 20 individuals from the midwestern USA with non-syndromic hearing loss and dilated vestibular aqueducts identified three people (15%) with PDS mutations. To determine whether PDS mutations in individuals with Pendred syndrome differ functionally from PDS mutations in individuals with non-syndromic hearing loss, we compared three common Pendred syndrome allele variants (L236P, T416P and E384G), with three PDS mutations reported only in individuals with non-syndromic hearing loss (V480D, V653A and I490L/G497S). The mutations associated with Pendred syndrome have complete loss of pendrin-induced chloride and iodide transport, while alleles unique to people with DFNB4 are able to transport both iodide and chloride, albeit at a much lower level than wild-type pendrin. We hypothesize that this residual level of anion transport is sufficient to eliminate or postpone the onset of goiter in individuals with DFNB4. We propose a model for pendrin function in the thyroid in which pendrin transports iodide across the apical membrane of the thyrocyte into the colloid space.