Development of Active Packaging Based on Agar-Agar Incorporated with Bacteriocin of Lactobacillus sakei.

In the search for new biodegradable materials and greater microbiological safety and stability of perishable food products, this study aimed to develop a bioplastic antibacterial film incorporating bacteriocin for application in commercial curd cheese and monitoring of microbiological stability. Films with good handling characteristics as well as physical, barrier, and mechanical properties were obtained. Regarding the antibacterial activity, the microbial reduction was demonstrated in a food matrix, obtaining a reduction of 3 logarithmic cycles for the group of coagulase positive staphylococci and from 1100 to <3.00 MPN/g in the analysis of thermotolerant coliforms. Therefore, the film presented food barrier characteristics with the external environment and adequate migration of the antibacterial compound to the product, contributing to the reduction of contamination of a food with high initial microbial load.

In vitro activity of dalbavancin and other anti-staphylococcal agents against infecting isolates of methicillin-resistant coagulase-negative staphylococci.

Introduction. Dalbavancin was approved in Europe in 2015 for skin and soft tissue infections.Hypothesis/Gap Statement. Data on methicillin-resistant coagulase-negative staphylococci (MR-CNS) dalbavancin susceptibility are scarce.Aim. To assess the susceptibility of MR-CNS to dalbavancin and other anti-staphylococcal agents.Methodology. A total of 443 MR-CNS clinical isolates from patients hospitalized in a Greek university hospital during a 2.5-year period (January 2018 to June 2020) were included. The MICs for vancomycin, teicoplanin, linezolid and daptomycin were investigated by Etest and the MIC for dalbavancin was determined according to European Committee on Antimicrobial Susceptibility Testing (EUCAST) guidelines in 196 isolates. The consumption of the aforementioned antimicrobials was calculated.Results. In total, 51 isolates were resistant to teicoplanin (11.5 %) and 211 (47.6 %) to linezolid; all were susceptible to vancomycin and daptomycin. Among 196 isolates tested, 32 (16.3 %) were resistant to dalbavancin. A significant increase of MIC during the study period was found for vancomycin, teicoplanin and daptomycin, while a decrease in linezolid's MIC was observed. Dalbavancin's MIC remained stable. No difference in consumption was observed among the studied anti-staphylococcal agents.Conclusion. An increase of vancomycin, teicoplanin and daptomycin MICs among MR-CNS was observed, whereas 47.6 % of isolates were non-susceptible to linezolid. Dalbavancin retains excellent potency against MR-CNS, even in the presence of non-susceptibility to other anti-staphylococcal antibiotics.

Evolutionary and Functional Analysis of Coagulase Positivity among the Staphylococci.

The bacterial genus Staphylococcus comprises a large group of pathogenic and nonpathogenic species associated with an array of host species. Staphylococci are differentiated into coagulase-positive or coagulase-negative groups based on the capacity to promote clotting of plasma, a phenotype historically associated with the ability to cause disease. However, the genetic basis of this important diagnostic and pathogenic trait across the genus has not been examined to date. Here, we selected 54 representative staphylococcal species and subspecies to examine coagulation of plasma derived from six representative host species. In total, 13 staphylococcal species mediated coagulation of plasma from at least one host species including one previously identified as coagulase negative (Staphylococcus condimenti). Comparative genomic analysis revealed that coagulase activity correlated with the presence of a gene (vwb) encoding the von Willebrand binding protein (vWbp) whereas only the Staphylococcus aureus complex contained a gene encoding staphylocoagulase (Coa), the classical mediator of coagulation. Importantly, S. aureus retained vwb-dependent coagulase activity in an S. aureus strain deleted for coa whereas deletion of vwb in Staphylococcus pseudintermedius resulted in loss of coagulase activity. Whole-genome-based phylogenetic reconstruction of the Staphylococcus genus revealed that the vwb gene has been acquired on at least four different occasions during the evolution of the Staphylococcus genus followed by allelic diversification via mutation and recombination. Allelic variants of vWbp from selected coagulase-positive staphylococci mediated coagulation in a host-dependent manner indicative of host-adaptive evolution. Taken together, we have determined the genetic and evolutionary basis of staphylococcal coagulation, revealing vWbp to be its archetypal determinant. IMPORTANCE The ability of some species of staphylococci to promote coagulation of plasma is a key pathogenic and diagnostic trait. Here, we provide a comprehensive analysis of the coagulase positivity of the staphylococci and its evolutionary genetic basis. We demonstrate that the von Willebrand binding protein rather than staphylocoagulase is the archetypal coagulation factor of the staphylococci and that the vwb gene has been acquired several times independently during the evolution of the staphylococci. Subsequently, vwb has undergone adaptive diversification to facilitate host-specific functionality. Our findings provide important insights into the evolution of pathogenicity among the staphylococci and the genetic basis for a defining diagnostic phenotype.

