Antimicrobial Wound Dressings against Fluorescent and Methicillin-Sensitive Intracellular Pathogenic Bacteria.

There is limited evidence indicating that drug-eluting dressings are clinically more effective than simple conventional dressings. To shed light on this concern, we have performed evidence-based research to evaluate the antimicrobial action of thymol (THY)-loaded antimicrobial dressings having antibiofilm forming ability, able to eradicate intracellular and extracellular pathogenic bacteria. We have used four different Staphylococcus aureus strains, including the ATCC 25923 strain, the Newman strain (methicillin-sensitive strain, MSSA) expressing the coral green fluorescent protein from the vector pCN47, and two clinical reference strains, Newman-(MSSA) and USA300-(methicillin-resistant strain), as traceable models of pathogenic bacteria commonly infecting skin and soft tissues. Compared to non-loaded dressings, THY-loaded polycaprolactone-based electrospun dressings were also able to eliminate pathogenic bacteria in coculture models based on infected murine macrophages. In addition, by using confocal microscopy and the conventional microdilution plating method, we corroborated the successful ability of THY in preventing also biofilm formation. Herein, we demonstrated that the use of wound dressings loaded with the natural monoterpenoid phenol derivative THY are able to eliminate biofilm formation and intracellular methicillin-sensitive S aureus more efficiently than with their corresponding THY-free counterparts.

Chitosan derivatives co-delivering nitric oxide and methicillin for the effective therapy to the methicillin-resistant S. aureus infection.

We developed a co-delivery system of nitric oxide (NO) and antibiotic for the antibiotic-resistant bacterial infection therapy. The NO could disperse the bacterial biofilms and convert the bacteria into an antibiotic-susceptible planktonic form. Using the chitosan-graft-poly(amidoamine) dendrimer (CS-PAMAM) as the co-delivery system, methicillin (MET) and NO were conjugated successively to form CS-PAMAM-MET/NONOate. The positive CS-PAMAM could efficiently capture the negatively charged bacteria and PAMAM provide abundant reaction points for high payloads of NO and MET. The CS-PAMAM-MET/NONOate displayed effective and combined antibacterial activity to the E. coli and S. aureus. Particularly, for the MET-resistant S. aureus (MRSA), the CS-PAMAM-MET/NONOate displayed the synergistic antibacterial activity. In vivo wound healing assays also confirmed that CS-PAMAM-MET/NONOate could heal the infection formed by MRSA and then accelerate the wound healing effectively. Moreover, CS-PAMAM-MET/NONOate showed no toxicity towards 3T3 cells in vitro and rats in vivo, providing a readily but high-efficient strategy to drug-resistant bacterial infection therapy.

Lysozyme-Assisted Photothermal Eradication of Methicillin-Resistant Staphylococcus aureus Infection and Accelerated Tissue Repair with Natural Melanosome Nanostructures.

Patients often face the challenge of antibiotic-resistant bacterial infections and lengthy tissue reconstruction after surgery. Herein, human hair-melanosome derivatives (HHMs), comprising keratins and melanins, are developed using a simple "low-temperature alkali heat" method for potentially personalized therapy. The mulberry-shaped HHMs have an average width of ∼270 nm and an average length of ∼700 nm, and the negatively charged HHMs can absorb positively charged Lysozyme (Lyso) to form the HHMs-Lyso composites through electrostatic interaction. These naturally derived biodegradable nanostructures act as exogenous killers to eliminate methicillin-resistant Staphylococcus aureus (MRSA) infection with a high antibacterial efficacy (97.19 ± 2.39%) by synergistic action of photothermy and "Lyso-assisted anti-infection" in vivo. Additionally, HHMs also serve as endogenous regulators of collagen alpha chain proteins through the "protein digestion and absorption" signaling pathway to promote tissue reconstruction, which was confirmed by quantitative proteomic analysis in vivo. Notably, the 13 upregulated collagen alpha chain proteins in the extracellular matrix (ECM) after HHMs treatment demonstrated that keratin from HHMs in collagen-dependent regulatory processes serves as a notable contributor to augmented wound closure. The current paradigm of natural material-tissue interaction regulates the cell-ECM interaction by targeting cell signaling pathways to accelerate tissue repair. This work may provide insight into the protein-level pathways and the potential mechanisms involved in tissue repair.

