Clinical practice recommendations for infectious disease management of diabetic foot infection (DFI) - 2023 SPILF.

In march 2020, the International Working Group on the Diabetic Foot (IWGDF) published an update of the 2015 guidelines on the diagnosis and management of diabetic foot infection (DFI). While we (the French ID society, SPILF) endorsed some of these recommendations, we wanted to update our own 2006 guidelines and specifically provide informative elements on modalities of microbiological diagnosis and antibiotic treatment (especially first- and second-line regiments, oral switch and duration). The recommendations put forward in the present guidelines are addressed to healthcare professionals managing patients with DFI and more specifically focused on infectious disease management of this type of infection, which clearly needs a multidisciplinary approach. Staging of the severity of the infection is mandatory using the classification drawn up by the IWGDF. Microbiological samples should be taken only in the event of clinical signs suggesting infection in accordance with a strict preliminarily established protocol. Empirical antibiotic therapy should be chosen according to the IWGDF grade of infection and duration of the wound, but must always cover methicillin-sensitive Staphylococcus aureus. Early reevaluation of the patient is a fundamental step, and duration of antibiotic therapy can be shortened in many situations. When osteomyelitis is suspected, standard foot radiograph is the first-line imagery examination and a bone biopsy should be performed for microbiological documentation. Histological analysis of the bone sample is no longer recommended. High dosages of antibiotics are recommended in cases of confirmed osteomyelitis.

Link between nasal carriage of Staphylococcus aureus and infected diabetic foot ulcers.

AIMS: Nasal carriage of Staphylococcus aureus in diabetic patients may be a risk factor for diabetic foot lesion infections. The aims of this study were to compare the genotypic profiles of S. aureus strains isolated from nares and diabetic foot ulcers (DFUs) using microarray technology. METHODS: Patients were included if they were admitted for diabetic foot infection (DFI) at any of three diabetology departments of Montpellier and Nîmes University Hospitals between 1 September 2010 to 30 June 2012. All S. aureus isolates were analyzed using oligonucleotides arrays; S. aureus resistance and virulence genes were determined and each isolate was affiliated to a clonal complex. RESULTS: The prevalence of S. aureus nasal carriage among the 276 included patients was 39.5% (n=109), while 36.6% (n=101) had S. aureus at both sites (nares and foot wounds) and, of these patients, 65.3% of patients harboured the same strain at both sites. In addition, the spread of the methicillin-resistant S. aureus (MRSA) ST398 clone in DFI and its tropism for bone were also further confirmed. CONCLUSION: These findings appear to provide new arguments in favour of the systematic detection of nasal S. aureus carriage to anticipate the management of DFI.

Polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE): a promising tool to diagnose bacterial infections in diabetic foot ulcers.

AIM: The diagnosis of diabetic foot infections is difficult due to limitations of conventional culture-based techniques. The objective of this study was to evaluate the contribution of denaturing gradient gel electrophoresis (DGGE) in the microbiological diagnosis of diabetic foot ulcers in comparison to conventional techniques, and also to evaluate the need to perform a biopsy sample for this diagnosis. METHODS: Twenty diabetic patients (types 1 and 2) with foot ulcers (grades 1-4) were included. After debridement of their wounds, samples were taken in duplicate by surface swabbing and deep-tissue biopsy. The samples were analyzed by conventional culture and by a new molecular biology tool, DGGE technology. RESULTS: Polymerase chain reaction (PCR)-DGGE led to the identification of more bacteria than did conventional cultures (mean: 2.35 vs 0.80, respectively). In 11 cases, the technology detected pathogenic species not isolated by classical cultures. PCR-DGGE also identified significantly more pathogenic species at deep levels compared with species detected at superficial levels (87% vs 58%, respectively; P = 0.03). In 9/20 cases, pathogenic bacteria were detected only in deep samples, revealing the need to perform tissue biopsy sampling. CONCLUSION: DGGE, achievable in 48h, could be a useful technique for the bacteriological diagnosis of diabetic foot infections. It may help to identify pathogenic bacteria in deeply infected ulcers, thereby contributing to a more appropriate use of antibiotics.

Management of diabetic foot ulcers with a TLC-NOSF wound dressing.