The road to success of coagulase-negative staphylococci: clinical significance of small colony variants and their pathogenic role in persistent infections.

Bacterial small colony variants represent an important aspect of bacterial variability. They are naturally occurring microbial subpopulations with distinctive phenotypic and pathogenic traits, reported for many clinically important bacteria. In clinical terms, SCVs tend to be associated with persistence in host cells and tissues and are less susceptible to antibiotics than their wild-type (WT) counterparts. The increased tendency of SCVs to reside intracellularly where they are protected against the host immune responses and antimicrobial drugs is one of the crucial aspects linking SCVs to recurrent or chronic infections, which are difficult to treat. An important aspect of the SCV ability to persist in the host is the quiescent metabolic state, reduced immune response and expression a changed pattern of virulence factors, including a reduced expression of exotoxins and an increased expression of adhesins facilitating host cell uptake. The purpose of this review is to describe in greater detail the currently available data regarding CoNS SCV and, in particular, their clinical significance and possible mechanisms by which SCVs contribute to the pathogenesis of the chronic infections. It should be emphasized that in spite of an increasing clinical significance of this group of staphylococci, the number of studies unraveling the mechanisms of CoNS SCVs formation and their impact on the course of the infectious process is still scarce, lagging behind the studies on S. aureus SCVs.

Improved CRISPR genome editing using small highly active and specific engineered RNA-guided nucleases.

Streptococcus pyogenes (Spy) Cas9 has potential as a component of gene therapeutics for incurable diseases. One of its limitations is its large size, which impedes its formulation and delivery in therapeutic applications. Smaller Cas9s are an alternative, but lack robust activity or specificity and frequently recognize longer PAMs. Here, we investigated four uncharacterized, smaller Cas9s and found three employing a "GG" dinucleotide PAM similar to SpyCas9. Protein engineering generated synthetic RNA-guided nucleases (sRGNs) with editing efficiencies and specificities exceeding even SpyCas9 in vitro and in human cell lines on disease-relevant targets. sRGN mRNA lipid nanoparticles displayed manufacturing advantages and high in vivo editing efficiency in the mouse liver. Finally, sRGNs, but not SpyCas9, could be packaged into all-in-one AAV particles with a gRNA and effected robust in vivo editing of non-human primate (NHP) retina photoreceptors. Human gene therapy efforts are expected to benefit from these improved alternatives to existing CRISPR nucleases.

Reverse Protonation of Buried Ion-Pairs in Staphylococcal Nuclease Mutants.

Although buried titratable residues in protein cavities are often of major functional importance, it is generally challenging to understand their properties such as the ionization state and factors of stabilization based on experimental studies alone. A specific set of examples involve buried Glu-Lys pairs in a series of variants of Staphylococcal nuclease, for which recent structural and thermodynamic studies appeared to suggest that both the stability and the ionization state of the buried Glu-Lys pair are sensitive to its orientation (i.e., Glu23-Lys36 vs Lys23-Glu36). To further clarify the situation, especially ionization states of the buried Glu-Lys pairs, we have conducted extensive molecular dynamics simulations and free energy computations. Microsecond molecular dynamics simulations show that the hydration level of the cavity depends on the orientation of the buried ion-pair therein as well as its ionization state; free energy simulations recapitulate the relative stability of Glu23-Lys36 (EK) vs Lys23-Glu36 (KE) mutants measured experimentally, although the difference is similar in magnitude regardless of the ionization state of the Glu-Lys pair. A complementary set of free energy simulations strongly suggests that, in contrast to the original suggestion in the experimental analysis, the Glu and Lys residues prefer to adopt their charge-neutral rather than the ionized states. This result is consistent with the low dielectric constant computed for water in the cavity, which makes it difficult for the protein cavity to stabilize a pair of charged Glu-Lys residues, even with water penetration. The current study highlights the role of free energy simulations in understanding the ionization state of buried titratable residues and the relevant energetic contributions, forming the basis for the rational design of buried charge networks in proteins.

