Evolutionary Pathways and Trajectories in Antibiotic Resistance.

Evolution is the hallmark of life. Descriptions of the evolution of microorganisms have provided a wealth of information, but knowledge regarding "what happened" has precluded a deeper understanding of "how" evolution has proceeded, as in the case of antimicrobial resistance. The difficulty in answering the "how" question lies in the multihierarchical dimensions of evolutionary processes, nested in complex networks, encompassing all units of selection, from genes to communities and ecosystems. At the simplest ontological level (as resistance genes), evolution proceeds by random (mutation and drift) and directional (natural selection) processes; however, sequential pathways of adaptive variation can occasionally be observed, and under fixed circumstances (particular fitness landscapes), evolution is predictable. At the highest level (such as that of plasmids, clones, species, microbiotas), the systems' degrees of freedom increase dramatically, related to the variable dispersal, fragmentation, relatedness, or coalescence of bacterial populations, depending on heterogeneous and changing niches and selective gradients in complex environments. Evolutionary trajectories of antibiotic resistance find their way in these changing landscapes subjected to random variations, becoming highly entropic and therefore unpredictable. However, experimental, phylogenetic, and ecogenetic analyses reveal preferential frequented paths (highways) where antibiotic resistance flows and propagates, allowing some understanding of evolutionary dynamics, modeling and designing interventions. Studies on antibiotic resistance have an applied aspect in improving individual health, One Health, and Global Health, as well as an academic value for understanding evolution. Most importantly, they have a heuristic significance as a model to reduce the negative influence of anthropogenic effects on the environment.

Biolog Phenotype Microarray Is a Tool for the Identification of Multidrug Resistance Efflux Pump Inducers.

Multidrug resistance efflux pumps frequently present low levels of basal expression. However, antibiotic-resistant mutants that overexpress these resistance determinants are selected during infection. In addition, increased expression of efflux pumps can be induced by environmental signals/cues, which can lead to situations of transient antibiotic resistance. In this study, we have applied a novel high-throughput methodology in order to identify inducers able to trigger the expression of the Stenotrophomonas maltophilia SmeVWX and SmeYZ efflux pumps. To that end, bioreporters in which the expression of the yellow fluorescent protein (YFP) is linked to the activity of either smeVWX or smeYZ promoters were developed and used for the screening of potential inducers of the expression of these efflux pumps using Biolog phenotype microarrays. YFP production was also measured by flow cytometry, and the levels of expression of smeV and smeY in the presence of a set of selected compounds were also determined by real-time reverse transcription-PCR (RT-PCR). The expression of smeVWX was induced by iodoacetate, clioquinol, and selenite, while boric acid, erythromycin, chloramphenicol, and lincomycin triggered smeYZ expression. The susceptibility to antibiotics that are known substrates of the efflux pumps decreased in the presence of the inducers. However, the analyzed multidrug efflux systems did not contribute to S. maltophilia resistance to the studied inducers. To sum up, the use of fluorescent bioreporters in combination with Biolog plates is a valuable tool for identifying inducers of the expression of bacterial multidrug resistance efflux pumps, and likely of other bacterial systems whose expression is regulated in response to signals/cues.

Surgical Ligation Versus Percutaneous Closure of Patent Ductus Arteriosus in Very Low-Weight Preterm Infants: Which are the Real Benefits of the Percutaneous Approach?

Percutaneous treatment of patent ductus arteriosus (PDA) in extreme premature infants is technically difficult, and therefore, often not consider as an alternative to surgery. The main objective of our work was to compare respiratory status prior and post ductal closure and morbi-mortality, in our series of preterm infants with percutaneous PDA closure versus surgical ligation in the same time-period. Retrospective review of all premature infants submitted to percutaneous and surgical PDA closure from January 2011 to December 2016. All the antenatal, perinatal, and postnatal characteristics were collected. The main outcome was the assessment of the pulmonary status before and after ductal closure using a pulmonary score. Secondary outcomes included moderate-severe disability in neurodevelopment, death before discharge, moderate-severe chronic lung disease, and morbidity at discharge. 25 patients with a mean weight of 1330 g (± 280) underwent percutaneous closure of PDA with ADO-II-AS, and a total of 53 underwent surgical ligation. 28/53 with similar gestational age, birth weight, and procedure weight to those in the percutaneous group, were selected to perform the comparative study. Ductal closure (percutaneous and surgical) resulted in improved respiratory status. However, percutaneous group achieved a fastest respiratory improvement, than surgical group. The surgical closure group associated higher morbidity among survivors (HIV, number of sepsis, need, and duration of inotropics post-interventionism). The incidence of recurrent laryngeal nerve palsy among the surgical group was 17%. Percutaneous closure of PDA in carefully selected low-weight preterm infants is a safe and reliable alternative to surgical ligation.