OBJECTIVE: To evaluate the efficacy, tolerance and acceptability of UrgoStart Contact (Laboratoires Urgo), a new wound dressing impregnated with NOSF, as an MMP regulator in the management of neuropathic diabetic foot ulcers. METHOD: A multicentre, pilot, prospective, non-controlled open-label clinical trial. Adult patients with type 1 or 2 diabetes mellitus, who had a grade 1A (Texas classification), uninfected, neuropathic foot ulcer, 1-15cm2 in size and of 1-20 months' duration (mean 6.7 ± 5.2 months) were included in the study. The primary endpoint was the relative reduction of the wound surface area (%) at the end of the study. Secondary endpoints included rate of complete healing, and tolerability and acceptability of the dressing. The wound dressing was changed regularly at the investigator's discretion, in accordance with the wound status and exudate level. Patients were followed up every 2 weeks for a 12-week period. At each visit, patients underwent clinical assessments, and ulcer surface area was measured by planimetry and photographs. RESULTS: Thirty-four diabetic patients with a neuropathic foot ulcer were included but only 33 cases were analysed, as data were completely lost for one patient. At baseline, mean surface area was 2.7±2.4cm2. At the 12-week follow-up, the median surface area reduction was 82.7% (mean reduction 62.7 ± 49.9%) and in 10 of the 33 analysed patients (30%) the wound was healed. Only two of the seven documented local adverse events were deemed to be dressing related. According to the nursing staff, acceptability was considered very satisfactory, particularly in term of conformability and ease of use. CONCLUSION: This pilot study indicates that use of the new UrgoStart Contact dressing, combined with offloading and debridement,may help promote the healing process of the neuropathic diabetic foot ulcers, and was well tolerated and accepted.

Treatment of acute Charcot foot with bisphosphonates: a systematic review of the literature.

AIM/HYPOTHESIS: We undertook a systematic review of the literature concerning the efficacy and safety of bisphosphonates in acute Charcot neuropathic osteoarthropathy. METHODS: MEDLINE, PubMed, the Cochrane Database of Systematic Reviews, and abstracts presented during the meetings of the American Diabetes Association and the European Association of Diabetes were searched for relevant publications from the period January 1990 to September 2011. RESULTS: A total of ten studies on the treatment of acute Charcot osteoarthropathy with bisphosphonates were identified and included in the analysis. Only four clinical trials were published, three of which were randomised. Bisphosphonates appeared to induce significant reductions in skin temperature and bone turnover markers compared with placebo, without serious adverse events. Nevertheless, bisphosphonates did not shorten the immobilisation time. Moreover, no data were available regarding their long-term effects. CONCLUSIONS/INTERPRETATIONS: Bisphosphonates have been shown to be effective for reducing bone turnover markers and skin temperature in some studies. Nevertheless, the long-term efficacy, specifically that regarding the occurrence of deformities and ulcerations, remains to be demonstrated as no follow-up studies have been published. Moreover, some studies have suggested that bisphosphonates may lengthen the resolution phase of the disease. In our opinion, the data are too weak to support the use of bisphosphonates as a routine treatment for acute Charcot neuroarthropathy.

Beneficial effects of implementing guidelines on microbiology and costs of infected diabetic foot ulcers.

AIMS/HYPOTHESIS: In 2003, guidelines for management of diabetic foot infection (DFI) were written by the authors' team according to the guidelines of the International Working Group on the Diabetic Foot. The effects of implementing these guidelines on the microbiology and costs of infected diabetic foot ulcers were assessed. METHODS: From 2003 to 2007, potential beneficial effects of implementing these guidelines were assessed by comparison over time of bacteriological data (number of bacterial samples, number of microorganisms isolated in cultures, prevalence of multidrug-resistant organisms [MDRO] and colonising flora), and costs related to use of antimicrobial agents and microbiology laboratory workload. RESULTS: The study included 405 consecutive diabetic patients referred to the Diabetic Foot Unit for a suspected DFI. From 2003 to 2007, a significant decrease was observed in the median number of bacteria species per sample (from 4.1 to 1.6), prevalence of MDRO (35.2% vs 16.3%) and methicillin-resistant Staphylococcus aureus (52.2% vs 18.9%) (p < 0.001). Moreover, prevalence of pathogens considered as colonisers dramatically fell from 23.1% to 5.8% of all isolates (p < 0.001). In parallel, implementation of guidelines was associated with a saving of euro14,914 (US$20,046) related to a reduced microbiology laboratory workload and euro109,305 (US$147,536) due to reduced prescription of extended-spectrum antibiotic agents. CONCLUSIONS/INTERPRETATION: Implementation of guidelines for obtaining specimens for culture from patients with DFI is cost-saving and provides interesting quality indicators in the global management of DFI.