Biosynthesized silver nanoparticles for inhibition of antibacterial resistance and biofilm formation of methicillin-resistant coagulase negative Staphylococci.

The ability of a natural stabilizing and reducing agent on the synthesis of silver nanoparticles (Ag NPs) was explored using a rapid and single-pot biological reduction method using Nocardiopsis sp. GRG1 (KT235640) biomass. The UV-visible spectral analysis of Ag NPs was found to show a maximum absorption peak located at a wavelength position of ∼422 nm for initial conformation. The major peaks in the XRD pattern were found to be in excellent agreement with the standard values of metallic Ag NPs. No other peaks of impurity phases were observed. The morphology of Ag NPs was confirmed through TEM observation, demonstrating that the particle size distribution of Ag NPs entrenched in spherical particles is in a range between 20 and 50 nm. AFM analysis further supported the nanosized morphology of the synthesized Ag NPs and allowed quantifying the Ag NPs surface roughness. The synthesized Ag NPs showed significant antibacterial and antibiofilm activity against biofilm positive methicillin-resistant coagulase negative Staphylococci (MR-CoNS), which were isolated from urinary tract infection as determined by spectroscopic methods in the concentration range of 5-60 µg/ml. The inhibition of biofilm formation with coloring stain was morphologically imaged by confocal laser scanning microscopy (CLSM). Morphological alteration of treated bacteria was observed by SEM analysis. The results clearly indicate that these biologically synthesized Ag NPs could provide a safer alternative to conventional antibiofilm agents against uropathogen of MR-CoNS.

N-(1,3,4-oxadiazol-2-yl)benzamide analogs, bacteriostatic agents against methicillin- and vancomycin-resistant bacteria.

Various reports of multidrug-resistant bacteria that are immune to all available FDA-approved drugs demand the development of novel chemical scaffolds as antibiotics. From screening a chemical library, we identified compounds with antibacterial activity. The most potent compounds, F6-5 and F6, inhibited growth of various drug-resistant Gram-positive bacterial pathogens at concentrations ranging from 1 µg/mL to 2 µg/mL. Both compounds were active against clinical isolates of methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-intermediate and vancomycin-resistant S. aureus (VISA and VRSA respectively) and vancomycin-resistant Enterococcus faecalis (VRE). Resistance generation experiments revealed that MRSA could develop resistance to the antibiotic ciprofloxacin but not to F6. Excitingly, F6 was found to be non-toxic against mammalian cells. In a mouse skin wound infection model, F6 was equipotent to the antibiotic fusidic acid in reducing MRSA burden.

[Molecular basis of methicillin-resistance in Staphylococcus aureus]. / Bases moleculares de la resistencia a meticilina en Staphylococcus aureus.

Staphylococcus aureus isolates resistant to several antimicrobials have been gradually emerged since the beginning of the antibiotic era. Consequently, the first isolation of methicillin-resistant S. aureus occurred in 1960, which was described a few years later in Chile. Currently, S. aureus resistant to antistaphylococcal penicillins is endemic in Chilean hospitals and worldwide, being responsible for a high burden of morbidity and mortality. This resistance is mediated by the expression of a new transpeptidase, named PBP2a or PBP2', which possesses lower affinity for the ß-lactam antibiotics, allowing the synthesis of peptidoglycan even in presence of these antimicrobial agents. This new enzyme is encoded by the mecA gene, itself embedded in a chromosomal cassette displaying a genomic island structure, of which there are several types and subtypes. Methicillin resistance is mainly regulated by an induction mechanism activated in the presence of ß-lactams, through a membrane receptor and a repressor of the gene expression. Although mec-independent methicillin resistance mechanisms have been described, they are clearly infrequent.