Vitamin K3 Induces the Expression of the Stenotrophomonas maltophilia SmeVWX Multidrug Efflux Pump.

Stenotrophomonas maltophilia is an opportunistic pathogen with increasing prevalence, which is able to cause infections in immunocompromised patients or in those with a previous pathology. The treatment of the infections caused by this bacterium is often complicated due to the several intrinsic antibiotic resistance mechanisms that it presents. Multidrug efflux pumps are among the best-studied mechanisms of S. maltophilia antibiotic resistance. Some of these efflux pumps have a basal expression level but, in general, their expression is often low and only reaches high levels when the local regulator is mutated or bacteria are in the presence of an effector. In the current work, we have developed a yellow fluorescent protein (YFP)-based sensor with the aim to identify effectors able to trigger the expression of SmeVWX, an efflux pump that confers resistance to quinolones, chloramphenicol, and tetracycline when it is expressed at high levels. With this purpose in mind, we tested a variety of different compounds and analyzed the fluorescence signal given by the expression of YFP under the control of the smeVWX promoter. Among the tested compounds, vitamin K3, which is a compound belonging to the 2-methyl-1,4-naphthoquinone family, is produced by plants in defense against infection, and has increasing importance in human therapy, was able to induce the expression of the SmeVWX efflux pump. In addition, a decrease in the susceptibility of S. maltophilia to ofloxacin and chloramphenicol was observed in the presence of vitamin K3, in both wild-type and smeW-deficient strains.

Atomically Resolved Short-Range Order at the Nanoscale in the Ca-Mn-O System.

The elucidation of the reaction mechanisms involving redox processes in functional transition-metal oxides, which usually start in areas of very few nanometers in size, is yet a challenge to be satisfactorily achieved. Atomically resolved HAADF and EELS have provided both chemical and structural information at the nanoscale, which reveal the preservation of short-range cationic order in areas of 2-3 nm length as the driving force behind the reversibility of the Ca2Mn3O8-Ca2Mn3O5 redox process. Oxygen evolution is accommodated by cationic diffusion along the Ca and Mn layers of the cation-deficient Ca2Mn3O8 delafossite related structure, whereas Mn remains octahedrally coordinated.

Effect of oral nutritional supplementation on wound healing in diabetic foot ulcers: a prospective randomized controlled trial.

AIMS: Among people with diabetes, 10-25% will experience a foot ulcer. Research has shown that supplementation with arginine, glutamine and ß-hydroxy-ß-methylbutyrate may improve wound repair. This study tested whether such supplementation would improve healing of foot ulcers in persons with diabetes. METHODS: Along with standard of care, 270 subjects received, in a double-blinded fashion, (twice per day) either arginine, glutamine and ß-hydroxy-ß-methylbutyrate or a control drink for 16 weeks. The proportion of subjects with total wound closure and time to complete healing was assessed. In a post-hoc analysis, the interaction of serum albumin or limb perfusion, as measured by ankle-brachial index, and supplementation on healing was investigated. RESULTS: Overall, there were no group differences in wound closure or time to wound healing at week 16. However, in subjects with an albumin level of ≤ 40 g/l and/or an ankle-brachial index of < 1.0, a significantly greater proportion of subjects in the arginine, glutamine and ß-hydroxy-ß-methylbutyrate group healed at week 16 compared with control subjects (P = 0.03 and 0.008, respectively). Those with low albumin or decreased limb perfusion in the supplementation group were 1.70 (95% CI 1.04-2.79) and 1.66 (95% CI 1.15-2.38) times more likely to heal. CONCLUSIONS: While no differences in healing were identified with supplementation in non-ischaemic patients or those with normal albumin, addition of arginine, glutamine and ß-hydroxy-ß-methylbutyrate as an adjunct to standard of care may improve healing of diabetic foot ulcers in patients with risk of poor limb perfusion and/or low albumin levels. Further investigation involving arginine, glutamine and ß-hydroxy-ß-methylbutyrate in these high-risk subgroups might prove clinically valuable.