Human skin microbiota-friendly lysostaphin.

Growing antibiotic resistance of bacteria is a burning problem of human and veterinary medicine. Expansion and introduction of novel microbicidal therapeutics is highly desirable. However, antibiotic treatment disturbs the balance of physiological microbiota by changing its qualitative and/or quantitative composition, resulting in a number of adverse effects that include secondary infections. Although such dysbiosis may be reversed by the treatment with probiotics, a more attractive alternative is the use of antibiotics that target only pathogens, while sparing the commensals. Here, we describe lysostaphin LSp222, an enzyme produced naturally by Staphylococcus pseudintermedius 222. LSp222 is highly effective against S. aureus, including its multi-drug resistant strains. Importantly, the inhibitory concentration for S. epidermidis, the predominant commensal in healthy human skin, is at least two orders of magnitude higher compared to S. aureus. Such significant therapeutic window makes LSp222 a microbiota-friendly antibacterial agent with a potential application in the treatment of S. aureus-driven skin infections.

Coagulase negative Staphylococcus bacteremia in hematopoietic stem cell transplant recipients: Clinical features and molecular characterization.

The purpose of our study was to investigate the epidemiology of coagulase negative staphylococci (CoNS) responsible for bacteremia in hematopoietic stem cell transplant (HSCT) recipients and to determine the prevalence and the genetic background of methicillin resistance. The prevalence of CoNS bacteremia was 7.4% (54/728), higher in allograft (10.7%) than in autograft (4.7%) recipients. A sepsis or a septic shock were observed in 9% of cases. No deaths were attributable to CoNS bacteremia. The methicillin resistance rate was 81%. All MR-CoNS, harbored mecA gene and 90% were typeable with SCCmec typing using PCR amplification. The SCCmec type IV was the most frequent (44%). Clonal dissemination of MR- Staphylococcus epidermidis strains was limited. Our study showed a low prevalence and favorable outcome of CoNS bacteremia in HSCT recipients with limited clonal diffusion. However, they were associated with a significant rate of severe infections and a high rate of methicillin resistance, mediated by SCCmec IV element in most cases.

The role of S126 in the Staphylococcus equorum MnSOD activity and stability.

The dimeric form of manganese superoxide dismutase is instrumental for activity because each of the monomers provides amino acid residues participating in the enzymatic reaction. Hence, preventing dissociation of the dimer would maintain the enzymatic activity in detrimental conditions e.g. high temperature. To prevent dissociation of the dimer, a disulphide (S-S) bond was introduced at the dimer interface. In the wild type structure, S126 interacts with S126 of the other monomer. In the presented work, a mutant was designed with an S126C substitution. The crystal structure of the S126C mutant showed that only 50-70% of monomers formed the S-S bond. This observed imperfect S-S bonding was likely caused by photolytic S-S bond breakage mediated by the neighbouring tryptophan residue. In the wild type, S126 is located facing W163 and forms a water-mediated hydrogen bond with E164; W163 and E164 are crucial in the enzyme's activity. The replacement of S126 by a cysteine residue lowered the activity of the enzyme by ~70%. S126 has never been considered to play a role in the enzyme's activity or stability, thus the finding showed the importance of this residue.

Biochemical properties of extracellular protease from Staphylococcus carnosus RT6 isolated from Harbin dry sausages, and its hydrolysis of meat proteins.

The characteristics of the extracellular protease, produced by Staphylococcus carnosus RT6 isolated from Harbin dry sausages, and its hydrolysis of meat proteins were investigated. The protease was purified by ammonium sulfate, ion exchange, and gel filtration chromatography to obtain a 20.0 kDa extracellular protease. The protease reached maximal activity at pH 9.0 and 50 °C and was stable at pH 7.0 to 11.0 and 20 to 40 °C. Its protease activity was easily inhibited in the presence of Zn2+ , Fe2+ , and Fe3+ . The enzymatic characterization of the protease revealed a Vmax 49.50 U/ml·min, Km 8.19 mg/ml, and the half-life = 28.06 min, ΔH* d  = 114.11 kJ/mol, ΔG* d  = 89.24 kJ/mol, and ΔS* d  = 77.00 J/mol·K at 50 °C. In addition, the protease hydrolyzed meat protein into small particles and produced soluble peptides. This study provides a basis for understanding the biochemical characteristics of the S. carnosus RT6 protease and its future application for fermented meat products.

