Critically important antimicrobial resistant Enterobacteriaceae in Irish farm effluent and their removal in integrated constructed wetlands.

This study investigated the ability of Integrated Constructed Wetlands (ICWs) to remove critically important antimicrobial resistant organisms (AROs) from farm wastewater. Influent samples from the untreated farm waste and effluent samples taken at the end of the ICW system were collected monthly from four ICWs, serving four different farm types (suckler, dairy, dairy & poultry and pig). Using selective media to screen for the presence of carbapenemase resistant organisms, plasmid mediated and AmpC ß-Lactamase producing organisms (ESBL/pAmpC) and fluoroquinolone resistant organisms, a total of 82 AROs were obtained with the majority being E. coli (n = 79). Statistically significant were the differences on the number of AROs isolated from influent (higher) compared to effluent, as well as a seasonal effect, with less AROs recovered during winter in comparison to other seasons (P < 0.05). On the other hand, there was no significant differences in the recovery of AROs on different farms. The majority of isolates from each of the farms (99%) were multi drug resistant, with 65% resistant to seven or more antimicrobials. A high incidence of tetracycline, trimethoprim/sulfamethoxazole, and ampicillin resistance was common to the isolates from all four farms but there were differences in ESBL levels with 63% of the isolates recovered from Farm 4 (piggery) being ESBLs compared to 18%, 36% and 4.5% recovered from Farms 1 (suckler), 2 (dairy) and 3 (dairy & poultry), respectively. No carbapenemase producing organisms were isolated. Our results showed that ICWs are effective in removing critically important AROs from farm wastewater on all four farm types.

Dining On Call: Outcomes of a hospital patient dining model.

Dining On Call (DOC) is a hospital foodservice model allowing patients to order meals any time throughout the day and is delivered within 45 minutes of the order. It is positively correlated with patient satisfaction, improvements in malnutrition, and reducing costs. Pre- and post-DOC data were collected from BC Children's Hospital, BC Women's Hospital, and North York General Hospital (NYGH) using patient satisfaction surveys and tray waste audits to measure outcomes. Patient satisfaction scores increased at all hospitals. BC Children's and Women's hospitals demonstrated reductions in tray waste, food cost/meal/day, and labour cost/meal/day post-DOC. North York General Hospital observed decreases in tray waste; however, food cost/meal/day and labour cost/meal/day increased post-DOC. This research provides convincing evidence into the achievable benefits associated with DOC on mother and paediatric units in hospital settings. DOC may prove to be an effective dining model for hospitals seeking to improve patient outcomes and reduce overall costs.

The rule of 10s versus the rule of 2s: High complication rates after conventional excision with postoperative margin assessment of specialty site versus trunk and proximal extremity melanomas.

Specialty site melanomas on the head and neck, hands and feet, genitalia, and pretibial leg have higher rates of surgical complications after conventional excision with postoperative margin assessment (CE-POMA) compared with trunk and proximal extremity melanomas. The rule of 10s describes complication rates after CE-POMA of specialty site melanomas: ∼10% risk for upstaging, ∼10% risk for positive excision margins, ∼10% risk for local recurrence, and ∼10-fold increased likelihood of reconstruction with a flap or graft. Trunk and proximal extremity melanomas encounter these complications at a lower rate, according to the rule of 2s. Mohs micrographic surgery (MMS) with frozen section melanocytic immunostains (MMS-I) and slow Mohs with paraffin sections decrease complications of surgery of specialty site melanomas by detecting upstaging and confirming complete tumor removal with comprehensive microscopic margin assessment before reconstruction. This article reviews information important for counseling melanoma patients about surgical treatment options and for developing consensus guidelines with clear indications for MMS-I or slow Mohs.

Melanomas of the head and neck have high-local recurrence risk features and require tissue-rearranging reconstruction more commonly than basal cell carcinoma and squamous cell carcinoma: A comparison of indications for microscopic margin control prior to reconstruction in 13,664 tumors.