Super resolution imaging achieved by using on-axis interferometry based on a Spatial Light Modulator.

An interferometry based method to achieve resolution beyond the diffraction barrier is proposed. Object is illuminated with different tilted beams, generated by using a Spatial Light Modulator (SLM). In addition, some constant phases are also assigned to each tilted beam with the SLM display. Then, the object is simultaneously illuminated with all tilted beams, producing an on-axis interferometry scheme. An interferogram at the image plane is formed for each set of constant phases added to the tilted beams. Using proper selection of constant phases for each of the interferograms, the synthetic aperture can be calculated. During the post processing, we take the Fourier transforms of the each image and the portions of the spectrum are spatially shifted and combined to obtain synthesized spectrum whose inverse Fourier transform gives high resolution image.

Analysis of transfer lesions in patients who underwent surgery for diabetic foot ulcers located on the plantar aspect of the metatarsal heads.

AIMS: To analyse the risk of reulceration caused by the transfer of lesions in patients with diabetes, undergoing resection of at least one metatarsal head. METHODS: A total of 119 patients with diabetes from the Diabetic Foot Unit (Complutense University, Madrid, Spain), who underwent resection of at least one metatarsal head were analysed prospectively from November 2006 to December 2011 to assess reulceration in the other metatarsal head. RESULTS: Seven patients were excluded for being subjected to a pan-metatarsal head resection and 11 patients dropped out. During a median follow-up period of 13.1 months (interquartile range 6.1-22.8 months), 41% of patients suffered from reulcerations. Reulceration frequency in patients operated on the 1st, 2nd, 3rd, 4th, 5th and several metatarsal heads was 9 (69%), 8 (44%), 12 (52%), 2 (25%), 6 (19%) and 4 (50%) events, respectively. The Cox regression model showed hazard ratios that were significant for the location of the metatarsal resection. The first metatarsal showed the highest risk for reulceration (hazard ratio 3.307; 1.472-7.430) and the fifth metatarsal showed the lowest risk (hazard ratio 0.339; 0.138-0.832). CONCLUSIONS: Reulceration is a frequent event following resection of a metatarsal head and should be regarded as an implicit complication of the intervention. The location of the resection determines the risk of reulceration, which is highest for patients operated on the first metatarsal head and lowest for patients operated on the fifth metatarsal head.

Charcot neuroarthropathy triggered and complicated by osteomyelitis. How limb salvage can be achieved.

BACKGROUND: Charcot neuroarthropathy is a severe complication in the feet of patients with diabetes, which can lead to a major amputation. Osteomyelitis and surgery for osteomyelitis have been reported as trigger mechanisms of developing Charcot neuroarthropathy. However, the development of acute Charcot neuroarthropathy triggered by osteomyelitis during conservative antibiotic treatment is not well outlined in the medical literature. CASE REPORTS: Two patients apparently developed mid and rear foot Charcot neuroarthropathy, which was clinically suspected while being treated with antibiotics for osteomyelitis. One of them presented osteomyelitis of the navicular bone and subsequently developed acute Charcot neuroarthropathy of the tarsometatarsal joints. The other presented calcaneal osteomyelitis with pathological fracture and developed Charcot neuroarthropathy of the transverse tarsal joint. No offloading had been implemented in either case. A major amputation had been indicated in both cases in their teaching hospitals. Limb salvage was achieved in both cases by means of surgery, culture-guided post-operative antibiotics, intraosseus instillation of super-oxidized solution, bed rest before placing a total contact cast and stabilization of the unstable foot with a total contact cast with an opening for checking the healing course and to detect any complications. The mechanisms of the development of acute Charcot neuroarthropathy in a patient with osteomyelitis are discussed. CONCLUSIONS: Osteomyelitis in the feet of patients with diabetes and neuropathy may trigger the development of acute Charcot neuroarthropathy. Fractures and dislocated joints may subsequently become infected from the index focus, producing a severe infected and unstable foot that may require a major amputation. Limb salvage can be achieved in specialized departments.