[Epidemiology of diabetic foot problems]. / Epidémiologie du pied diabétique.

Since diabetes mellitus is growing at epidemic proportions worldwide, the prevalence of diabetes-related complications is bound to increase. Diabetic foot disorders, a major source of disability and morbidity, are a significant burden for the community and a true public health problem. Many epidemiological data have been published on the diabetic foot but they are difficult to interpret because of variability in the methodology and in the definitions used in these studies. Moreover, there is a lack of consistency in population characteristics (ethnicity, social level, accessibility to care) and how results are expressed. In westernized countries, two of 100 diabetic patients are estimated to suffer from a foot ulcer every year. Amputation rates vary considerably: incidence ranges from 1 per thousand in the Madrid area and in Japan to up to 20 per thousand in some Indian tribes in North America. In metropolitan France, the incidence of lower-limb amputation is approximately 2 per thousand but with marked regional differences, and in French overseas territories, the incidence rate is much higher. Nevertheless, the risk for ulceration and amputation is much higher in diabetics compared to the nondiabetic population: the lifetime risk of a diabetic individual developing an ulcer is as high as 25% and it is estimated that every 30s an amputation is performed for a diabetic somewhere in the world. As reviewed in this paper, peripheral neuropathy, arterial disease, and foot deformities are the main factors accounting for this increased risk. Age and sex as well as social and cultural status are contributing factors. Knowing these factors is essential to classify every diabetic using a risk grading system and to take preventive measures accordingly.

The Internet and the diabetic foot: quality of online information in French language.

UNLABELLED: The Internet has become a major source of health information for consumers. Nevertheless the quality of medical information varies widely and is generally poor. AIM: This study aimed to evaluate the quality of information delivered on French-speaking Internet about the diabetic foot. METHODS: Websites were selected using three popular search engines and introducing "foot+diabetes" and "foot+diabetic" as keywords. Two diabetologists independently evaluated the quality of information using a specially created scoring grid (range 0-52) based on acknowledged and published criteria with items relevant to general characteristics of the site and to information content. One hundred and twenty websites were selected but only 27 were included for analysis. RESULTS: Agreement between the two raters was close for global score and site content but lower for site characteristics. Averaged global score ranged from 8 to 44. Only five sites were assessed as very advisable with a score higher than 39; in contrast 18 sites were judged as not advisable at all (score lower than 26). CONCLUSION: This study, the first to be devoted to information about the diabetic foot on the Internet, clearly shows the variability and the general poor quality of information delivered by the great majority of French-speaking websites. Regulation organisms are urgently needed for checking and labelling public oriented health information in order to make the Internet a performing tool for patient information.

In vitro monomer swapping in EmrE, a multidrug transporter from Escherichia coli, reveals that the oligomer is the functional unit.

EmrE is a small multidrug transporter, 110 amino acids long that extrudes various drugs in exchange with protons, thereby rendering Escherichia coli cells resistant to these compounds. Negative dominance studies and radiolabeled substrate-binding studies suggested that EmrE functions as an oligomer. Projection structure of two-dimensional crystals of the protein revealed an asymmetric dimer. To identify the functional unit of EmrE, a novel approach was developed. In this method, quantitative monomer swapping is induced in detergent-solubilized EmrE by exposure to 80 degrees C, a treatment that does not impair transport activity. Oligomer formation is highly specific as judged by several criteria, among them the fact that (35)S-EmrE can be "pulled out" from a mixture prepared from generally labeled cells. Using this technique, we show that inactive mutant subunits are functionally complemented when mixed with wild type subunits. The hetero-oligomers thus formed display a decreased affinity to substrates. In addition, sulfhydryl reagents inhibit the above hetero-oligomer even though Cys residues are present only in the inactive monomer. It is concluded that, in EmrE, the oligomer is the functional unit.