BACKGROUND: On the basis of high-local recurrence risk features and tissue-rearranging reconstruction, consensus guidelines recommend microscopic margin control for keratinocyte carcinomas (KCs) but not for cutaneous melanoma. OBJECTIVE: To compare high-local recurrence risk features and frequency of tissue-rearranging reconstruction for head and neck KC with those for melanoma. METHODS: Retrospective cohort study of KC versus melanoma treated at the Hospital of the University of Pennsylvania with Mohs micrographic surgery. RESULTS: A total of 12,189 KCs (8743 basal cell carcinomas and 3343 squamous cell carcinomas) and 1475 melanomas (1065 melanomas in situ and 410 invasive melanomas) were identified from a prospectively updated Mohs micrographic surgery database. Compared with KCs, melanomas were significantly more likely to have high-local recurrence risk features, including larger preoperative size (2.10 cm vs 1.30 cm [P < .0001]), recurrent status (5.08% vs 3.91% [P = .031]), and subclinical spread (31.73% vs 26.52% [P < .0001]). Tissue-rearranging reconstruction was significantly more common for melanoma than for KCs (44.68% vs 33.02% [P < .0001]; odds ratio, 1.98 [P < .0001]). LIMITATIONS: This was a retrospective study, and it did not compare outcomes with those of other treatment methods, such as slow Mohs or conventional excision. CONCLUSION: Melanomas of the head and neck have high-local recurrence risk features and require tissue-rearranging reconstruction more frequently than KCs do.

Comparison of Accuracy of Patient and Physician Scar Length Estimates Before Mohs Micrographic Surgery for Facial Skin Cancers.

Importance: Patients are satisfied when surgical outcomes meet their expectations. Dissatisfaction with surgical scars is one of the most common reasons that patients sue surgeons who perform Mohs micrographic surgery (MMS). Objective: To measure the accuracy of patient and physician estimations of scar length prior to skin cancer removal with MMS. Design, Setting, and Participants: This cross-sectional study was conducted between December 1, 2017, and February 28, 2018, at the MMS clinic of a single tertiary referral center health system. A total of 101 adults presenting for MMS for treatment of facial skin cancers volunteered for this study, and 86 surgeons who performed the MMS procedure participated. Main Outcomes and Measures: Patients and physicians independently drew the anticipated scar length on the patients' skin prior to surgery. Preoperative estimates by patients and surgeons were compared with actual postoperative scar length. Results: Of the 101 patients who participated, 57 patients (56.4%) were men and 57 patients (56.4%) were aged 65 years or older. Eighty-four patients (83.2%) underestimated scar length, whereas 67 of the 86 surgeons (77.9%) correctly estimated the scar length (P < .001). The actual postoperative scar length was 2.2 (interquartile range, 1.5-3.6) times larger than the patients' preoperative estimate but only 1.1 (interquartile range, 1.0-1.2) times larger than the surgeons' preoperative estimate (P < .001). Preoperative consultation with the surgeon, a personal history of MMS, or patient-directed research about MMS were not associated with improvement of patients' estimations of scar length. Conclusions and Relevance: This study's findings suggest that patients with facial skin cancers have unrealistic expectations regarding scars that measure, on average, less than half the length of the actual postoperative scars. Surgeons appear to accurately estimate the length of most surgical scars and have an opportunity to set realistic patient expectations about scar length before surgery.

Variation in Prescribing and Factors Associated With the Use of Prophylactic Antibiotics for Mohs Surgery: A Single-Institution Retrospective Study.