[Treatment of infected diabetic foot ulcers clinical effectiveness of a dressing of alginate and hydrocolloid, with silver fiber. Analysis of results of a series of cases]. / Tratamiento de úlceras infectadas de pie diabético.

INTRODUCTION: High levels of bacterial load have shown a deleterious influence on wound healing. Using antimicrobial dressings can control ulcers' bioburden. The aim of our study was to evaluate the improving of infected diabetic foot ulcers due an alginate's fiber and hydrocolloid silver dressing. MATERIAL AND METHODS: We analysed a case series of 6 patients with diabetic foot ulcers without peripheral vascular disease and diagnosed from critical colonization and/or local infection according the presence of inflammation clinical signs. Patients were treated for a minimum period of two weeks. We analysed the percentage reduction in ulcer area from the day of enrolment to antimicrobial dressing removal. RESULTS: The duration of treatment had a median of 5 weeks with a minimum of 2 weeks and up to 6. The median percentage of area reduction of the wounds was 47.7% (range: 0.5%-90%). The mean percentage reduction on the lesion was 58% from 2 weeks and 67.14% at 3 weeks. All patients had reduced significantly their size at 3 weeks from beginning of treatment (p < 0.05). CONCLUSION: The use of an alginate's fiber and hydrocolloid silver dressing promotes healing on diabetic foot ulcers with local infection, reducing the inflammatory clinical signs significantly over a period of three weeks.

Genetic monitoring on the world's first MSC eco-labeled common octopus (O. vulgaris) fishery in western Asturias, Spain.

Octopus vulgaris (Cuvier, 1797) is a cephalopod species with great economic value. In western Asturias (northwest of Spain), O. vulgaris artisanal fisheries are relatively well monitored and conditionally eco-labeled by the Marine Stewardship Council (MSC). Despite this, the Asturian octopus stocks have not been genetically assessed so far. In order to improve the current fishery plan and contrast the octopus eco-label validity in Asturias, 539 individuals from five regions of the O. vulgaris geographic distribution, including temporal samplings in Asturias, were collected and genotyped at thirteen microsatellite loci. All the samples under analysis were in agreement with Hardy-Weinberg expectations. Spatial levels of genetic differentiation were estimated using F-statistics, multidimensional scaling, and Bayesian analyses. Results suggested that the O. vulgaris consists of at least four genetically different stocks coming from two ancestral lineages. In addition, temporal analyses showed stability in terms of genetic variation and high NE (> 50) for several generations in different localities within Asturias, pointing out to indeed sustainable fishery exploitation levels. Even though, the current Asturias fishery plan shows no significant genetic damages to the stocks, the regional-specific management plans need systematic genetic monitoring schemes as part of an efficient and preventive regional fishery regulation strategy.

Does osteomyelitis in the feet of patients with diabetes really recur after surgical treatment? Natural history of a surgical series.

AIMS/HYPOTHESIS: The aim of this study was to determine the rate of recurrence, reulceration and new episodes of osteomyelitis and the duration of postoperative antibiotic treatment in a prospective cohort of patients with diabetes who underwent conservative surgery for osteomyelitis. METHODS: The prospective cohort included patients with diabetes and a definitive diagnosis of osteomyelitis who were admitted to the Diabetic Foot Unit (Surgery Department, La Paloma Hospital, Las Palmas de Gran Canaria, Spain) and underwent surgical treatment from 1 November 2007 to 30 May 2010. RESULTS: Eighty-one patients were operated on for osteomyelitis during the study period. Seven patients were lost to follow-up at different stages of the study. The median duration of follow-up was 101.8 weeks (quartile 1 = 56.6, quartile 3 = 126.7). Forty-eight patients (59.3%) underwent conservative surgery, 32 (39.5%) had minor amputations and there was one (1.2%) major amputation. Twenty patients (24.7%) required reoperation because of persistent infection. Postoperative antibiotic treatment over a median period of 36 days was provided. Wound healing was achieved by secondary intention for a median of 8 weeks. Sixty-five patients were available for follow-up after healing. The percentage of recurrence, reulceration, and new episodes of osteomyelitis was 4.6% (3/65), 43% (28/65) and 16.9% (11/65), respectively. Mortality during follow-up (excluding in-hospital deaths and patients lost to follow-up) was 13% (9/69). CONCLUSION: A low rate of recurrence of osteomyelitis after surgical treatment for osteomyelitis was achieved. Despite new episodes, our approach to managing this cohort of patients with diabetes and foot osteomyelitis achieved 98.8% limb salvage.

