Phenylthiazoles with potent & optimum selectivity toward Clostridium difficile.

Clostridium difficile (C. difficile) is one of the most threatening bacteria globally, causing high mortality and morbidity in humans and animals, and is considered a public health threat that requires urgent and aggressive action. Interruption of the human gut microbiome and the development of antibiotic resistance urgently require development and synthesis of effective alternative antibiotics with minimal effects on the normal gut microbial flora. In this study, cyclization of the aminoguanidine head to the thiazole nucleus while maintaining its other pharmacophoric features leads to selective targeting of Clostridioides difficile as shown in the graphical abstract. The most promising compound, 5, was significantly more efficient than vancomycin and metronidazole against six strains of C. diff with MIC values as low as 0.030 µg mL-1. Additionally, compound 5 was superior to vancomycin and metronidazole, showing no inhibition toward nine tested strains of the normal human gut microbiota (>64 µg mL-1). The high safety profile of compound 5 was also observed with two cell lines HRT-18 and Vero cells.

Novel phenylthiazoles with a tert-butyl moiety: promising antimicrobial activity against multidrug-resistant pathogens with enhanced ADME properties.

The structure-activity relationship of a new tert-butylphenylthiazole series, with a pyrimidine linker, was investigated. We wished to expand knowledge of this novel class of antibiotics by generating 21 new derivatives bearing ≥2 heteroatoms in their side chains. Their activity was examined against isolates of methicillin-resistant Staphylococcus aureus (MRSA), Clostridium difficile, Escherichia coli, Neisseria gonorrhoeae, and Candida albicans. Two compounds with 1,2-diaminocyclohexane as a nitrogenous side chain showed promising activity against the highly infectious MRSA USA300 strain, with a minimum inhibitory concentration (MIC) of 4 µg mL-1. One of these two compounds demonstrated potent activity against C. difficile, with a MIC of 4 µg mL-1. Moderate activities against a C. difficile strain with a MIC of 8 µg mL-1 were noted. Some new compounds possessed antifungal activity against a wild fluconazole-resistant C. albicans strain, with MIC values of 4-16 µg mL-1. ADME and metabolism-simulation studies were performed for the most promising compound and compared with lead compounds. Our results revealed that one compound possessed greater penetration of bacterial membranes and metabolic resistance, which aided a longer duration of action against MRSA.

Anion-Exchange Electrospun Mixed-Matrix Polymer Fibers of Colesevelam for Water Treatment.

Novel anion-exchange electrospun fiber membranes of polycaprolactone doped with the cationic, cross-linked colesevelam polymer are reported. The weight fraction of cross-linked cationic colesevelam polymer, as the active phase within the PCL matrix, can readily be controlled in the synthesis of the mixed-matrix fibers (Cole@PCL), enabling optimization of the ion-exchange properties of the resulted membranes. This approach enabled adaptation of anion-exchange resins to a permeable, flexible membrane form, which is a significant advancement toward futuristic water treatment applications, demonstrated herein for the removal of trace contaminants, including nitrates and phosphates, as well as anionic dyes. The Cole@PCL membranes demonstrated the dependence of contaminant uptake on the weight percentage of colesevelam in the mixed-matrix membrane. An optimal 10 wt % of colesevelam was identified, demonstrating a staggering ion removal capacity of 155.8 mg/g for nitrate, 177.6 mg/g for phosphate, and 70 mg/g for Methyl Orange.

Naphthylthiazoles: a class of broad-spectrum antifungals.

Cryptococcal infections remain a major cause of mortality worldwide due to the ability of Cryptococci to pass through the blood-brain barrier (BBB) causing lethal meningitis. The limited number of available therapeutics, which exhibit limited availability, severe toxicity and low tolerability, necessitates the development of new therapeutics. Investigating the antifungal activity of a novel series of naphthylthiazoles provided trans-diaminocyclohexyl derivative 18 with many advantageous attributes as a potential therapeutic for cryptococcal meningitis. Briefly, the antimycotic activity of 18 against cryptococcal strains was highly comparable to that of amphotericin-B and fluconazole with MIC values as low as 1 µg mL-1. Moreover, compound 18 possessed additional advantages over fluconazole; it significantly reduced the intracellular burden of Cryptococci and markedly inhibited cryptococcal biofilm formation. Initial PK assessment of 18 indicated its ability to reach the CNS after oral administration with high permeability, and it maintained therapeutic plasma concentrations for 18 h. Its antifungal activity extended to other clinically relevant strains, such as fluconazole-resistant C. auris.