Bases moleculares de la resistencia a meticilina en Staphylococcus aureus / Molecular basis of methicillin-resistance in Staphylococcus aureus

Resumen Desde el inicio de la era antimicrobiana se han ido seleccionando gradualmente cepas de Staphylococcus aureus resistentes a antimicrobianos de amplio uso clínico. Es así como en 1960 se describen en Inglaterra las primeras cepas resistentes a meticilina, y algunos años después son informadas en hospitales de Chile. Actualmente, S. aureus resistente a penicilinas antiestafilocóccicas es endémico en los hospitales de nuestro país y del mundo, siendo responsable de una alta morbimortalidad. La resistencia es mediada habitualmente por la síntesis de una nueva transpeptidasa, denominada PBP2a o PBP2' que posee menos afinidad por el β-lactámico, y es la que mantiene la síntesis de peptidoglicano en presencia del antimicrobiano. Esta nueva enzima se encuentra codificada en el gen mecA, a su vez inserto en un cassette cromosomal con estructura de isla genómica, de los cuales existen varios tipos y subtipos. La resistencia a meticilina se encuentra regulada, principalmente, por un mecanismo de inducción de la expresión del gen en presencia del β-lactámico, a través de un receptor de membrana y un represor de la expresión. Si bien se han descrito mecanismos generadores de resistencia a meticilina mec independientes, son categóricamente menos frecuentes.

Staphylococcus aureus isolates resistant to several antimicrobials have been gradually emerged since the beginning of the antibiotic era. Consequently, the first isolation of methicillin-resistant S. aureus occurred in 1960, which was described a few years later in Chile. Currently, S. aureus resistant to antistaphylococcal penicillins is endemic in Chilean hospitals and worldwide, being responsible for a high burden of morbidity and mortality. This resistance is mediated by the expression of a new transpeptidase, named PBP2a or PBP2', which possesses lower affinity for the β-lactam antibiotics, allowing the synthesis of peptidoglycan even in presence of these antimicrobial agents. This new enzyme is encoded by the mecA gene, itself embedded in a chromosomal cassette displaying a genomic island structure, of which there are several types and subtypes. Methicillin resistance is mainly regulated by an induction mechanism activated in the presence of β-lactams, through a membrane receptor and a repressor of the gene expression. Although mec-independent methicillin resistance mechanisms have been described, they are clearly infrequent.

Cold plasma inactivation of chronic wound bacteria.

Cold plasma is partly ionized non-thermal plasma generated at atmospheric pressure. It has been recognized as an alternative approach in medicine for sterilization of wounds, promotion of wound healing, topical treatment of skin diseases with microbial involvement and treatment of cancer. Cold plasma used in wound therapy inhibits microbes in chronic wound due to its antiseptic effects, while promoting healing by stimulation of cell proliferation and migration of wound relating skin cells. In this study, two types of plasma systems are employed to generate cold plasma: a parallel plate dielectric barrier discharge and a capillary-guided corona discharge. Parameters such as applied voltage, discharge frequency, treatment time and the flow of the carrier gas influence the cold plasma chemistry and therefore change the composition and concentration of plasma species that react with the target sample. Chronic wound that fails to heal often infected by multidrug resistant organisms makes them recalcitrant to healing. Methicillin-resistant Staphylococcus aureus (MRSA) and Pseudomonas aeruginosa (Pseudomonas aeruginosa) are two common bacteria in infected and clinically non-infected wounds. The efficacies of the cold plasma generated by the two designs on the inactivation of three different isolates of MRSA and four isolates of P. aeruginosa are reported here.

In Vitro Activity of Epigallocatechin Gallate and Quercetin Alone and in Combination versus Clinical Isolates of Methicillin-Resistant Staphylococcus aureus.

Topical infections can become life threatening in immunocompromised patients. However, fewer treatments are available as multi-drug-resistant bacteria become more common. The natural compounds epigallocatechin gallate (1) and quercetin (2) alone and in combination were tested as potential antimicrobial clinical therapies. Strong antimicrobial activity was produced by 1 alone against methicillin-resistant Staphylococcus aureus, and activity was significantly increased in the presence of 2. A synergistic interaction was observed between the two compounds. Kill kinetics indicate the combination is bactericidal over 24 h.

Methicillin-Resistant Staphylococcus aureus in Raw Milk: Prevalence, SCCmec Typing, Enterotoxin Characterization, and Antimicrobial Resistance Patterns.