Vancomycin-resistant enterococci and coagulase-negative staphylococci prevalence among patients attending at Felege Hiwot Comprehensive Specialized Hospital, Bahir Dar, Ethiopia.

BACKGROUND: Vancomycin resistant enterococci (VRE) and vancomycin resistance coagulase negative staphylococci (VRCoNS) are common pathogens causing difficult to treat health care associated infections (HAI). Hence, the World Health Organization listed VRE as one of the high priority pathogens for new antibiotic discovery and antimicrobial resistance surveillance. Despite this, data on the prevalence of VRE and VRCoNS in Ethiopia is scarce. Thus, the present study determined prevalence of VRE and VRCoNS among patients attending Felege-Hiwot comprehensive specialized hospital, Ethiopia. METHODS: A hospital based cross-sectional study was conducted on 384 patients selected conveniently from February to March 2020. Data on demographic and clinical variables were collected using a structured questionnaire by face-to-face interview. Simultaneously urine, venous blood and wound swab were collected and processed following standard bacteriological technique. Antimicrobial susceptibility test was performed by minimum inhibitory concentration method using E-test for vancomycin and Kirby-Bauer disc diffusion method for other classes of antibiotics. Data was entered and analyzed using SPSS version 23. Logistic regression was performed to identify factors associated with VRE infection. P. value < 0.05 was considered as statistically significant. RESULTS: The prevalence of enterococci and CoNS were 6.8% and 12% respectively. The prevalence of VRE was 34.61% (9/26), while all CoNS (46 isolates) were susceptible to vancomycin. The majority (66.7%) of VRE was isolated from blood samples. Furthermore all VRE (100%), 58.8% of vancomycin susceptible enterococci and 45.7% of CoNS were multidrug resistant (MDR). Having educational level of secondary school and below (AOR = 12.80, CI = 1.149-142.5), previous exposure to catheterization (AOR = 56.0, CI = 4.331-724.0) and previous antibiotic use practice (AOR = 26.25, CI = 3.041-226.2) were a significant associated explanatory factor for VRE infection. CONCLUSIONS: The prevalence of vancomycin resistance enterococci, which is also multidrug resistant, was significantly high. Though no vancomycin resistance CoNS detected, the MDR level of CoNS was high. Thus to limit enterococci and CoNS infections and MDR development, focused infection prevention measures should be implemented.

Genomic investigation of clinically significant coagulase-negative staphylococci.

Introduction. Coagulase-negative staphylococci have been recognized both as emerging pathogens and contaminants of clinical samples. High-resolution genomic investigation may provide insights into their clinical significance.Aims. To review the literature regarding coagulase-negative staphylococcal infection and the utility of genomic methods to aid diagnosis and management, and to identify promising areas for future research.Methodology. We searched Google Scholar with the terms (Staphylococcus) AND (sequencing OR (infection)). We prioritized papers that addressed coagulase-negative staphylococci, genomic analysis, or infection.Results. A number of studies have investigated specimen-related, phenotypic and genetic factors associated with colonization, infection and virulence, but diagnosis remains problematic.Conclusion. Genomic investigation provides insights into the genetic diversity and natural history of colonization and infection. Such information allows the development of new methodologies to identify and compare relatedness and predict antimicrobial resistance. Future clinical studies that employ suitable sampling frames coupled with the application of high-resolution whole-genome sequencing may aid the development of more discriminatory diagnostic approaches to coagulase-staphylococcal infection.

Whole genome sequencing of coagulase positive staphylococci from a dog-and-owner screening survey.