Functional analysis of novel multidrug transporters from human pathogens.

Proteins of the Smr family are the smallest multidrug transporters, about 110 amino acids long, that extrude various drugs in exchange with protons, thereby rendering bacteria resistant to these compounds. One of these proteins, EmrE, is an Escherichia coli protein, which has been cloned based on its ability to confer resistance to ethidium and methyl viologen and which has been extensively characterized. More than 60 genes coding for Smr proteins have been identified in several bacteria based on amino acid sequence similarity to the emrE gene. In this work we have analyzed the sequence similarity among these homologues and identified some distinct signature sequence elements and several fully conserved residues. Five of these homologues, from human pathogens Mycobacterium tuberculosis, Bordetella pertussis, and Pseudomonas aeruginosa and from Escherichia coli, were cloned into an E. coli expression system. The proteins were further characterized and show varying degrees of methyl viologen uptake into proteoliposomes and [(3)H]TPP binding in solubilized membranes. The homologues can also form mixed oligomers with EmrE that exhibit intermediate binding characteristics. A comparative study of various homologous proteins provides a tool for deciphering structure-function relationship and monomer-monomer interaction in multidrug transporters and in membrane proteins in general.

Small is mighty: EmrE, a multidrug transporter as an experimental paradigm.

EmrE is a multidrug transporter from Escherichia coli that functions as a homooligomer and is unique in its small size. In each monomer there are four tightly packed transmembrane segments and one membrane-embedded charged residue. This residue provides the basis for the coupling mechanism as part of a binding site "time shared" by substrates and protons.

Vesicular monoamine transporters heterologously expressed in the yeast Saccharomyces cerevisiae display high-affinity tetrabenazine binding.

A mammalian vesicular neurotransmitter transporter has been expressed in the yeast Saccharomyces cerevisiae. The gene encoding the rat vesicular monoamine transporter (rVMAT(1)) was cloned in several expression plasmids. The transporter was expressed at detectable levels only when short sequences using codons favored by S. cerevisiae were fused preceding the start of translation of rVMAT(1). The scarce expression of the wild-type protein was, most likely, due to the fact that part of the N-terminus of the protein is encoded by codons not preferred in S. cerevisiae. Furthermore, low growth temperatures increased rVMAT(1) expression and altered its processing. Whereas at 30 degrees C the protein is not glycosylated, at lower temperatures ( approximately 16 degrees C) half of the expressed transporters undergo core glycosylation. In addition, under these conditions the levels of protein expression significantly increase. Using a functional chimeric protein composed by VMAT and the green fluorescent protein (GFP), it is shown that the punctate pattern of intracellular distribution remains invariable at the different temperatures. Using a similar fusion sequence, the bovine VMAT isoform 2 (bVMAT(2)) was also expressed in yeast. The yeast-expressed bVMAT(2) binds [(3)H]dihydrotetrabenazine ([(3)H]TBZOH) with the same characteristics found in the native protein from bovine chromaffin granules. Dodecyl maltoside-solubilized bVMAT(2) retains the conformation required for [(3)H]TBZOH binding. We exploited the robust binding to follow the transporter during purification assays on a Ni(2+)-chelating column. In this report we describe for the first time the heterologous expression of a neurotransmitter transporter in the yeast S. cerevisiae.

Precious things come in little packages.