New Phenylthiazoles: Design, Synthesis, and Biological Evaluation as Antibacterial, Antifungal, and Anti-COVID-19 Candidates.

The combination of antibacterial and antiviral agents is becoming a very important aspect of dealing with resistant bacterial and viral infections. The N-phenylthiazole scaffold was found to possess significant anti-MRSA, antifungal, and anti-COVID-19 activities as previously published; hence, a slight refinement was proposed to attach various alkyne lipophilic tails to this promising scaffold, to investigate their effects on the antimicrobial activity of the newly synthesized compounds and to provide a valuable structure-activity relationship. Phenylthiazole 4 m exhibited the most potent anti-MRSA activity with 8 µg/mL MIC value. Compounds 4 k and 4 m demonstrated potent activity against Clostridium difficile with MIC values of 2 µg/mL and moderate activity against Candida albicans with MIC value of 4 µg/mL. When analyzed for their anti-COVID-19 inhibitory effect, compound 4 b emerged with IC50 =1269 nM and the highest selectivity of 138.86 and this was supported by its binding score of -5.21 kcal mol-1 when docked against SARS-CoV-2 M pro . Two H-bonds were formed, one with His164 and the other with Met49 stabilizing phenylthiazole derivative 4 b, inside the binding pocket. Additionally, it created two arene-H bonds with Asn142 and Glu166, through the phenylthiazole scaffold and one arene-H bond with Leu141 via the phenyl ring of the lipophilic tail.

Exploring novel aryl/heteroaryl-isosteres of phenylthiazole against multidrug-resistant bacteria.

Antimicrobial resistance has become a concern as a worldwide threat. A novel scaffold of phenylthiazoles was recently evaluated against multidrug-resistant Staphylococci to control the emergence and spread of antimicrobial resistance, showing good results. Several structural modifications are needed based on the structure-activity relationships (SARs) of this new antibiotic class. Previous studies revealed the existence of two key structural features essential for the antibacterial activity, the guanidine head and lipophilic tail. In this study, a new series of twenty-three phenylthiazole derivatives were synthesized utilizing the Suzuki coupling reaction to explore the lipophilic part. The in vitro antibacterial activity was evaluated against a range of clinical isolates. The three most promising compounds, 7d, 15d and 17d, with potent MIC values against MRSA USA300 were selected for further antimicrobial evaluation. The tested compounds exhibited potent results against the tested MSSA, MRSA, and VRSA strains (concentration: 0.5 to 4 µg mL-1). Compound 15d inhibited MRSA USA400 at a concentration of 0.5 µg mL-1 (one-fold more potent than vancomycin) and showed low MIC values against ten clinical isolates, including linezolid-resistant strain MRSA NRS119 and three vancomycin-resistant isolates VRSA 9/10/12. Moreover, compound 15d retained its potent antibacterial activity using the in vivo model by the burden reduction of MRSA USA300 in skin-infected mice. The tested compounds also showed good toxicity profiles and were found to be highly tolerable to Caco-2 cells at concentrations of up to 16 µg mL-1, with 100% of the cells remaining viable.

Expanding the structure-activity relationships of alkynyl diphenylurea scaffold as promising antibacterial agents.

With the continuous and alarming threat of exhausting the current antimicrobial arsenals, efforts are urgently needed to develop new effective ones. In this study, the antibacterial efficacy of a set of structurally related acetylenic-diphenylurea derivatives carrying the aminoguanidine moiety was tested against a panel of multidrug-resistant Gram-positive clinical isolates. Compound 18 was identified with a superior bacteriological profile than the lead compound I. Compound 18 demonstrated an excellent antibacterial profile in vitro: low MIC values, extended post-antibiotic effect, refractory ability to resistance development upon extended repeated exposure, and high tolerability towards mammalian cells. Finally, when assessed in a MRSA skin infection animal model, compound 18 showed considerable healing and less inflammation, decrease in the bacterial loads in skin lesions, and it surpassed fusidic acid in controlling the systemic dissemination of S. aureus. Collectively, compound 18 represents a promising lead anti-MRSA agent that merits further investigation for the development of new anti-staphylococcal therapeutics.