BACKGROUND: Antibiotic use associated with Mohs surgery is increasing. OBJECTIVE: To understand variation in practice patterns and factors associated with antibiotic use. MATERIALS AND METHODS: The authors conducted a retrospective cohort study of antibiotic use among patients treated with Mohs micrographic surgery between July 1, 2013, and June 30, 2017, at an academic medical center. Multivariate logistic regression was used to evaluate for associations between antibiotic prescribing and the surgeon, site, reconstruction, and patient characteristics. RESULTS: The odds of prescribing antibiotics differed significantly between each surgeon evaluated; 3.35-fold variation in postoperative antibiotic use was observed. Increasing number of surgical sites (odds ratio [OR] 1.24; 95% confidence interval [CI] 1.09-1.41), number of Mohs stages required (OR 1.18; 95% CI 1.08-1.28), and defect area (OR 1.31; 95% CI 1.25-1.37), as well as patient female sex (OR 1.14; 95% CI 1.03-1.27), were associated with increased postoperative antibiotic use, whereas age >80 was associated with decreased use (OR 0.75; 95% CI 0.64-0.87) compared with age <60. CONCLUSION: Antibiotics are more commonly prescribed for repairs that are considered higher risk for infection. However, significant variation exists between surgeons, even within a single institution, suggesting a need for prospective trials and consensus guideline development.

Evolution of Excisional Surgery Practices for Melanoma in the United States.

IMPORTANCE: National Comprehensive Cancer Network guidelines for melanoma have consistently recommended wide local excision as the standard of care since their inception. Although surgery with more comprehensive margin assessment (eg, Mohs surgery) has been advocated for certain subsets of melanoma, how often these techniques are used in clinical practice is uncertain. OBJECTIVE: To examine trends in the use of comprehensive margin assessment surgery for melanoma by tracking claims data for Mohs surgery. DESIGN, SETTING, AND PARTICIPANTS: This national cross-sectional analysis examined claims data from the Optum Clinformatics Data Mart, a nationally representative database. The study cohort consisted of 79 108 patients undergoing surgical excision for melanoma from January 1, 2001, through December 31, 2016. Data were analyzed from January 1, 2001, through December 31, 2016. MAIN OUTCOMES AND MEASURES: The primary outcome was the likelihood of a melanoma being treated with Mohs surgery over time, evaluated by multivariable logistic regression and expressed as the odds of treatment per additional calendar year. RESULTS: Among 79 108 patients with melanoma (median age, 63 years [interquartile range {IQR}, 51-73]; 47 407 men [59.9%]), 75 047 were treated with conventional excision (median age, 62 years [IQR, 50-73 years]; 44 786 men [59.7%]) and 4061 with Mohs surgery (median age, 67 years [IQR, 56-76 years]; 2621 men [64.5%]). Mohs surgery was used in 5.1% of all surgical cases, with the rate of Mohs surgery increasing 304% from 2.6% in 2001 to 7.9% in 2016. Odds of receiving Mohs surgery for melanoma increased significantly in more recent calendar years (odds ratio [OR], 1.02 per calendar year; 95% CI, 1.01-1.03; P < .001). Immunohistochemistry (IHC) use was only coded with Mohs surgery in 1087 cases (26.8%), and the odds of receiving Mohs surgery with IHC increased in more recent calendar years (OR, 1.13 per calendar year; 95% CI, 1.10-1.15; P < .001). Use of Mohs surgery and Mohs surgery with IHC for melanoma differed widely across geographic census divisions with greater than 3-fold variation between the regions with highest and lowest use in every period (eg, for 2013 through 2016, the East South Central region used Mohs surgery in 8.8% of melanoma excisions compared with 2.6 in the New England region). CONCLUSIONS AND RELEVANCE: Despite stable guidelines for melanoma surgery, the results of this study suggest that surgical practices for melanoma are evolving. Wide variations in surgical practice patterns for melanoma are present in the United States. This study's findings suggest that the effect of variations in surgical techniques on outcomes requires scrutiny and further study.

Crescentic Modification to Island Pedicle Rotation Flaps for Defects of the Distal Nose.

BACKGROUND: Island pedicle flaps based on a lateral sling of the nasalis have difficulty reaching distal nasal defects due to tethering of the muscle to its insertion point. The authors hypothesized that reach could be improved by modifying a crescentic flap to rotate around a pivot point equidistant to the flap and the defect. OBJECTIVE: To describe the design, execution, and results with a modified crescentic island pedicle rotation flap for repair of distal nasal defects after Mohs micrographic surgery. METHODS: The authors performed a retrospective analysis of patients who had distal nasal defects repaired with a modified crescentic island pedicle rotation flap over an 8-year period. All charts and photographs were examined. Sex and age of the patient, type of cutaneous carcinoma, location and size of the defect, and complications were recorded. RESULTS: Forty-eight patients were included. The flap was used to successfully reconstruct defects on the distal nose ranging in size from 0.25 cm to 3.8 cm. Complications involved one wound infection and one episode of postoperative bleeding. CONCLUSION: The authors' modifications to the island pedicle flap may be reliably used to reconstruct small- to medium-sized defects of the distal nose in a single stage with minimal risk of complications.