Staphylococcus aureus is a known major cause of foodborne illnesses, and raw milk and dairy products are often contaminated by enterotoxigenic and antimicrobial-resistant S. aureus strains. In the present study, 35 S. aureus strains were isolated from 383 raw milk samples collected from various dairy herds in the province of Milan (northern Italy). The isolates were characterized based on their antimicrobial susceptibility patterns and the presence of genes encoding staphylococcal enterotoxins (sea, seb, sec, sed, and see). About half (45.7%) of the strains were enterotoxigenic, and 37.1% were resistant to at least one of the antimicrobial drugs tested. Seven (20%) of 35 isolates were identified as methicillin-resistant S. aureus (MRSA), and SCCmec typing performed with a multiplex PCR assay revealed the presence of gene cassettes IV and V, typical of community-acquired MRSA, and I and II, characteristic of health care-associated MRSA. The MRSA strains were evaluated for the presence of the Panton-Valentine leukocidin gene, but this gene was not found. The results of the present study revealed the presence of toxin-producing S. aureus and MRSA strains in raw milk. MRSA and enterotoxigenic S. aureus in dairy farms are an important risk factor for the spread of staphylococcal infections; therefore, further studies are needed to find strategies for monitoring and controlling the presence of S. aureus, especially MRSA, in dairy products.

Antimicrobial potential of phylogenetically unique actinomycete, Streptomyces sp. JRG-04 from marine origin.

Due to the emergence of severe infectious diseases and thriving antibiotic resistance, there is a need to explore microbial-derived bioactive secondary metabolites from unexplored regions. Present study deals with a mangrove estuary derived strain of Streptomyces sp. with potent antimicrobial activity against various pathogens, including methicillin resistant Staphylococcus aureus. Bioactive compound was effective even at low MIC level, damages the membrane of methicillin resistant S. aureus and causes cell death, however it has no cytotoxic effect on H9C2 cells. 16S rRNA shared 99.5% sequence similarity to Streptomyces longispororuber. Optimum biomass and antimicrobial compound production were observed in production medium supplemented with 1.0% maltose and 0.5% yeast extract. The active compound purified from the chloroform extract of the cell-free supernatant was studied by FT-IR, 1H NMR, 13C NMR and LC ESI-MS and identified as aromatic polyketide. ß-ketosynthase (KS) domain of the Streptomyces strain revealed 93.2% sequence similarity to the benzoisochromanequinone, an actinorhodin biosynthetic gene cluster of Streptomyces coelicolor A3(2). However, the region synthesizing the secondary metabolite produced by the S. longispororuber was not related to the KS domain of the strain, due to the phenomenon of horizontal gene transfer over the period of evolutionary process, thus generating metabolic compound diversity.

Staphylococcus aureus is a major pathogen in severe acute bacterial rhinosinusitis.

PURPOSE: The purpose of this study was to investigate the epidemiology and microbiology of severe acute bacterial rhinosinusitis in patients admitted to a Danish tertiary hospital. METHODS: A retrospective study was performed including all cases of acute rhinosinusitis admitted to the ENT-department of Aarhus University Hospital, in the period 2001 to 2011. RESULTS: In total, 143 cases of acute rhinosinusitis were identified of which 51% were children. The most prevalent bacterial strains were Staphylococcus aureus followed by group A streptococcus, Haemophilus influenzae and Streptococcus pneumonia. Morexella catarrhalis was only rarely cultured. Anerobes were not assessed routinely. Of all patients, 47.8% presented with orbital complications and 2.1% developed intracranial complications. Patients infected with group A streptococcus had significantly higher leukocyte and neutrophil counts than other patients. All cultured S. aureus were resistant to penicillin, but sensitive to methicillin. CONCLUSION: Our results imply that S. aureus and group A streptococcus are important pathogens in severe and complex cases of ABRS, in addition to the accepted pathogens S. pneumoniae and H. influenzae. On the contrary M. catarrhalis appears less significant. These findings have important implications regarding the selection of relevant treatment strategy in secondary care, which may currently be underestimating the role of S. aureus.

Computational studies of bacterial resistance to ß-lactam antibiotics: mechanism of covalent inhibition of the penicillin-binding protein 2a (PBP2a).

