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.

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.

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.

Scanning cysteine accessibility of EmrE, an H+-coupled multidrug transporter from Escherichia coli, reveals a hydrophobic pathway for solutes.

EmrE is a 12-kDa Escherichia coli multidrug transporter that confers resistance to a wide variety of toxic reagents by actively removing them in exchange for hydrogen ions. The three native Cys residues in EmrE are inaccessible to N-ethylmaleimide (NEM) and a series of other sulfhydryls. In addition, each of the three residues can be replaced with Ser without significant loss of activity. A protein without all the three Cys residues (Cys-less) has been generated and shown to be functional. Using this Cys-less protein, we have now generated a series of 48 single Cys replacements throughout the protein. The majority of them (43) show transport activity as judged from the ability of the mutant proteins to confer resistance against toxic compounds and from in vitro analysis of their activity in proteoliposomes. Here we describe the use of these mutants to study the accessibility to NEM, a membrane permeant sulfhydryl reagent. The study has been done systematically so that in one transmembrane segment (TMS2) each single residue was replaced. In each of the other three transmembrane segments, at least four residues covering one turn of the helix were replaced. The results show that although the residues in putative hydrophilic loops readily react with NEM, none of the residues in putative transmembrane domains are accessible to the reagent. The results imply very tight packing of the protein without any continuous aqueous domain. Based on the findings described in this work, we conclude that in EmrE the substrates are translocated through a hydrophobic pathway.

Identification of residues in the translocation pathway of EmrE, a multidrug antiporter from Escherichia coli.

EmrE is a small, 12-kDa, highly polyspecific antiporter, which exchanges hydrogen ions with aromatic cations such as methyl viologen. EmrE-mediated transport is inhibited by the sulfhydryl-reactive reagent 4-(chloromercuri)benzoic acid (PCMB) but not by a variety of other sulfhydryl reagents. This differential effect is due to the fact that the organic mercurial is a substrate of the transporter and can reach domains otherwise inaccessible to the different reagents. To find out which of the three cysteine residues in EmrE is reacting with PCMB, each was replaced with serine and it was shown that none of them is essential for transport activity. A protein completely devoid of Cys residues (CL) is also capable of substrate accumulation albeit at a slower rate. Mutated proteins in which only one of the native cysteines was left whereas the other changed to serine were also constructed. The use of these proteins demonstrated that two of the three Cys in EmrE, Cys-41 and Cys-95, but not Cys-39, react with PCMB. A related mercurial, 4-(chloromercuri)benzenesulfonic acid (PCMBS), is only a very poor inhibitor, probably because of the negative charge it bears. PCMBS reacts with EmrE in an asymmetric and unique way. It reacts with the mutant bearing a single Cys residue in position 95 (CL-C95) only when the reagent is present in the outside face of the membrane and with the mutant CL-C41 only when allowed to permeate to the cell interior; as expected, it does not react with the mutant protein bearing a single Cys at position 39 (CL-C39). It is concluded that PCMB permeates through the substrate pathway of EmrE and covalently reacts with the two exposed residues, Cys-95 and Cys-41, but not with Cys-39, located on the opposite face of the helix relative to residue 41. In addition, because of the asymmetric reactivity to PCMBS, an inhibitor that does not permeate through the protein, it is concluded that Cys-41 is closer to the cytoplasmic face than Cys-95. The results demonstrate the existence of a domain accessible only to substrates and provide a unique tool for studying the substrate permeation pathway of an ion-coupled transporter.

Replacements of histidine 226 of NhaA-Na+/H+ antiporter of Escherichia coli. Cysteine (H226C) or serine (H226S) retain both normal activity and pH sensitivity, aspartate (H226D) shifts the pH profile toward basic pH, and alanine (H226A) inactivates the carrier at all pH values.