The Anti-MRSA Activity of Phenylthiazoles: A Comprehensive Review.

Antimicrobial resistance is an aggravating global issue therefore it has been under extensive research in an attempt to reduce the number of antibiotics that are constantly reported as obsolete jeopardizing the lives of millions worldwide. Thiazoles possess a reputation as one of the most diverse biologically active nuclei, and phenylthiazoles are no less exceptional with an assorted array of biological activities such as anthelmintic, insecticidal, antimicrobial, antibacterial, and antifungal activity. Recently phenyl thiazoles came under the spotlight as a scaffold having strong potential as an anti-MRSA lead compound. It is a prominent pharmacophore in designing and synthesizing new compounds with antibacterial activity against multidrug-resistant bacteria such as MRSA, which is categorized as a serious threat pathogen, that exhibited concomitant resistance to most of the first-line antibiotics. MRSA has been associated with soft tissue and skin infections resulting in high death rates, rapid dissemination, and loss of millions of dollars of additional health care costs. In this brief review, we have focused on the advances of phenylthiazole derivatives as potential anti-MRSA from 2014 to 2021. The review encompasses the effect on biological activity due to combining this molecule with various synthetic pharmacophores. The physicochemical aspects were correlated with the pharmacokinetic properties of the reviewed compounds to reach a structure-activity relationship profile. Lead optimization of phenyl thiazole derivatives has additionally been outlined where the lipophilicity of the compounds was balanced with the metabolic stability and oral solubility to aid the researchers in medicinal chemistry, design, and synthesizing effective anti- MRSA phenylthiazoles in the future.

Exploring the structure-activity relationships of diphenylurea as an antibacterial scaffold active against methicillin- and vancomycin-resistant Staphylococcus aureus.

A set of structurally related diphenylurea derivatives bearing aminoguanidine moiety were synthesized, and their antibacterial activity was assessed against a panel of multi-drug resistant Gram-positive clinical isolates. Two compounds 6 and 24 were identified with better bacteriological profile than the lead compound I. The multi-step resistance development studies indicated that MRSA are less likely to develop resistance toward diphenylurea compounds. Moreover, these compounds demonstrated a prolonged post-antibiotic effect than that of vancomycin. Furthermore, compounds 6 and 24 were able to re-sensitize VRSA to vancomycin, resulting in 8- to more than 32-fold improvement in vancomycin MIC values against clinical VRSA isolates. Finally, when assessed in an in vivo skin infection mouse model, the efficacy of compound 24 was very comparable to that of the commercially available fusidic acid ointment. Additionally, the diphenylurea 24 did not have a pronounced effect on the animal weights along the experiment indicating its safety and tolerability to mice. Taken together, these results indicate that the diphenylurea scaffold merits further investigation as a promising anti-staphylococcal treatment option.

Evaluation of bisphenylthiazoles as a promising class for combating multidrug-resistant fungal infections.

To minimize the intrinsic toxicity of the antibacterial agent hydrazinyloxadiazole 1, the hydrazine moiety was replaced with ethylenediamine (compound 7). This replacement generated a potent antifungal agent with no antibacterial activity. Notably, use of a 1,2-diaminocyclohexane moiety, as a conformationally-restricted isostere for ethylenediamine, potentiated the antifungal activity in both the cis and trans forms of N-(5-(2-([1,1'-biphenyl]-4-yl)-4-methylthiazol-5-yl)-1,3,4-oxadiazol-2-yl)cyclohexane-1,2-diamine (compounds 16 and 17). Both compounds 16 and 17 were void of any antibacterial activity; nonetheless, they showed equipotent antifungal activity in vitro to that of the most potent approved antifungal agent, amphotericin B. The promising antifungal effects of compounds 16 and 17 were maintained when assessed against an additional panel of 26 yeast and mold clinical isolates, including the Candida auris and C. krusei. Furthermore, compound 17 showed superior activity to amphotericin B in vitro against Candida glabrata and Cryptococcus gattii. Additionally, neither compound inhibited the normal human microbiota, and both possessed excellent safety profiles and were 16 times more tolerable than amphotericin B.

Development of Biphenylthiazoles Exhibiting Improved Pharmacokinetics and Potent Activity Against Intracellular Staphylococcus aureus.