Diagnosing diabetic foot osteomyelitis: is the combination of probe-to-bone test and plain radiography sufficient for high-risk inpatients?

AIMS: To investigate the accuracy of the sequential combination of the probe-to-bone test and plain X-rays for diagnosing osteomyelitis in the foot of patients with diabetes. METHODS: We prospectively compiled data on a series of 338 patients with diabetes with 356 episodes of foot infection who were hospitalized in the Diabetic Foot Unit of La Paloma Hospital from 1 October 2002 to 31 April 2010. For each patient we did a probe-to-bone test at the time of the initial evaluation and then obtained plain X-rays of the involved foot. All patients with positive results on either the probe-to-bone test or plain X-ray underwent an appropriate surgical procedure, which included obtaining a bone specimen that was processed for histology and culture. We calculated the sensitivity, specificity, predictive values and likelihood ratios of the procedures, using the histopathological diagnosis of osteomyelitis as the criterion standard. RESULTS: Overall, 72.4% of patients had histologically proven osteomyelitis, 85.2% of whom had positive bone culture. The performance characteristics of both the probe-to-bone test and plain X-rays were excellent. The sequential diagnostic approach had a sensitivity of 0.97, specificity of 0.92, positive predictive value of 0.97, negative predictive value of 0.93, positive likelihood ratio of 12.8 and negative likelihood ratio of 0.02. Only 6.6% of patients with negative results on both diagnostic studies had osteomyelitis. CONCLUSIONS: Clinicians seeing patients in a setting similar to ours (specialized diabetic foot unit with a high prevalence of osteomyelitis) can confidently diagnose diabetic foot osteomyelitis when either the probe-to-bone test or a plain X-ray, or especially both, are positive.

Inter-observer reproducibility of probing to bone in the diagnosis of diabetic foot osteomyelitis.

AIMS: The objectives of our study were (i) to analyse the inter-observer reproducibility or diagnostic variability of the probing-to-bone test, depending on the training of the professional involved, and (ii) to assess whether the probing-to-bone test can be extrapolated to any professional specialty that deals with these patients. METHODS: This was a cross-sectional study, involving 75 patients with diabetic foot ulcer and clinical suspicion of osteomyelitis. A registration sheet was completed for all patients involved in the research study, gathering data relative to the results of the probing-to-bone test performed by three observers. Observer 1 was a very experienced professional with several years of experience in the treatment of the diabetic foot; observer 2 was a medium-experienced professional whose experience ranges from 6 to 12 months in the treatment of the diabetic foot; observer 3 was a healthcare professional without experience in the treatment of the diabetic foot. Data were gathered confidentially by a fourth researcher. RESULTS: The results showed a kappa index of 0.593 (95% CI 0.407-0.778) between observer 1 and observer 2, 0.397 (95% CI 0.188-0.604) between observer 1 and observer 3 and 0.53 (95% CI 0.335-0.725) between observer 2 and observer 3. CONCLUSIONS: The probing-to-bone test demonstrated moderate to fair concordance with an experienced examiner, although the degree of concordance is not significant between groups.

AQP-1 association with body fluid loss in 10-km runners.

Intrinsic body fluid regulation is critical for optimizing endurance performance. Aquaporins (AQPs) are a family of transmembrane proteins that transport water and glycerol across cellular membranes. A recent report revealed an association between a single nucleotide polymorphism (SNP) in the 3' untranslated region of the aquaporin-1 (AQP1) gene and endurance performance. The purpose of the study was to explore the association between the AQP1 SNP and acute body fluid loss in long distance runners. The subjects (N=91, Age=26±3 yrs; Ht=170±11 cm; Wt=61±5 kg; mean±SD) were biologically unrelated male long distance runners. Data were collected before and after an international 10 km road race. Body fluid loss was determined by the difference between nude body weight before and after the 10 km run. The AQP1 (G→C) gene variation was detected by the ARMS-PCR procedure. Genotypes were determined by PCR product size. Carriers of the AQP1 SNP had a significantly greater adjusted body fluid loss (3.7±0.9 kg) than non-carriers (1.5±1.1 kg) (P<0.05). In conclusion, our study found an association between the AQP1 SNP and acute body fluid loss in long distance runners.