We have previously shown that replacement of His-226 in the NhaA Na+/H+ antiporter of Escherichia coli to Arg (H226R) shifts the pH profile of the antiporter toward acidic pH and as a result of delta nhaA delta nhaB strain bearing this mutation is Na+ sensitive at alkaline pH (Gerchman, Y., Olami, Y., Rimon, A., Taglicht, D., Schuldiner, S. and Padan, E. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 1212-1216). In the present work the role of His-226 in the response of NhaA to pH has been studied in detail. The Na+ sensitivity of the delta nhaA delta nhaB mutant bearing the H226R-NhaA plasmid at alkaline pH provided a very powerful tool to isolate revertants and suppressants of H226R growing on high Na+ at alkaline pH. With this approach cysteine (H226C) and serine (H226S) replacements were found to efficiently replace His-226 and yield an antiporter, which like the wild-type protein, is activated by pH between pH 7 and 8. These results imply that polarity and/or hydrogen bonding, the common properties shared by these amino acid residues, are essential at position 226 for pH regulation of NhaA. This suggestion was substantiated by site-directed mutagenesis of His-226 either to alanine (H226A) or aspartate (H226D). Whereas H226A-NhaA shows very low activity which is not activated by pH, H226D-NhaA is active and regulated by pH. The pH profile of H226D is shifted by half a pH unit toward alkaline pH, as opposed to the previously isolated mutant H226R which has a pH profile shift, to the same extent, but toward acidic pH. It is suggested that charge modifies the pH profile but is not essential for the pH regulation of NhaA.

Histidine-419 plays a role in energy coupling in the vesicular monoamine transporter from rat.

Vesicular monoamine transporters (VMAT) catalyze transport of serotonin, dopamine, epinephrine and norepinephrine into subcellular storage organelles in a variety of cells. Accumulation of the neurotransmitter depends on the proton electrochemical gradient across the organelle membrane and involves VMAT-mediated exchange of two lumenal protons with one cytoplasmic amine. It has been suggested in the past that His residues play a role in H+ movement or in its coupling to active transport in H(+)-symporters and antiporters. Indeed VMAT-mediated transport is inhibited by reagents specific for His residues. We have identified one His residue in VMAT1 from rat which is conserved in other vesicular neurotransmitter transporters. Mutagenesis of this His (H419) to either Arg or Cys completely inhibits [3H]serotonin and [3H]dopamine accumulation. Mutagenesis also inhibits other H(+)-dependent partial reactions of VMAT such as the acceleration of binding of the high affinity ligand reserpine, but does not inhibit the [3H]reserpine binding which is not dependent on H+ translocation. It is concluded that His-419 plays a role in energy coupling in r-VMAT1.

Kinetic properties of NhaB, a Na+/H+ antiporter from Escherichia coli.

NhaB, a Na+/H+ antiporter from Escherichia coli, was overproduced, purified, and reconstituted in a functional state, demonstrating that a single polypeptide, the product of the nhaB gene, can catalyze full activity. NhaB is a minor protein that accounts for less than 0.1% of the total membrane protein. The use of proteoliposomes made possible the determination of important kinetic and pharmacological properties in the absence of passive and mediated leaks. The activity of NhaB was found to have some pH dependence; the apparent Km for Na+ changes by 10-fold from 1.55 mM at pH 8.5 to 16.66 mM at pH 7.2, while the Vmax remains constant. It was demonstrated that NhaB is electrogenic and translocates more H+ than Na+ per cycle; the rate of sodium efflux from proteoliposomes was accelerated by a membrane potential, negative inside, and NhaB activity generated a membrane potential as monitored by two techniques. The stoichiometry of NhaB was estimated by a thermodynamic method in which the magnitude of delta psi generated by NhaB was measured at various Na+ gradients. A kinetic method, in which the electrophoretic movement of 86Rb+ (in the presence of valinomycin) was monitored in parallel with measurements of NhaB-mediated 22Na+ uptake, allowed us to determine a stoichiometry of 3H+/2Na+. The significance of the existence of two antiporters with different stoichiometries, NhaA and NhaB, active in the same cell, is discussed.

Homology of a vesicular amine transporter to a gene conferring resistance to 1-methyl-4-phenylpyridinium.