ß-Lactam resistance of methicillin-resistant Staphylococcus aureus (MRSA), a pathogenic bacterium that causes staph infections, represents a serious threat to public health. This arises primarily due to the inability of ß-lactam antibiotics to inhibit the transpeptidase activity of penicillin-binding protein 2a (PBP2a). Effective inhibition of PBP2a to prevent the bacterial cell wall biosynthesis is of great importance for the treatment of a variety of clinically challenging infectious diseases caused by MRSA. To gain fundamental insights into the mode of covalent inhibition of the enzyme, we have carried out computational studies of the acylation reactions between small ß-lactam molecules (methicilin and nitrocefin) and PBP2a using the B3LYP/6-31G* and ONIOM(B3LYP/6-31G*:AMBER) hybrid quantum mechanical/molecular mechanical methods. Our calculations show that the acylation involves two transition states and that methicilin and nitrocefin undergo acylation in slightly different manners. The acylation of nitrocefin is more facile, which is attributed to the larger release of ring strain and the larger resonance stabilization gained upon ring opening. We suggest that, in addition to the nonbonded interactions between the ligand and the protein, these quantum chemical factors, which are associated with efficiency of the acylation step, should be taken into account and carefully controlled in designing novel ß-lactam inhibitors of PBP2a.

Functional and morphological adaptation to peptidoglycan precursor alteration in Lactococcus lactis.

Cell wall peptidoglycan assembly is a tightly regulated process requiring the combined action of multienzyme complexes. In this study we provide direct evidence showing that substrate transformations occurring at the different stages of this process play a crucial role in the spatial and temporal coordination of the cell wall synthesis machinery. Peptidoglycan substrate alteration was investigated in the Gram-positive bacterium Lactococcus lactis by substituting the peptidoglycan precursor biosynthesis genes of this bacterium for those of the vancomycin-resistant bacterium Lactobacillus plantarum. A set of L. lactis mutant strains in which the normal d-Ala-ended precursors were partially or totally replaced by d-Lac-ended precursors was generated. Incorporation of the altered precursor into the cell wall induced morphological changes arising from a defect in cell elongation and cell separation. Structural analysis of the muropeptides confirmed that the activity of multiple enzymes involved in peptidoglycan synthesis was altered. Optimization of this altered pathway was necessary to increase the level of vancomycin resistance conferred by the utilization of d-Lac-ended peptidoglycan precursors in the mutant strains. The implications of these findings on the control of bacterial cell morphogenesis and the mechanisms of vancomycin resistance are discussed.

Synthesis and antibacterial activity of 5-deoxy-5-episubstituted arbekacin derivatives.

5-Deoxy-5-episubstituted arbekacin derivatives have been designed and efficiently synthesized. The synthetic compounds showed potent antibacterial activity against both Staphylococcus aureus, including methicillin-resistant S. aureus, and Pseudomonas aeruginosa. In particular, these derivatives were superior to arbekacin against MRSA strains expressing the bifunctional aminoglycoside-modifying enzyme AAC(6')-APH(2''). The antibacterial activity of the 5-deoxy-5-episubstituted arbekacin derivatives against Pseudomonas aeruginosa was markedly influenced by the efflux system of MexXY/OprM. The 6'-N-methyl derivative of the 5-epi arbekacin was effective against Pseudomonas aeruginosa expressing the aminoglycoside-modifying enzyme AAC(6').

Evaluation of mannitol salt agar, CHROMagar Staph aureus and CHROMagar MRSA for detection of meticillin-resistant Staphylococcus aureus from nasal swab specimens.

Mannitol salt agar (MSA), CHROMagar Staph aureus (CSA) and CHROMagar MRSA (CSA-MRSA) were evaluated with nasal surveillance specimens for their ability to detect Staphylococcus aureus and meticillin-resistant S. aureus (MRSA). CSA was found to be more sensitive than MSA in detecting S. aureus (98 versus 84.3 %; P=0.03). CSA and CSA-MRSA were equivalent in the ability to detect MRSA at 24 h (89.7 versus 87.2 %) and at 48 h (94.9 versus 94.9 %). When combined with Staphaurex slide confirmation testing, both CSA and CSA-MRSA were highly specific (100 %) media for detecting MRSA from nasal swab specimens.

Antibiotic resistance in exopolysaccharide-forming Staphylococcus epidermidis clinical isolates from orthopaedic implant infections.