Exploring the structure-activity relationship (SAR) at the cationic part of arylthiazole antibiotics revealed hydrazine as an active moiety. The main objective of the study is to overcome the inherited toxicity associated with the free hydrazine. A series of hydrocarbon bridges was inserted in between the groups, to separate the two amino groups. Hence, the aminomethylpiperidine-containing analog 16 was identified as a new promising antibacterial agent with efficient antibacterial and pharmacokinetic profiles. Briefly, compound 16 outperformed vancomycin in terms of the antibacterial spectrum against vancomycin-resistant staphylococcal and enterococcal strains with minimum inhibitory concentrations (MICs) ranging from 2 to 4 µg/mL, which is a faster bactericidal mode of action, completely eradicating the high staphylococcal burden within 6-8 h, and it has a unique ability to completely clear intracellular staphylococci. In addition, the initial pharmacokinetic assessment confirmed the high metabolic stability of compound 16 (biological half-life >4 h); it had a good extravascular distribution and maintained a plasma concentration higher than the average MIC value for over 12 h. Moreover, compound 16 significantly reduced MRSA burden in an in vivo MRSA skin infection mouse experiment. These attributes collectively suggest that compound 16 is a good therapeutic candidate for invasive staphylococcal and enterococcal infections. From a mechanistic point of view, compound 16 inhibited undecaprenyl diphosphate phosphatase (UppP) with an IC50 value of 29 µM.

Evaluation of N-phenyl-2-aminothiazoles for treatment of multi-drug resistant and intracellular Staphylococcus aureus infections.

The increasing emergence of antibiotic-resistant bacterial pathogens calls for additional urgency in the development of new antibacterial candidates. N-Phenyl-2-aminothiazoles are promising candidates that possess potent anti-MRSA activity and could potentially replenish the MRSA antibiotic pipeline. The initial screen of a series of compounds in this novel class against several bacterial strains revealed that the aminoguanidine analogues possessed promising activities and superior safety profiles. The determined MICs of these compounds were comparable to, if not better than, those of the control drugs (linezolid and vancomycin). Remarkably, compounds 3a, 3b, and 3e possessed potent activities against multidrug resistant staphylococcal isolates and several clinically important pathogens, such as vancomycin-resistant enterococci (VRE) and Streptococcus pneumoniae. In addition, the compounds were superior to vancomycin in the rapid killing of MRSA and the longer post-antibiotic effects. Furthermore, low concentrations of compounds 3a, 3b, and 3e reduced the intracellular burden of MRSA by greater than 90%. Initial in vitro PK/toxicity assessments revealed that compound 3e was highly tolerable and possessed a low metabolic clearance rate and a highly acceptable half-life.

Repurposing approach identifies pitavastatin as a potent azole chemosensitizing agent effective against azole-resistant Candida species.

The limited number of antifungals and the rising frequency of azole-resistant Candida species are growing challenges to human medicine. Drug repurposing signifies an appealing approach to enhance the activity of current antifungal drugs. Here, we evaluated the ability of Pharmakon 1600 drug library to sensitize an azole-resistant Candida albicans to the effect of fluconazole. The primary screen revealed 44 non-antifungal hits were able to act synergistically with fluconazole against the test strain. Of note, 21 compounds, showed aptness for systemic administration and limited toxic effects, were considered as potential fluconazole adjuvants and thus were termed as "repositionable hits". A follow-up analysis revealed pitavastatin displaying the most potent fluconazole chemosensitizing activity against the test strain (ΣFICI 0.05) and thus was further evaluated against 18 isolates of C. albicans (n = 9), C. glabrata (n = 4), and C. auris (n = 5). Pitavastatin displayed broad-spectrum synergistic interactions with both fluconazole and voriconazole against ~89% of the tested strains (ΣFICI 0.05-0.5). Additionally, the pitavastatin-fluconazole combination significantly reduced the biofilm-forming abilities of the tested Candida species by up to 73%, and successfully reduced the fungal burdens in a Caenorhabditis elegans infection model by up to 96%. This study presents pitavastatin as a potent azole chemosensitizing agent that warrant further investigation.

Ospemifene displays broad-spectrum synergistic interactions with itraconazole through potent interference with fungal efflux activities.