BACKGROUND: Staphylococcus aureus and S. pseudintermedius are the two most common coagulase positive staphylococci (CPS). S. aureus is more prevalent among humans, whereas S. pseudintermedius is more commonly isolated from dogs, however, both can cause various community and hospital acquired diseases in humans. METHODS: In the current study we screened 102 dogs and 84 owners in Hungary. We tested the antibiotic susceptibility of the strains and in order to get a better picture of the clonal relationship of the strains, we used pulsed-field gel electrophoresis. In addition, three pairs of isolates with identical PFGE patterns were whole genome sequenced, MLST and spa types were established. RESULTS: Carriage rate of S. aureus was 23.8% in humans and 4.9% in dogs and two cases of co-carriage were found among dogs and owners. S. pseudintermedius carriage rate was 2.4% and 34.3%, respectively, with only one co-carriage. The isolates were generally rather susceptible to the tested antibiotics, but high tetracycline resistance of S. pseudintermedius strains was noted. The co-carried isolates shared almost the same resistance genes (including tet(K), bla(Z), norA, mepR, lmrS, fosB) and virulence gene pattern. Apart from the common staphylococcal enzymes and cytotoxins, we found enterotoxins and exfoliative toxins as well. The two S. aureus pairs belonged to ST45-t630, ST45-t671 and ST15-t084, ST15-t084, respectively. The co-carried S. pseudintermedius isolates shared the same housekeeping gene alleles determining a novel sequence type ST1685. CONCLUSIONS: Based on the genomic data, dog-owner co-carried strains displayed only insignificant differences therefore provided evidence for potential human-to-dog and dog-to-human transmission.

The Card1 nuclease provides defence during type III CRISPR immunity.

In the type III CRISPR-Cas immune response of prokaryotes, infection triggers the production of cyclic oligoadenylates that bind and activate proteins that contain a CARF domain1,2. Many type III loci are associated with proteins in which the CRISPR-associated Rossman fold (CARF) domain is fused to a restriction  endonuclease-like domain3,4. However, with the exception of the well-characterized Csm6 and Csx1 ribonucleases5,6, whether and how these inducible effectors provide defence is not known. Here we investigated a type III CRISPR accessory protein, which we name cyclic-oligoadenylate-activated single-stranded ribonuclease and single-stranded deoxyribonuclease 1 (Card1). Card1 forms a symmetrical dimer that has a large central cavity between its CRISPR-associated Rossmann fold and restriction endonuclease domains that binds cyclic tetra-adenylate. The binding of ligand results in a conformational change comprising the rotation of individual monomers relative to each other to form a more compact dimeric scaffold, in which a manganese cation coordinates the catalytic residues and activates the cleavage of single-stranded-but not double-stranded-nucleic acids (both DNA and RNA). In vivo, activation of Card1 induces dormancy of the infected hosts to provide immunity against phage infection and plasmids. Our results highlight the diversity of strategies used in CRISPR systems to provide immunity.

Prevalence of methicillin-resistant coagulase-negative staphylococci among Egyptian patients after surgical interventions.

Coagulase-negative staphylococci (CoNS) are frequently isolated from wound infections. There are limited data examining the prevalence of methicillin-resistant CoNS (MRCoNS) among Egyptian patients after surgery. Thus, we studied 208 hospitalised patients, who had skin and soft tissue infections (SSTIs) due to various causes. Samples were cultured for isolation and identification of CoNS and isolates were screened for susceptibility against 23 different antimicrobials. Out of 241 Staphylococcal isolates, 114 (47.3%) were CoNS. The prevalence of MRCoNS among surgical site infection, diabetic foot, abscess, and burn patients was 13.4%, 11.5%, 15.6%, and 10.3%, respectively. The lowest resistance of the 27 identified MRCoNS isolates was to vancomycin, amikacin and gatifloxacin (7% each). We conclude that CoNS isolates are major pathogens associated with wound infections at our institution and MRCoNS probably poses a substantial threat for patients in Egypt, though most MRCoNS isolates demonstrated susceptibility to vancomycin.

Molecular Insights into Zn2+ Inhibition of the Antibacterial Endopeptidase Lysostaphin from Staphylococcus simulans.