The opportunistic pathogen Staphylococcus epidermidis is able to produce biofilm and to frequently cause implant infections. In recent years, it has also exhibited an increasing antimicrobial drug resistance. Here, the resistance to a panel of 16 different antibiotics in 342 clinical strains of S. epidermidis from orthopaedic implant infections has been investigated. The isolates were pheno- and genotyped for extracellular polysaccharide production, relevant to staphylococcal biofilm formation, in order to ascertain possible associations with antibiotic resistance. Approximately 10% of the isolates were found to be sensitive to all screened antibiotics. In all, 37-38% were resistant to beta-lactams such as oxacillin and imipenem, while the resistance to penicillin, ampicillin, cefazolin, cefamandole, was consistently observed in over 80% of the strains. Erythromycin- and clindamycin- resistant strains were approximately 41% and 16%, respectively. Of the isolates, 10% was resistant to chloramphenicol, 23% to sulfamethoxazole and 26% to ciprofloxacin. Resistance to vancomycin was never observed. Interestingly, exopolysaccharide-producing strains exhibited a significantly higher prevalence in the resistance to the four aminoglycosides (gentamicin, amikacin, netilmicin, tobramycin), to sulfamethoxazole and to ciprofloxacin with respect to non-producing isolates. Moreover, multiple resistance to antibiotics was more frequent among exopolysaccharide-forming strains.

A dynamic structure for the acyl-enzyme species of the antibiotic aztreonam with the Citrobacter freundii beta-lactamase revealed by infrared spectroscopy and molecular dynamics simulations.

Infrared difference spectra show that at least 4 conformations coexist for the ester carbonyl group of the stable acyl-enzyme species formed between the antibiotic aztreonam and the class C beta-lactamase from Citrobacter freundii. A novel method for the assignment of the bands that arise from the ester carbonyl group has been employed. This has made use of the finding that the infrared absorption intensity of aliphatic esters is surprisingly constant, so a direct comparison with simple model esters has been possible. This has allowed a clear distinction to be made between ester and amide (protein) absorptions. The polarity of the conformer environment varies from hexane-like to strongly hydrogen-bonded. We assume that the conformer with the lowest frequency (1,690 cm(-)(1)) and hence the strongest hydrogen-bonding is the singular conformer observed in the X-ray crystallographic structure, since a good interaction via two hydrogen bonds with the oxyanion hole is seen. Molecular dynamics simulation by the method of locally enhanced sampling revealed that the motion of the ester carbonyl of the acyl-enzyme species in and out of the oxyanion hole is facile. The simulation revealed two pathways for this motion that would go through intermediates that first break one or the other of the two hydrogen bonds to the oxyanion hole, prior to departure of the carbonyl moiety out of the active site. It is likely that such motion for the acyl-enzyme species might also occur with more typical beta-lactam substrates for beta-lactamases, but their detection in the more rapid time scale may prove a challenge.

Multiple conformations of the acylenzyme formed in the hydrolysis of methicillin by Citrobacter freundii beta-lactamase: a time-resolved FTIR spectroscopic study.

Time-resolved infrared difference spectroscopy has been used to show that the carbonyl group of the acylenzyme reaction intermediate in the Citrobacter freundii beta-lactamase-catalyzed hydrolysis of methicillin can assume at least four conformations. A single-turnover experiment shows that all four conformations decline during deacylation with essentially the same rate constant. The conformers are thus in exchange on the reaction time scale, assuming that deacylation takes place only from the conformation which is most strongly hydrogen bonded or from a more minor species not visible in these experiments. All conformers have the same (10 cm-1) narrow bandwidth compared with a model ethyl ester in deuterium oxide (37 cm-1) which shows that all conformers are well ordered relative to free solution. The polarity of the carbonyl group environment in the conformers varies from 'ether-like' to strongly hydrogen bonding (20 kJ/mol), presumably in the oxyanion hole of the enzyme. From the absorption intensities, it is estimated that the conformers are populated approximately proportional to the hydrogen bonding strength at the carbonyl oxygen. A change in the difference spectrum at 1628 cm-1 consistent with a perturbation (relaxation) of protein beta-sheet occurs slightly faster than deacylation. Consideration of chemical model reactions strongly suggests that neither enamine nor imine formation in the acyl group is a plausible explanation of the change seen at 1628 cm-1. A turnover reaction supports the above conclusions and shows that the conformational relaxation occurs as the substrate is exhausted and the acylenzymes decline. The observation of multiple conformers is discussed in relation to the poor specificity of methicillin as a substrate of this beta-lactamase and in terms of X-ray crystallographic structures of acylenzymes where multiple forms are not apparently observed (or modeled). Infrared spectroscopy has shown itself to be a useful method for assessment of the uniqueness of enzyme-substrate interactions in physiological turnover conditions as well as for determination of ordering, hydrogen bonding, and protein perturbation.