Azole antifungals are vital therapeutic options for treating invasive mycotic infections. However, the emergence of azole-resistant isolates combined with limited therapeutic options presents a growing challenge in medical mycology. To address this issue, we utilized microdilution checkerboard assays to evaluate nine stilbene compounds for their ability to interact synergistically with azole drugs, particularly against azole-resistant fungal isolates. Ospemifene displayed the most potent azole chemosensitizing activity, and its combination with itraconazole displayed broad-spectrum synergistic interactions against Candida albicans, Candida auris, Cryptococcus neoformans, and Aspergillus fumigatus (ΣFICI = 0.05-0.50). Additionally, in a Caenorhabditis elegans infection model, the ospemifene-itraconazole combination significantly reduced fungal CFU burdens in infected nematodes by ~75-96%. Nile Red efflux assays and RT-qPCR analysis suggest ospemifene interferes directly with fungal efflux systems, thus permitting entry of azole drugs into fungal cells. This study identifies ospemifene as a novel antifungal adjuvant that augments the antifungal activity of itraconazole against a broad range of fungal pathogens.

Screening for potent and selective anticlostridial leads among FDA-approved drugs.

Clostridium difficile is a leading cause of morbidity and mortality particularly in hospital settings. In addition, treatment is very challenging due to the scarcity of effective therapeutic options. Thus, there remains an unmet need to identify new therapeutic agents capable of treating C. difficile infections. In the current study, we screened two FDA-approved drug libraries against C. difficile. Out of almost 3200 drugs screened, 50 drugs were capable of inhibiting the growth of C. difficile. Remarkably, some of the potent inhibitors have never been reported before and showed activity in a clinically achievable range. Structure-activity relationship analysis of the active hits clustered the potent inhibitors into four chemical groups; nitroimidazoles (MIC50 = 0.06-2.7 µM), salicylanilides (MIC50 = 0.2-0.6 µM), imidazole antifungals (MIC50 = 4.8-11.6 µM), and miscellaneous group (MIC50 = 0.4-22.2 µM). The most potent drugs from the initial screening were further evaluated against additional clinically relevant strains of C. difficile. Moreover, we tested the activity of potent inhibitors against representative strains of human normal gut microbiota to investigate the selectivity of the inhibitors towards C. difficile. Overall, this study provides a platform that could be used for further development of potent and selective anticlostridial antibiotics.

Oxadiazolylthiazoles as novel and selective antifungal agents.

Studying the structure-activity relationships (SAR) of oxadiazolylthiazole antibiotics unexpectedly led us to identify ethylenediamine- and propylenediamine-analogs as potential antimycotic novel lead structures. Replacement of the ethylenediamine moiety for the lead compound 7 with cis-diaminocyclohexyl group (compound 18) significantly enhanced the antifungal activity. In addition to the high safety margin of 18 against mammalian cells, it showed highly selective broad-spectrum activity against fungal cells without inhibiting the human normal microbiota. The antifungal activity of 18 was investigated against 20 drug-resistant clinically important fungi, including Candida species, Cryptococcus, and Aspergillus fumigatus strains. In addition to the low MIC values that mostly ranged between 0.125 and 2.0 µg/mL, compound 18 outperformed fluconazole in disrupting mature Candida biofilm.

Balancing Physicochemical Properties of Phenylthiazole Compounds with Antibacterial Potency by Modifying the Lipophilic Side Chain.

Bacterial resistance to antibiotics is presently one of the most pressing healthcare challenges and necessitates the discovery of new antibacterials with unique chemical scaffolds. However, the determination of the optimal balance between structural requirements for pharmacological action and pharmacokinetic properties of novel antibacterial compounds is a significant challenge in drug development. The incorporation of lipophilic moieties within a compound's core structure can enhance biological activity but have a deleterious effect on drug-like properties. In this Article, the lipophilicity of alkynylphenylthiazoles, previously identified as novel antibacterial agents, was reduced by introducing cyclic amines to the lipophilic side chain. In this regard, substitution with methylpiperidine (compounds 14-16) and thiomorpholine (compound 19) substituents significantly enhanced the aqueous solubility profile of the new compounds more than 150-fold compared to the first-generation lead compound 1b. Consequently, the pharmacokinetic profile of compound 15 was significantly enhanced with a notable improvement in both half-life and the time the compound's plasma concentration remained above its minimum inhibitory concentration (MIC) against methicillin-resistant Staphylococcus aureus (MRSA). In addition, compounds 14-16 and 19 were found to exert a bactericidal mode of action against MRSA and were not susceptible to resistance formation after 14 serial passages. Moreover, these compounds (at 2× MIC) were superior to the antibiotic vancomycin in the disruption of the mature MRSA biofilm. The modifications to the alkynylphenylthiazoles reported herein successfully improved the pharmacokinetic profile of this new series while maintaining the compounds' biological activity against MRSA.