The vesicular amine transporter (VAT) catalyzes transport and storage of catechol and indolamines into subcellular organelles in a wide variety of cells. It plays a central role in neurotransmission and is the primary target for several pharmacological agents. One of the drugs, reserpine, binds very tightly to the transporter and remains bound even after solubilization, a finding that has proven useful for purification of the transporter from bovine adrenal medulla in a fully functional state. The sequences of 26 N-terminal amino acids and of an additional 7-amino acid internal peptide are presented. Antibodies against a synthetic peptide based on the above sequences immunoprecipitate the transporter, confirming the conclusion that the peptide sequence is derived from bovine VAT. To our knowledge, documentation of sequences of vesicular neurotransmitter transporters has not been presented previously. In addition, the sequences obtained are highly homologous to the predicted sequence of a protein from PC12 cells that confers to Chinese hamster ovary cells resistance to 1-methyl-4-phenylpyridinium (MPP+), an agent that causes parkinsonism in model systems, confirming the hypothesis that the protein conferring resistance to MPP+ is a VAT.

Cloning, sequencing and expression of the nhaA and nhaR genes from Salmonella enteritidis.

Na+/H+ antiporter activity is wide-spread and plays essential physiological roles. We found that several Enterobacteriaceae share conserved sequences with nhaA, the gene coding for an E. coli antiporter. A delta nhaA strain, which is sensitive to Na+ and Li+, was used to clone by complementation a DNA fragment from Salmonella enteritidis which confers resistance to the ions. The cloned fragment increased Na+/H+ antiport activity in membranes isolated from strains carrying the respective hybrid plasmid. DNA sequence analysis of the insert revealed two open reading frames. Both encode putative polypeptides which are closely homologous to the nhaA and nhaR gene products from Escherichia coli. The antiporter activity displays properties very similar to that of the E. coli NhaA, namely, it is activated by alkaline pH and recognizes Li+ with high affinity.

Covalent modification of the amine transporter with N,N'-dicyclohexylcarbodiimide.

N,N'-Dicyclohexylcarbodiimide (DCC) has been previously shown to inhibit the amine transporter from chromaffin granules [Gasnier, B., Scherman, D., & Henry, J.P. (1985) Biochemistry 24, 3660-3667]. A study of the mechanism of inhibition is presented together with the demonstration of covalent modification of the protein. DCC inhibits binding of R1 (reserpine) and R2 (tetrabenazine) types of ligands to the transporter as well as transport. Ligands of the R2 type, but not those of the R1 type, protect against inhibition of all the reactions by DCC, i.e., accumulation of serotonin, binding if reserpine (R1 ligand), and binding of ketanserine (R2 ligand). The ability of a given R2 ligand to protect the transporter correlates well with its binding constant. Water-soluble carbodiimides, such as 1-ethyl-3-[3-(diethylamino)propyl]carbodiimide (EDC), do not have any effect on the catalytic activity of the transporter. A fluorescent hydrophobic analogue of DCC, N-cyclohexyl-N'-[4-(dimethylamino)-alpha-naphthyl]carbodiimide (NCD-4), inhibits at about the same concentration range as DCC. [14C]DCC labels several polypeptides in the chromaffin granule membranes. Labeling of a polypeptide with an apparent Mr of 80K is inhibited in the presence of R2 ligands. The labeled polypeptide copurifies with the recently identified and isolated transporter [Stern-Bach, Y., Greenberg-Ofrath, N., Flechner, I., & Schuldiner, S. (1990) J. Biol. Chem. 256, 3961-3966].

Identification and purification of a functional amine transporter from bovine chromaffin granules.

The amine transporter from bovine chromaffin granules has been purified in a functional state. Two isoforms with different pI values have been separated and shown to be active. One with an unusually acidic pI (approximately 3.5) has been shown to be a glycoprotein with an apparent Mr of 80,000. The purified polypeptide catalyzes transport of serotonin upon reconstitution with an apparent Km of 2 microM and a Vmax of 140 nmol/mg/min, 150-200-fold higher than the one determined in the native system. Transport is inhibited by reserpine and tetrabenazine, ligands which bind to two distinct sites on the transporter. These findings suggest that the binding sites for both drugs reside on a single polypeptide. The reconstituted purified transporter binds [3H]reserpine with a biphasic kinetic behavior, KD values of 0.3 and 30 nM and Bmax of 310 and 4200 pmol/mg protein, respectively. In addition, binding of [3H]reserpine is accelerated upon imposition of a pH gradient across the proteoliposome. From these findings it is evident that a single polypeptide catalyzes the various functions of the transporter.