In situ serial crystallography for rapid de novo membrane protein structure determination.

De novo membrane protein structure determination is often limited by the availability of large crystals and the difficulties in obtaining accurate diffraction data for experimental phasing. Here we present a method that combines in situ serial crystallography with de novo phasing for fast, efficient membrane protein structure determination. The method enables systematic diffraction screening and rapid data collection from hundreds of microcrystals in in meso crystallization wells without the need for direct crystal harvesting. The requisite data quality for experimental phasing is achieved by accumulating diffraction signals from isomorphous crystals identified post-data collection. The method works in all experimental phasing scenarios and is particularly attractive with fragile, weakly diffracting microcrystals. The automated serial data collection approach can be readily adopted at most microfocus macromolecular crystallography beamlines.

Membrane (and Soluble) Protein Stability and Binding Measurements in the Lipid Cubic Phase Using Label-Free Differential Scanning Fluorimetry.

Label-free differential scanning fluorimetry (DSF) is a relatively new method for evaluating the stability of proteins. It can be used as a screening tool for downstream applications such as crystallization. The method is attractive in that it requires miniscule quantities of proteins, it can be performed using intrinsic tryptophan and tyrosine fluorescence, and, with the right equipment, it is easy to perform. To date, the method has been used with proteins in liquid solutions and dispersions. It was of interest to determine if DSF could be used with membrane proteins in the lipid cubic phase (LCP), which increasingly is being used for crystallization in support of structure-function studies. The cubic phase is viscous. Furthermore, in coexistence with excess aqueous solution, as happens during crystallization trials, it can become turbid and scatter light. The concern was that these features may render the mesophase unsuitable for DSF analysis. However, using lysozyme and four integral membrane proteins we demonstrate that the method works with all tested proteins in solution and in the LCP. Of note is the observation that some of the test membrane proteins are more stable while others are less so in the mesophase. The method also works in ligand binding measurements. Thus, DSF should prove useful as an analytical tool for identifying host and additive lipids, detergents, precipitants and chemical probes that support the generation of quality crystals by the cubic phase method. Microscale thermophoresis was used to supplement the DSF study and was also shown to work with proteins in the mesophase. Measurements with lysozyme highlight the utility of the cubic mesophase as a model system in which to perform confinement studies.

Crystal structure of undecaprenyl-pyrophosphate phosphatase and its role in peptidoglycan biosynthesis.

As a protective envelope surrounding the bacterial cell, the peptidoglycan sacculus is a site of vulnerability and an antibiotic target. Peptidoglycan components, assembled in the cytoplasm, are shuttled across the membrane in a cycle that uses undecaprenyl-phosphate. A product of peptidoglycan synthesis, undecaprenyl-pyrophosphate, is converted to undecaprenyl-phosphate for reuse in the cycle by the membrane integral pyrophosphatase, BacA. To understand how BacA functions, we determine its crystal structure at 2.6 Å resolution. The enzyme is open to the periplasm and to the periplasmic leaflet via a pocket that extends into the membrane. Conserved residues map to the pocket where pyrophosphorolysis occurs. BacA incorporates an interdigitated inverted topology repeat, a topology type thus far only reported in transporters and channels. This unique topology raises issues regarding the ancestry of BacA, the possibility that BacA has alternate active sites on either side of the membrane and its possible function as a flippase.

Structural insights into the mechanism of the membrane integral N-acyltransferase step in bacterial lipoprotein synthesis.