The 110-amino acid multidrug transporter from E. coli, EmrE, is a member of the family of MiniTexan or Smr drug transporters. EmrE can transport acriflavine, ethidium bromide, tetraphenylphosphonium (TPP+), benzalkonium and several other drugs with relatively high affinities. EmrE is an H+/drug antiporter, utilizing the proton electrochemical gradient generated across the bacterial cytoplasmic membrane by exchanging two protons with one substrate molecule. The EmrE multidrug transporter is unique in its small size and hydrophobic nature. Hydropathic analysis of the EmrE sequence predicts four alpha-helical transmembrane segments. This model is experimentally supported by FTIR studies that confirm the high alpha-helicity of the protein and by high-resolution heteronuclear NMR analysis of the protein structure. The TMS of EmrE are tightly packed in the membrane without any continuous aqueous domain, as was shown by Cysteine scanning experiments. These results suggest the existence of a hydrophobic pathway through which the substrates are translocated. EmrE is functional as a homo-oligomer as suggested by several lines of evidence, including co-reconstitution experiments of wild-type protein with inactive mutants in which negative dominance has been observed. EmrE has only one membrane embedded charged residue, Glu-14, that is conserved in more than fifty homologous proteins and it is a simple model system to study the role of carboxylic residues in ion-coupled transporters. We have used mutagenesis and chemical modification to show that Glu-14 is part of the substrate-binding site. Its role in proton binding and translocation was shown by a study of the effect of pH on ligand binding, uptake, efflux and exchange reactions. We conclude that Glu-14 is an essential part of a binding site, common to substrates and protons. The occupancy of this site is mutually exclusive and provides the basis of the simplest coupling of two fluxes. Because of some of its properties and its size, EmrE provides a unique system to understand mechanisms of substrate recognition and translocation.

A single carboxyl mutant of the multidrug transporter EmrE is fully functional.

EmrE, a multidrug transporter from Escherichia coli removes toxic compounds from the cell in exchange with protons. Glu-14 is the only charged residue in the putative membrane domains and is fully conserved in more than 50 homologues of the protein. This residue was shown to be an essential part of the binding site, common to protons and substrate. EmrE bearing a single carboxylic residue, Glu-14, shows uptake and binding properties similar to those of the wild type. This suggests that a small protein bearing only 110 amino acids with a single carboxyl in position 14 is the most basic structure that shows ion-coupled transport activity. The role of Glu-14 in substrate binding was examined by using dicyclohexylcarbodiimide, a hydrophobic carbodiimide that is known to react with carboxyls. Tetraphenylphosphonium binding to both wild type and the single carboxyl mutant is inhibited by dicyclohexylcarbodiimide in a dose-dependent manner. Ethidium and other substrates of EmrE prevent this inhibition with an order of potency in accord with their apparent affinities. This suggests that dicyclohexylcarbodiimide binding is sterically prevented by the substrate, supporting the contention that Glu-14, the reactive residue, is part of the substrate-binding site.

The projection structure of EmrE, a proton-linked multidrug transporter from Escherichia coli, at 7 A resolution.

EmrE belongs to a family of eubacterial multidrug transporters that confer resistance to a wide variety of toxins by coupling the influx of protons to toxin extrusion. EmrE was purified and crystallized in two dimensions by reconstitution with dimyristoylphosphatidylcholine into lipid bilayers. Images of frozen hydrated crystals were collected by cryo-electron microscopy and a projection structure of EmrE was calculated to 7 A resolution. The projection map shows an asymmetric EmrE dimer with overall dimensions approximately 31 x 40 A, comprising an arc of highly tilted helices separating two helices nearly perpendicular to the membrane from another two helices, one tilted and the other nearly perpendicular. There is no obvious 2-fold symmetry axis perpendicular to the membrane within the dimer, suggesting that the monomers may have different structures in the functional unit.

A model for coupling of H(+) and substrate fluxes based on "time-sharing" of a common binding site.

Both prokaryotic and eukaryotic cells contain an array of membrane transport systems maintaining the cellular homeostasis. Some of them (primary pumps) derive energy from redox reactions, ATP hydrolysis, or light absorption, whereas others (ion-coupled transporters) utilize ion electrochemical gradients for active transport. Remarkable progress has been made in understanding the molecular mechanism of coupling in some of these systems. In many cases carboxylic residues are essential for either binding or coupling. Here we suggest a model for the molecular mechanism of coupling in EmrE, an Escherichia coli 12-kDa multidrug transporter. EmrE confers resistance to a variety of toxic cations by removing them from the cell interior in exchange for two protons. EmrE has only one membrane-embedded charged residue, Glu-14, which is conserved in more than 50 homologous proteins. We have used mutagenesis and chemical modification to show that Glu-14 is part of the substrate-binding site. Its role in proton binding and translocation was shown by a study of the effect of pH on ligand binding, uptake, efflux, and exchange reactions. The studies suggest that Glu-14 is an essential part of a binding site, which is common to substrates and protons. The occupancy of this site by H(+) and substrate is mutually exclusive and provides the basis of the simplest coupling for two fluxes.