Energetics of reserpine binding and occlusion by the chromaffin granule biogenic amine transporter.

The energetics of reserpine binding to the bovine adrenal biogenic amine transporter suggest that H+ ion translocation converts the transporter to a form which binds reserpine essentially irreversibly. Reserpine binding to bovine adrenal chromaffin granule membrane vesicles is accelerated by generation of a transmembrane pH difference (delta pH) (interior acid) or electrical potential (delta psi) (interior positive). Both components of the electrochemical H+ potential (delta mu H+) must be dissipated to block reserpine binding, and generation of either one stimulates the binding rate. Reserpine binding is less dependent than amine transport on the delta pH, suggesting that translocation of fewer H+ ions is required to expose the high-affinity site than are required for net transport. Bound reserpine dissociates very slowly, if at all, from the transporter. Binding is stable to 1% cholate, 1.5% Triton X-100, 1 M SCN-, and 8 M urea, but sodium dodecyl sulfate (0.035%) and high temperatures (100 degrees C) released bound reserpine, indicating that binding is noncovalent. The results raise the possibility that the transporter, by translocating one H+ ion outward down its concentration gradient, is converted to a form that can either transport a neutral substrate molecule inward or occlude reserpine in a dead-end complex.

Volume regulation in Mycoplasma gallisepticum: evidence that Na+ is extruded via a primary Na+ pump.

The primary extrusion of Na+ from Mycoplasma gallisepticum cells was demonstrated by showing that when Na+-loaded cells were incubated with both glucose (10 mM) and the uncoupler SF6847 (0.4 microM), rapid acidification of the cell interior occurred, resulting in the quenching of acridine orange fluorescence. No acidification was obtained with Na+-depleted cells or with cells loaded with either KCl, RbCl, LiCl, or CsCl. Acidification was inhibited by dicyclohexylcarbodiimide (50 microM) and diethylstilbesterol (50 microM), but not by vanadate (100 microM). By collapsing delta chi with tetraphenylphosphonium (200 microM) or KCl (25 mM), the fluorescence was dequenched. The results are consistent with a delta chi-driven uncoupler-dependent proton gradient generated by an electrogenic ion pump specific for Na+. The ATPase activity of M. gallisepticum membranes was found to be Mg2+ dependent over the entire pH range tested (5.5 to 9.5). Na+ (greater than 10 mM) caused a threefold increase in the ATPase activity at pH 8.5, but had only a small effect at pH 5.5. In an Na+-free medium, the enzyme exhibited a pH optimum of 7.0 to 7.5, with a specific activity of 30 +/- 5 mumol of phosphate released per h per mg of membrane protein. In the presence of Na+, the optimum pH was between 8.5 and 9.0, with a specific activity of 52 +/- 6 mumol. The Na+-stimulated ATPase activity at pH 8.5 was much more stable to prolonged storage than the Na+-independent activity. Further evidence that two distinct ATPases exist was obtained by showing that M. gallisepticum membranes possess a 52-kilodalton (kDa) protein that reacts with antibodies raised against the beta-subunit of Escherichia coli ATPase as well as a 68-kDa protein that reacts with the anti-yeast plasma membrane ATPases antibodies. It is postulated that the Na+ -stimulated ATPases functions as the electrogenic Na+ pump.

Osmotic swelling allows fusion of sendai virions with membranes of desialyzed erythrocytes and chromaffin granules.

Sendai virus particles are able to fuse with Pronase-neuraminidase-treated human erythrocyte membranes as well as with vesicles obtained from chromaffin granules of bovine medulla. Fusion is inferred either from electron microscopic studies or from the observation that incubation of fluorescently labeled (bearing octadecyl Rhodamine B chloride) virions, with right-side-out erythrocyte vesicles (ROV) or with chromaffin granule membrane vesicles (CGMV), resulted in fluorescence dequenching. Fusion of Sendai virions with virus receptor depleted ROV was observed only under hypotonic conditions. Fusion with virus receptor depleted ROV required the presence of the two viral envelope glycoproteins, namely, the HN and F polypeptides. A 3-fold increase in the degree of fluorescence dequenching (virus-membrane fusion) was also obtained upon incubation of Sendai virions with CGMV in medium of low osmotic strength. This increase was not observed with inactivated, unfusogenic Sendai virions. The results of the present work demonstrate that, under hypotonic conditions, fusion between Sendai virions and biological membranes does not require the presence of specific receptors. Such fusion is characterized by the same features as fusion with and infection by Sendai virions of living cultured cells.