[Toxic epidermal necrolysis treated with cyclosporin A]. / Tratamiento de necrólisis epidérmica tóxica con ciclosporina A.

Lyell's syndrome or toxic epidermal necrolysis (TEN) is an extremely rare and dangerous severe skin disorder characterized by a high proportion of cutaneous lesions leading to necrosis and subsequent shedding of the epidermis over large areas of skin after an idiosyncratic reaction triggered by a drug. The patients who suffer it pathophysiologically have similar complications to those seen in major burns. TEN traditionally has been treated with immunomodulators such as glucocorticoids, intravenous gammaglobulin, cyclophosphamide, thalidomide or plasmapheresis. A variable, and sometimes contradictory response, has been obtained in some series. Cyclosporin A has been tested as a single immunomodulator in patients with TEN since the end of the 90 s in a limited number series. The results have improved in regards to survival compared with studies with other drugs. We report three consecutive cases of toxic epidermal necrolysis treated with cyclosporin A in this article.

Strongly reduced lattice thermal conductivity in Sn-doped rare-earth (M) filled skutterudites M x Co4Sb12-y Sn y , promoted by Sb-Sn disordering and phase segregation.

CoSb3 thermoelectric skutterudite has been filled with rare-earth metals (M = La, Ce, Yb) and partially doped with Sn in specimens of M x Co4Sb12-y Sn y stoichiometry. This has been achieved under high-pressure conditions at 3.5 GPa in a piston-cylinder hydrostatic press. A structural investigation using synchrotron X-ray diffraction data reveals a phase segregation in twin skutterudite phases with filling fraction fluctuation and different unit-cell sizes. As a result of three effects acting as phonon scatterers, namely the rattling effect of M at the wide 8a cages of the cubic Im3̄ structure, the phase segregation, and the intrinsic disorder introduced by Sn substitution at the Sb sublattice, the total thermal conductivity (κ) dramatically falls to reach minimum values under 2 W m-1 K-1, well below those typically exhibited by other thermoelectric materials based upon single-filled skutterudites. The power factor is substantially enhanced to 1.11 mW m-1 K-2 in Yb0.5Co4Sb11.6Sn0.4 with respect to the unfilled composition, as a result of the charge transfer promoted by the filler.

A wide-ranging Pseudomonas aeruginosa PeptideAtlas build: A useful proteomic resource for a versatile pathogen.

Pseudomonas aeruginosa is an important opportunistic human pathogen with high prevalence in nosocomial infections. This microorganism is a good model for understanding biological processes such as the quorum-sensing response, the metabolic integration of virulence, the mechanisms of global regulation of bacterial physiology, and the evolution of antibiotic resistance. Till now, P. aeruginosa proteomic data, although available in several on-line repositories, were dispersed and difficult to access. In the present work, proteomes of the PAO1 strain grown under different conditions and from diverse cellular compartments have been joined to build the Pseudomonas PeptideAtlas. This resource is a comprehensive mass spectrometry-derived peptide and inferred protein database with 71.3% coverage of the total predicted proteome of P. aeruginosa PAO1, the highest coverage among bacterial PeptideAtlas datasets. The proteins included cover 89% of metabolic proteins, 72% of proteins involved in genetic information processing, 83% of proteins responsible for environmental information processing, more than 88% of the ones related to quorum sensing and biofilm formation, and 89% of proteins responsible for antimicrobial resistance. It exemplifies a necessary tool for targeted proteomics studies, system-wide observations, and cross-species observational studies. The manuscript describes the building of the PeptideAtlas and the contribution of the different proteomic data used. SIGNIFICANCE: Pseudomonas aeruginosa is among the most versatile human bacterial pathogens. Studies of its proteome are very important as they can reveal virulence factors and mechanisms of antibiotic resistance. The construction of a proteomic resource such as the PeptideAtlas enables targeted proteomics studies, system-wide observations, and cross-species observational studies.