Lipoproteins serve essential roles in the bacterial cell envelope. The posttranslational modification pathway leading to lipoprotein synthesis involves three enzymes. All are potential targets for the development of new antibiotics. Here we report the crystal structure of the last enzyme in the pathway, apolipoprotein N-acyltransferase, Lnt, responsible for adding a third acyl chain to the lipoprotein's invariant diacylated N-terminal cysteine. Structures of Lnt from Pseudomonas aeruginosa and Escherichia coli have been solved; they are remarkably similar. Both consist of a membrane domain on which sits a globular periplasmic domain. The active site resides above the membrane interface where the domains meet facing into the periplasm. The structures are consistent with the proposed ping-pong reaction mechanism and suggest plausible routes by which substrates and products enter and leave the active site. While Lnt may present challenges for antibiotic development, the structures described should facilitate design of therapeutics with reduced off-target effects.

Crystal structure and biochemical characterization of the transmembrane PAP2 type phosphatidylglycerol phosphate phosphatase from Bacillus subtilis.

Type 2 phosphatidic acid phosphatases (PAP2s) can be either soluble or integral membrane enzymes. In bacteria, integral membrane PAP2s play major roles in the metabolisms of glycerophospholipids, undecaprenyl-phosphate (C55-P) lipid carrier and lipopolysaccharides. By in vivo functional experiments and biochemical characterization we show that the membrane PAP2 coded by the Bacillus subtilis yodM gene is the principal phosphatidylglycerol phosphate (PGP) phosphatase of B. subtilis. We also confirm that this enzyme, renamed bsPgpB, has a weaker activity on C55-PP. Moreover, we solved the crystal structure of bsPgpB at 2.25 Å resolution, with tungstate (a phosphate analog) in the active site. The structure reveals two lipid chains in the active site vicinity, allowing for PGP substrate modeling and molecular dynamic simulation. Site-directed mutagenesis confirmed the residues important for substrate specificity, providing a basis for predicting the lipids preferentially dephosphorylated by membrane PAP2s.

Comparing the efficacies of alginate, foam, hydrocolloid, hydrofiber, and hydrogel dressings in the management of diabetic foot ulcers and venous leg ulcers: a systematic review and meta-analysis examining how to dress for success.

Diabetic foot ulcers and venous leg ulcers are chronic wounds frequently encountered by dermatologists. Choosing appropriate wound dressings can effectively promote wound healing and potentially reduce morbidity and financial burden experienced by patients. The objective of our systematic review and meta-analysis was to evaluate wound healing efficacies of synthetic active dressings in diabetic foot ulcer and venous leg ulcer management. For data collection, PubMed, Embase, Cochrane Library, CINAHL, and clinicaltrials.gov online databases were searched from database inception to 10 May 2015. Fixed and random effects modeling were used to calculate pooled risk ratios for complete ulcer healing from pairwise dressing comparisons. The results of our review showed moderate-quality level evidence that hydrogels were more effective in healing diabetic foot ulcers than basic wound contact dressings (RR 1.80 [95% CI, 1.27-2.56]). The other dressing comparisons showed no statistically significant differences between the interventions examined in terms of achieving complete diabetic foot ulcer healing. Non-adherent dressings were more cost-effective than hydrofiber dressings for diabetic foot ulcers in terms of mean total cost per patient of the dressings themselves. All venous leg ulcer pairwise dressing comparisons showed equivalent dressing efficacies in terms of promoting complete ulcer healing. Overall, most synthetic active dressings and traditional wound dressings are equally efficacious in treating diabetic foot ulcers and venous leg ulcers. For treating diabetic foot ulcers, hydrogels are more efficacious than basic wound contact dressings, and non-adherent dressings are more cost-effective than hydrofiber dressings. Ultimately, dressing choice should be tailored to the wound and the patient.

In meso in situ serial X-ray crystallography of soluble and membrane proteins at cryogenic temperatures.