Modifying the lipophilic part of phenylthiazole antibiotics to control their drug-likeness.

Compounds with high lipophilic properties are often associated with bad physicochemical properties, triggering many off-targets, and less likely to pass clinical trials. Two metabolically stable phenylthiazole antibiotic scaffolds having notable high lipophilic characters, one with alkoxy side chain and the other one with alkynyl moiety, were derivatized by inserting a cyclic amine at the lipophilic tail with the objective of improving physicochemical properties and the overall pharmacokinetic behavior. Only alkynyl derivatives with 4- or 5-membered rings showed remarkable antibacterial activity. The azetidine-containing compound 8 was the most effective and it revealed a potent antibacterial effect against 15 multi-drug resistant (MDR)-Gram positive pathogens including Staphylococcus aureus, Streptococcus pneumoniae, Staphylococcus epidermidis and enterococci. Compound 8 was also highly effective in clearing 99.7% of the intracellular methicillin-resistant S. aureus (MRSA) harbored inside macrophages. In addition to the remarkable enhancement in aqueous solubility, the in vivo pharmacokinetic study in rats indicated that compound 8 can penetrate gut cells and reach plasma at a therapeutic concentration within 15 min and maintain effective plasma concentration for around 12 h. Interestingly, the main potential metabolite (compound 9) was also active as an antibacterial agent with potent antibiofilm activity.

Identification of a Phenylthiazole Small Molecule with Dual Antifungal and Antibiofilm Activity Against Candida albicans and Candida auris.

Candida species are a leading source of healthcare infections globally. The limited number of antifungal drugs combined with the isolation of Candida species, namely C. albicans and C. auris, exhibiting resistance to current antifungals necessitates the development of new therapeutics. The present study tested 85 synthetic phenylthiazole small molecules for antifungal activity against drug-resistant C. albicans. Compound 1 emerged as the most potent molecule, inhibiting growth of C. albicans and C. auris strains at concentrations ranging from 0.25-2 µg/mL. Additionally, compound 1 inhibited growth of other clinically-relevant yeast (Cryptococcus) and molds (Aspergillus) at a concentration as low as 0.50 µg/mL. Compound 1 exhibited rapid fungicidal activity, reducing the burden of C. albicans and C. auris below the limit of detection within 30 minutes. Compound 1 exhibited potent antibiofilm activity, similar to amphotericin B, reducing the metabolic activity of adherent C. albicans and C. auris biofilms by more than 66% and 50%, respectively. Furthermore, compound 1 prolonged survival of Caenorhabditis elegans infected with strains of C. albicans and C. auris, relative to the untreated control. The present study highlights phenylthiazole small molecules, such as compound 1, warrant further investigation as novel antifungal agents for drug-resistant Candida infections.

Phenylthiazoles with nitrogenous side chain: An approach to overcome molecular obesity.

A novel series of phenylthiazoles bearing cyclic amines at the phenyl-4 position was prepared with the objective of decreasing lipophilicity and improving the overall physicochemical properties and pharmacokinetic profile of the compounds. Briefly, the piperidine ring (compounds 10 and 12) provided the best ring size in terms of antibacterial activity when tested against 16 multidrug-resistant clinical isolates. Both compounds were superior to vancomycin in the ability to eliminate methicillin-resistant Staphylococcus aureus (MRSA), residing within infected macrophages and to disrupt mature MRSA biofilm. Additionally, compounds 10 and 12 exhibited a fast-bactericidal mode of action in vitro. Furthermore, the new derivatives were 160-times more soluble in water than the previous lead compound 1b. Consequently, compound 10 was orally bioavailable with a highly-acceptable pharmacokinetic profile in vivo that exhibited a half-life of 4 h and achieved a maximum plasma concentration that exceeded the minimum inhibitory concentration (MIC) values against all tested bacterial isolates.