Control of sodium fluxes in Mycoplasma gallisepticum.

Swelling of Mycoplasma gallisepticum cells when incubated in a glucose-free isoosmotic NaCl buffer was shown to be due to the entrance of NaCl into the cell. Volume regulation therefore depends on Na+ extrusion. The mechanism of Na+ extrusion in cells and proteoliposomes, prepared from M. gallisepticum membrane fragments, was investigated by following both 22Na+ efflux and pH changes. Our results indicate that Na+ is expelled from cells via two separate mechanisms, an Na+/cation exchange mechanism and an Na+-ATPase. The possible association of these mechanisms with K+ accumulation is suggested.

The amine transporter from bovine chromaffin granules. Partial purification.

We have partially purified the amine transporter from bovine adrenal chromaffin granules in a single step utilizing affinity chromatography. A 5-hydroxytryptamine moiety has been coupled to a Sepharose 4B matrix in a position ortho to the hydroxyl group. When membranes solubilized with sodium cholate are chromatographed on the above matrix a 45,000 Mr polypeptide is highly enriched. The enrichment is dependent on the presence of the proper ligand on the matrix and is inhibited if the column is previously equilibrated with a soluble ligand. Enrichment of the above polypeptide is accompanied by an increase in the specific activity of the transporter as measured by its labeling by 4-azido-3-nitrophenylazo(5-hydroxytryptamine). The ability of reserpine, a competitive inhibitor of binding and transport, to inhibit labeling of the purified transporter correlates well with its known kinetic constants in the native membranes. The polypeptide purified is identical to the one previously identified as the putative transporter based on specific labeling by a photoaffinity label (Gabizon, R., Yetinzon, T., and Schuldiner, S. (1982) J. Biol. Chem. 257, 15145-15150). The results clearly support the contention that the 45,000 Mr peptide is the amine transporter or one of its subunits.

Functional asymmetry of the amine transporter from chromaffin granules.

The studies presented in this communication describe the existence of a pH-dependent kinetic barrier in amine translocation in membrane vesicles isolated from chromaffin granules from bovine adrenal medulla. This barrier prevents efflux of amines previously accumulated in the membrane vesicles. In these preparations, once the amine is accumulated, there is no further need for ATP. This would suggest a low permeability of the membrane, both to ions and to the amine. In addition, we show that under conditions which thermodynamically favour efflux this is kinetically blocked. This block is due to a rapid protonation of the transporter in the interior of the vesicle. Net efflux can be induced by agents that bring upon an alkalinization of the internal pH such as ammonium salts or nigericin. On the other hand, the rate of exchange with extravesicular substrate is almost identical at the various pH values tested. The physiological implications of this mechanism are discussed.

Photoinactivation and identification of the biogenic amine transporter in chromaffin granules from bovine adrenal medulla.

We have synthesized a new derivative of 5-hydroxytryptamine, a substrate of the biogenic amine transporter in bovine chromaffin granules. This new compound, 4-azido,3-nitrophenyl-azo-(5-hydroxytryptamine), is a competitive, reversible, and specific inhibitor of the biogenic amine transporter in the dark (Ki = 2 microM)., Upon illumination, the inactivation of the transporter becomes irreversible and follows an apparent first order rate kinetics (KINAC = 4 microM). The transporter can be protected if other substrates are present during illumination. When [3H]4-azido,3-nitrophenyl-azo-(5-hydroxytryptamine) is used, preferential labeling of one polypeptide (Mr approximately 45,000) is observed in the light, provided no other substrates of the transporter are present during illumination. We have tentatively identified this polypeptide as the transporter itself or one of its subunits.