BACKGROUND: Mature lysostaphin (~28-kDa Lss) from Staphylococcus simulans proves effective in killing methicillin-resistant Staphylococcus aureus (MRSA) which is endemic in hospitals worldwide. Lss is Zn2+-dependent endopeptidase, but its bacteriolytic activity could be affected by exogenously added Zn2+. OBJECTIVE: To gain greater insights into structural and functional impacts of Zn2+and Ni2+on Lss-induced bioactivity. METHODS: Lss purified via immobilized metal ion-affinity chromatography was assessed for bioactivity using turbidity reduction assays. Conformational change of metal ion-treated Lss was examined by circular dichroism and intrinsic fluorescence spectroscopy. Co-sedimentation assay was performed to study interactions between Zn2+-treated Lss and S. aureus peptidoglycans. Metal ionbinding prediction and intermolecular docking were used to locate an extraneous Zn2+-binding site. RESULTS: A drastic decrease in Lss bioactivity against S. aureus and MRSA was revealed only when treated with Zn2+, but not Ni2+, albeit no negative effect of diethyldithiocarbamate-Zn2+-chelator on Lss-induced bioactivity. No severe conformational change was observed for Lss incubated with exogenous Zn2+ or Ni2+. Lss pre-treated with Zn2+ efficiently bound to S. aureus cell-wall peptidoglycans, suggesting non-interfering effect of exogenous metal ions on cell-wall targeting (CWT) activity. In silico analysis revealed that exogenous Zn2+, but not Ni2+, preferably interacted with a potential extraneous Zn2+-binding site (His253, Glu318 and His323) placed near the Zn2+-coordinating Lssactive site within the catalytic (CAT) domain. CONCLUSION: Our present data signify the adverse influence of exogenous Zn2+ ions on Lss-induced staphylolytic activity through the exclusive presence within the CAT domain of an extraneous inhibitory Zn2+-binding site, without affecting the CWT activity.

Production, purification and biochemical characterisation of a novel lipase from a newly identified lipolytic bacterium Staphylococcus caprae NCU S6.

A novel lipase, SCNL, was isolated from Staphylococcus caprae NCU S6 strain in the study. The lipase was purified to homogeneity with a yield of 6.13% and specific activity of 502.76 U/mg, and its molecular weight was determined to be approximately 87 kDa. SCNL maintained above 80% of its initial activity at a wide range of temperatures (20-50 °C) and pH values (6-11), with an optimal temperature at 40 °C and optimal pH at 9.0 with p-nitrophenyl palmitate as a substrate. SCNL exhibited a higher residual activity than the other staphylococcal lipases in the presence of common enzyme inhibitors and commercial detergents. The lipase activity was enhanced by organic solvents (isooctane, glycerol, DMSO and methanol) and metal ions (Na+, Ba2+, Ca2+, and Mn2+). The Km and Vmax values of SCNL were 0.695 mM and 262.66 s-1 mM-1, respectively. The enzyme showed a preference for p-NP stearate, tributyrin and canola oil. These biochemical features of SCNL suggested that it may be an excellent novel lipase candidate for industrial and biotechnological applications.

Multidrug resistance bacteremia in neonates and its association with late-onset sepsis and Coagulase-negative Staphylococci.

INTRODUCTION: This study aimed to assess the association between multidrug resistance (MDR) and late-onset sepsis (LOS) among newborns with bloodstream infection (BSI). METHODOLOGY: In this cross-sectional study, we routinely tested every newborn with a presumptive diagnosis of sepsis admitted to the largest reference maternity hospital in Lima, Peru for BSI over an 18-month period. We tested every isolate for MDR by using the disk-diffusion method and assessed its associated factors by using a robust Poisson regression analysis with a particular focus on its association with LOS (vs. early-onset sepsis, EOS). RESULTS: We analyzed a total of 489 subjects, including 340 (69%) newborns with LOS, and estimated an MDR rate of 80% (95% confidence interval, CI: 76%-83%), which was significantly higher (p-value < 0.001) among LOS (85%; 95% CI: 81%-89%) than EOS cases (67%; 95% CI: 59%-75%). The primary isolate was coagulase-negative Staphylococci (CoNS) (60%), which exhibited a limited subset of antibiotic MDR patterns, most of which were characterized by their resistance to cefoxitin, gentamicin, and clindamycin and levofloxacin. Overall, the prevalence of MDR was higher among LOS compared to EOS cases (adjusted prevalence ratio [aPR] = 1.28; 95% CI: 1.14-1.45), and among BSI due to CoNS compared to other bacteria (Apr = 1.10; 95% CI: 1.01-1.20). CONCLUSIONS: MDR among newborns with sepsis is exceptionally high, being even higher among those with LOS than newborns with EOS, and among those infected with CoNS compared to other bacteria. Furthermore, CoNS exhibited a limited subset of MDR patterns, which could be used to guide therapeutic decisions.