Here, a method for presenting crystals of soluble and membrane proteins growing in the lipid cubic or sponge phase for in situ diffraction data collection at cryogenic temperatures is introduced. The method dispenses with the need for the technically demanding and inefficient crystal-harvesting step that is an integral part of the lipid cubic phase or in meso method of growing crystals. Crystals are dispersed in a bolus of mesophase sandwiched between thin plastic windows. The bolus contains tens to hundreds of crystals, visible with an in-line microscope at macromolecular crystallography synchrotron beamlines and suitably disposed for conventional or serial crystallographic data collection. Wells containing the crystal-laden boluses are removed individually from hermetically sealed glass plates in which crystallization occurs, affixed to pins on goniometer bases and excess precipitant is removed from around the mesophase. The wells are snap-cooled in liquid nitrogen, stored and shipped in Dewars, and manually or robotically mounted on a goniometer in a cryostream for diffraction data collection at 100 K, as is performed routinely with standard, loop-harvested crystals. The method is a variant on the recently introduced in meso in situ serial crystallography (IMISX) method that enables crystallographic measurements at cryogenic temperatures where crystal lifetimes are enormously enhanced whilst reducing protein consumption dramatically. The new approach has been used to generate high-resolution crystal structures of a G-protein-coupled receptor, α-helical and ß-barrel transporters and an enzyme as model integral membrane proteins. Insulin and lysozyme were used as test soluble proteins. The quality of the data that can be generated by this method was attested to by performing sulfur and bromine SAD phasing with two of the test proteins.

Ternary structure reveals mechanism of a membrane diacylglycerol kinase.

Diacylglycerol kinase catalyses the ATP-dependent conversion of diacylglycerol to phosphatidic acid in the plasma membrane of Escherichia coli. The small size of this integral membrane trimer, which has 121 residues per subunit, means that available protein must be used economically to craft three catalytic and substrate-binding sites centred about the membrane/cytosol interface. How nature has accomplished this extraordinary feat is revealed here in a crystal structure of the kinase captured as a ternary complex with bound lipid substrate and an ATP analogue. Residues, identified as essential for activity by mutagenesis, decorate the active site and are rationalized by the ternary structure. The γ-phosphate of the ATP analogue is positioned for direct transfer to the primary hydroxyl of the lipid whose acyl chain is in the membrane. A catalytic mechanism for this unique enzyme is proposed. The active site architecture shows clear evidence of having arisen by convergent evolution.

Fast native-SAD phasing for routine macromolecular structure determination.

We describe a data collection method that uses a single crystal to solve X-ray structures by native SAD (single-wavelength anomalous diffraction). We solved the structures of 11 real-life examples, including a human membrane protein, a protein-DNA complex and a 266-kDa multiprotein-ligand complex, using this method. The data collection strategy is suitable for routine structure determination and can be implemented at most macromolecular crystallography synchrotron beamlines.

Topical antibiotic prophylaxis for prevention of surgical wound infections from dermatologic procedures: a systematic review and meta-analysis.

IMPORTANCE: Topical antibiotics are not indicated for postsurgical wound infection prophylaxis in clean and clean-contaminated dermatologic surgeries, yet many dermatologists continue to prescribe them. The objective of our systematic review and meta-analysis was to critically assess the efficacy of topical antibiotics in terms of preventing postsurgical wound infections in the dermatology outpatient setting. METHODS: PubMed, Embase, MD Consult, Science Direct, Springer Link, DynaMed and Cochrane online medical databases were searched from 1980 to 2013. RESULTS: Using random effects modeling, the pooled odds ratio of developing a postsurgical wound infection was 0.71 (95% CI, 0.42-1.19). DISCUSSION: Pooled data of the four trials in the meta-analysis did not show a statistically significant difference in incidence of postsurgical wound infections between topical antibiotics and petrolatum/paraffin. In the setting of moist occlusive dressings, there is no statistically significant difference in prophylactic efficacy between applying and not applying ointment to surgical wounds. Wounds at increased risk of developing surgical site infections include wounds in diabetics, wounds located in certain anatomic regions, and wounds created by some surgical procedures. CONCLUSIONS: Petrolatum should be used instead of topical antibiotics as a prophylactic measure to prevent postsurgical wound infections in the outpatient dermatologic setting.