31P-CP-MAS NMR studies on TPP+ bound to the ion-coupled multidrug transport protein EmrE.

The binding of tetraphenylphosphonium (TPP+) to EmrE, a membrane-bound, 110 residue Escherichia coli multidrug transport protein, has been observed by 31P cross-polarisation-magic-angle spinning nuclear magnetic resonance spectroscopy (CP-MAS NMR). EmrE has been reconstituted into dimyristoyl phosphatidylcholine bilayers. CP-MAS could selectively distinguish binding of TPP+ to EmrE in the fluid membrane. A population of bound ligand appears shifted 4 ppm to lower frequency compared to free ligand in solution, which suggests a rather direct and specific type of interaction of the ligand with the protein. This is also supported by the observed restricted motion of the bound ligand. The observation of another weakly bound substrate population arises from ligand binding to negatively charged residues in the protein loop regions.

A common binding site for substrates and protons in EmrE, an ion-coupled multidrug transporter.

EmrE is an Escherichia coli 12-kDa multidrug transporter, which confers resistance to a variety of toxic cations by removing them from the cell interior in exchange with two protons. EmrE has only one membrane-embedded charged residue, Glu-14, that is conserved in more than 50 homologous proteins and it is a simple model system to study the role of carboxylic residues in ion-coupled transporters. We have used mutagenesis and chemical modification to show that Glu-14 is part of the substrate binding site. Its role in proton binding and translocation was shown by a study of the effect of pH on ligand binding, uptake, efflux and exchange reactions. We conclude that Glu-14 is an essential part of a binding site, common to substrates and protons. The occupancy of this site is mutually exclusive and provides the basis of the simplest coupling of two fluxes.

An essential glutamyl residue in EmrE, a multidrug antiporter from Escherichia coli.

EmrE is an Escherichia coli 12-kDa protein that confers resistance to toxic compounds, by actively removing them in exchange with protons. The protein includes eight charged residues. Seven of these residues are located in the hydrophilic loops and can be replaced with either Cys or another amino acid bearing the same charge, without impairing transport activity. Glu-14 is the only charged residue in the membrane domain and is conserved in all the proteins of the family. We show here that this residue is the site of action of dicyclohexylcarbodiimide, a carbodiimide known to act in hydrophobic environments. When Glu-14 was replaced with either Cys or Asp, resistance was abolished. Whereas the E14C mutant displays no transport activity, the E14D protein shows efflux and exchange at rates about 30-50% that of the wild type. The maximal DeltapH-driven uptake rate of E14D is only 10% that of the wild type. The mutant shows a different pH profile in all the transport modes. Our results support the notion that Glu-14 is an essential part of a binding domain shared by substrates and protons but mutually exclusive in time. This notion provides the molecular basis for the obligatory exchange catalyzed by EmrE.

A membrane-embedded glutamate is required for ligand binding to the multidrug transporter EmrE.

EmrE is an Escherichia coli multidrug transporter that confers resistance to a variety of toxins by removing them in exchange for hydrogen ions. The detergent-solubilized protein binds tetraphenylphosphonium (TPP(+)) with a K(D) of 10 nM. One mole of ligand is bound per approximately 3 mol of EmrE, suggesting that there is one binding site per trimer. The steep pH dependence of binding suggests that one or more residues, with an apparent pK of approximately 7.5, release protons prior to ligand binding. A conservative Asp replacement (E14D) at position 14 of the only membrane-embedded charged residue shows little transport activity, but binds TPP(+) at levels similar to those of the wild-type protein. The apparent pK of the Asp shifts to <5.0. The data are consistent with a mechanism requiring Glu14 for both substrate and proton recognition. We propose a model in which two of the three Glu14s in the postulated trimeric EmrE homooligomer deprotonate upon ligand binding. The ligand is released on the other face of the membrane after binding of protons to Glu14.