Dactylosporangium fulvum, founda “hybrid” biosynthetic gene cluster responsible forthe production of both an antibiotic, pyridomycin, and a novel chlorocatechol-containingsiderophore named chlorodactyloferrin. While both of these naturalproducts were synthesized independently, analysis of the culture supernatantalso identified a conjugate of both molecules. We then found thatthe addition of ferric iron to purified chlorodactyloferrin and pyridomycininstigated their conjugation, leading to the formation of a covalentbond between the siderophore-catechol and the pyridomycin-pyridinegroups. Using model reactants, this iron-based reaction was foundto proceed through a Michael-type addition reaction, where ferriciron oxidizes the siderophore-catechol group into its quinone form,which is then attacked by the antibiotic pyridyl-nitrogen to formthe catechol–pyridinium linkage. These findings prompted usto explore if other “cargo” molecules could be attachedto chlorodactyloferrin in a similar manner, and this was indeed confirmedwith a pyridine-substituted TAMRA fluorophore as well as with pyridine-substitutedpenicillin, rifampicin, and norfloxacin antibiotic analogues. Theresultant biomimetic conjugates were demonstrated to effectively entera number of bacteria, with TAMRA–chlorodactyloferrin conjugatescausing fluorescent labeling of the bacteria, and with penicillinand rifampicin conjugates eliciting antibiotic activity. These findingsopen up new opportunities for the design and facile synthesis of anovel class of biomimetic siderophore conjugates with antibiotic activity.Inspired by naturalsideromycins, the conjugation of antibioticsto siderophores is an attractive strategy to facilitate “Trojanhorse” delivery of antibiotics into bacteria. Genome analysisof a soil bacterium,  Iron can mediate coupling of catechol-containingsiderophoreswith pyridine-containing antibiotics in a process that can be exploitedto design custom siderophore conjugates. This innate penetration barrier is often reinforcedby efficient antibiotic efflux pumps that further prevent intracellularaccumulation. The development of novel technologies to facilitatethe delivery of antibiotics to their targets would therefore openup new doors to the development of future antibiotics.Antibioticdrug resistant bacterial infections pose an ever-growingburden to global healthcare systems worldwide, with research and developmentinto novel antibiotic innovations classed as a critical priority bythe World Health Organization to help fight such drug resistant bacterialinfections.4 Inspiredby the properties of these natural products, major developments inmedicinal chemistry allow for rational approaches to designing andimproving antibiotic entry across the bacterial envelope of Gram-negative E. coli through the implementation of eNTRy rules.5 As an alternative approach for improved antibioticpenetration, a select number of natural products enter bacteria byhijacking bacterially active uptake systems for siderophore-mediatediron acquisition. Such “Trojan horse” antibiotics, alsoknown as sideromycins, are often conjugates between an antibioticand a siderophore  that were thus naturally selectedfor activity and hence capable of penetrating bacteria to reach theirtargets. These natural antibiotics often have optimal physicochemicalproperties to enter bacteria by passive diffusion or though alternativeroutes such as through porins. in refs −11), butbaumannii) or anti15 salmycins,16 and ferrimycins,18 as well as naturalmonoglucosylated enterobactin linked class IIb microcins19 . When considered alone in their nonconjugated form, the antibacterialwarheads of such conjugates often display low antibiotic activitiesbecause they are unable to effectively enter bacteria. The correspondingantibiotic conjugates show greatly enhanced penetration capacity asa result of hijacking bacterial siderophore-iron uptake systems.22 In the case of albomycin and salmycin, it wasdemonstrated that in addition to bacterial penetration, these antibioticmoieties are liberated (decoupled) from their respective siderophoresonce inside the bacteria, either by an intracellular peptidase foralbomycin,23 and through intramolecularcyclization following iron reduction for salmycin.24 These entry and decoupling properties of natural sideromycinsprovide important blueprints for the rational design and synthesisof alternative synthetic siderophore–antibiotic conjugates.To date, three major natural sideromycinfamilies have been described,namely albomycins,11 Despite these tremendous efforts, few examples resulted in favorableantibiotic activity. In particular, synthetic conjugates of siderophoreswith β-lactam antibiotics have been frequently found to leadto active molecules.27 It should,however, be noted that β-lactam antibiotics target penicillin-bindingproteins of the bacterial periplasmic space and are likely activewithout the need for decoupling, meaning that these molecules possesspartial attributes of the natural sideromycins. While it remains challenging,a number of intracellularly active siderophore–antibiotic conjugateshave been described based on siderophores such as desferrioxamine,29 enterobactin,32 pyochelin,33 and pyoverdin35 . ResDactylosporangiumfulvum. Atypically, this siderophore–antibiotic conjugatewas found to be generated through a unique iron-mediated conjugationof the individual siderophore and antibiotic units. We then investigatethe likely mechanism of this siderophore–antibiotic conjugationand demonstrate that this strategy can be exploited for the constructionof other siderophore–antibiotic and siderophore–fluorophoreconjugates. Finally, we present data supporting the bacterial uptakeof these conjugates, clearly demonstrating that they reach their antibiotictargets in D. fulvum. Together, these findings notonly enrich our knowledge on siderophore–antibiotic conjugatesbut also provide an important blueprint to facilitate their futuredesign.In this paper, we report the discovery and characterization ofa novel natural siderophore–antibiotic conjugate with its biosynthesisencoded by a hybrid biosynthetic gene cluster in Dactylosporangium fulvum (accession number CP073720.11)followed by the annotation and classification of biosynthetic geneclusters (BGCs) using AntiSMASH38 identified20 BGCs. One of these BGCs appeared to be structured as a hybrid cluster(Table S1), with half encoding for machinery neededfor the production of the antibiotic pyridomycin (Pyr 1), similar to that in S. pyridomyceticus,(2). The presence of a Pyr BGC was unsurprising,as D. fulvum is a known producer of this antibioticthat targets bacterial ACP-enoyl reductase of the fatty acid synthesistype II (FASII) system42 (further characterization of the Pyr BGC is to bepresented in a future manuscript). Transcriptome analysis of D. fulvum by RNA-Seq found that both halves of this “hybrid”BGC were actively coexpressed . Genetic disruption of the ClDaf BGC through inactivation of thefirst NRPS gene, dafA (D. fulvum ΔdafA), did not impact Pyr 1 production but led to the lossof a secondary metabolite, with the mass around [M + H]+1 = 1001 and an isotopic profile of a chlorinated molecule . In addition to ClDaf 2, a minor metabolite with the mass of [M + H]+1 = 967was also no longer produced in D. fulvum ΔdafA, a compound later identified as a non-halogenated derivative ofClDaf 2, named dactyloferrin (Daf 3). Inreciprocal experiments, genetic inactivation of a core pyridomycinbiosynthetic gene pyrA (D. fulvum ΔpyrA) prevented Pyr 1 production, as previously demonstratedin S. pyridomyceticus,. To confirm thatClDaf 2 had iron binding capacity as a siderophore, UHPLC-MSanalysis confirmed that addition of ferric iron to the bacterial supernatantled to the appearance of the expected iron bond complex 2:iron  . In addition, purified ClDaf 2 and Daf 3 were both shown to bind iron using the Chrome Azurol S assay. Unlike typical siderophores,the ClDaf 2 BGC was found to be transcribed under ironnormal conditions , and this transcription appeared not to be coupled to iron availability,as addition of the iron-chelator 2,2′-dipyridyl (DIP) did notinduce upregulation of the ClDaf 2 BGC , while it did induce another predicted D. fulvum siderophore BGC (predicted to produce deferoxamine).In addition, ClDaf 2 production by D. fulvum appeared unaffected by iron DIP mediated iron depletion .Whole genome sequencingof yceticus, and the yceticus, but did 2 and Daf 3 indicated a mass of [M + H]+ = 1001.4590 and 967.4956,corresponding to the predicted formulas of C40H66N14O14Cl  and C40H67N14O14  respectively. NMR characterization of ClDaf 2 inwater confirmed it to be a linear NRPS derived molecule composed ofeight amide linked monomers, specifically: 4-chloro-2,3-dihydroxybenzoicacid, glycine, arginine, ornithine, N-hydroxyl-N-acetyl-ornithine, ornithine, serine, and cyclo-N-hydroxy-ornithine . Consistent with previous work,44 NMR analysis of the purified diamagnetic gallium(III) complex ofClDaf, 2:gallium, in water confirmed the expected metalcoordination by the catecholate and by the hydroxamate moieties ofthe N-hydroxyl-N-acetyl-ornithineand cyclo-N-hydroxy-ornithine units . The structural elucidationof Daf 3 by NMR confirmed it to be the non-chlorinatedanalogue of ClDaf 2 . The assigned 1H, 13C, and 15N chemical shifts of 2, 2:gallium, and 3 are reported in Table S2.HRMS analysis of purified ClDaf 2, analysis of the ClDaf BGC showed two large multimodularNRPS proteins(DafA and B), with DafA comprising a starting condensation domain(for the addition of the first monomer) and four modules with adenylationdomains, and where DafB included three additional modules with adenylationdomains . This analysis also confirmed the incorporation of both d- and l-ornithine-based amino acids into ClDaf 2, but as acid hydrolysis removes ornithine modifications47 and because the molecule is predicted to contain both two d- and two l-ornithines, an absolute assignment could notbe established by this approach. Nonetheless, in line with this supportingdata, it appears reasonable to assume that the structure of ClDaf 2 is consistent with the arrangement of epimerase domainsin the ClDaf NRPS modules (2 (Table S1). The DHB unit itself is expected to be biosynthesized from chorismateby DafC, DafD, DafE, and DafF (Table S1), that bearhigh homology to the proteins required for DHB biosynthesis for enterobactinin E. coli. and the acetyl transferase dafJ (expected to mediate ornithine N-acetylation) , where DafT1–4 share high homology with enterobactinuptake transporters FepC, G, D, and B respectively,50 likely involved in 2:iron uptake, and whereDafT5/6 (predicted ABC efflux transporters) are likely involved inClDaf 2 efflux.In line with the eight identified monomers of ClDaf domains 1.Four od-isomer 1C. The itandards S9. This tandards S9. This tandards S9. This  E. coli. Regardintandards S9. This tandards S9. This 2 and Pyr 1 were demonstrated to be biosynthesized independently in D. fulvum using the genetic inactivation of their respectiveBGCs, their proximity in this hybrid BGC invited the investigationof a possible hybrid siderophore-antibiotic molecule, especially inthe context of previously described natural sideromycins such as albomycin,salmycin, and ferrimycin.18 To this end, it was found that when D. fulvum wasgrown in a modified medium containing mannitol as a carbon source,a secondary metabolite was identified with the isotopic profile ofa chlorinated molecule and with a mass matching a potential conjugateof ClDaf 2 and Pyr 1 , later named ClDaf-Pyr 4. It was then found that ClDaf-Pyr 4 was not detectedin cultures of D. fulvum mutants not producing Pyr 1 (D. fulvum:ΔpyrA) or ClDaf 2 (D. fulvum:ΔdafA) . Rather unexpectedly it was then found that supplementationof D. fulvum:ΔpyrA with exogenous pyridomycin(100 μM) complemented the detection of ClDaf-Pyr 4 . Togetherthese results supported that the detected molecule consists of bothClDaf 2 and Pyr 1 and that its formationoccurs following the synthesis of the individual natural products.While ClDaf 4 could be formed from ClDaf 2 and Pyr 1 in the absence of biological material(bacteria/enzymes), we first attempted to mix purified Pyr 1 and ClDaf 2, but no product was observed by eitherMALDI-TOF or LC-MS. Taking into account the role of siderophores inchelating ferric iron, and that the latter is a known redox-activemetal in a number of biological systems,54 we then evaluated whether theaddition of iron to the mixture of Pyr 1 and ClDaf 2 had any impact. Significantly, this experiment showed thatthe addition of noncatalytic quantities of ferric iron . The formation of noncomplexed ClDaf-Pyr 4 was also observed with CuSO4 (10 equiv) but notwith ferrous iron (FeSO4), CoCl3, or GaCl3  . Together these data indicate the notion that onlyredox-active transition metals acting as oxidation reagents supportthe formation of ClDaf-Pyr 4.To determine if ClDaf-Pyr 4:iron, wasshown to have a HRMS of [M + H]+ = 1592.5743 . Mass spectrometry fragmentation of ClDaf-Pyr 4 suggested that the molecules were covalently linked at thechloro-DHB moiety of ClDaf 2 but could not provide furtherresolution (data not shown). Solution NMR analysis of the purified 4:gallium complex was carried out to verify the conjugatestructure. 1H and 13C NMR analysis in acetonitrile-d3/D2O (50% v/v) confirmed the detectionof the majority of the signals from both Pyr 1 and ClDaf 2 (Table S3). Important differenceswere, however, observed for the signals corresponding to the chloro-DHBmoiety of ClDaf 2, where only a single aromatic protonin the 6.5–7.0 ppm range was detected, indicating that theother proton is lost to form the covalent bond. 13C-HMBCdata of this remaining proton had scalar correlations through twoand three bonds with C-1, 3, 4, and 6 of the chloro-DHB, but not withthe carbonyl (CO), confirming that it corresponds to position 5 (H-5),and provided additional evidence that the covalent bond was formedwith C-6  as described further below.The iron complexedform of ClDaf-Pyr, with C-6 2D.NOESYe moiety 2D. Toget4 represents a rare molecular feature, witha similar bond previously only identified in the natural siderophorechryseomonin. The retrosynthesis of chryseomonin from chrysobactinby Adolphs and colleagues55 was achievedusing chemistry described by Saxena et al.,56 where pyridine was linked to the catechol moiety of chrysobactinusing molecular iodine as an oxidant. Neiland and co-workers57 then showed that the mechanism of this conjugationinvolved a formal oxidation of the catechol to its quinone form, followedby the Michael-type addition of the nucleophilic pyridine nitrogenon the C-6 electrophilic carbon of the quinone. Several publicationshave appeared in recent years documenting the same mechanism withvarious other nucleophiles.58 In parallel,ferric iron is a known oxidation agent of catechols, which can mediatethe concomitant formation of semiquinone radicals, and ultimatelyquinones when excess ferric iron is provided.59 Therefore, it seemed plausible that the newly identified conjugationstrategy based on the use of iron(III) also proceeded through a Michael-typeaddition reaction. In our attempt to validate this prediction, a numberof experiments were conducted and cross-referenced for the final results.The catechol–pyridinium linkagefound in the structure ofClDaf-Pyr 4. For this goal, 4-chloro-2,3-dihydroxybenzoatemethyl ester 5 and 3-methylpyridine 12 , molecular iodine (1 equiv),and silver oxide resulted in the formation of product 16, with identical regiospecific pyridinium coupling of ClDaf-Pyr 4, as confirmed by NMR NOESY/HMBC correlations . In a similar manner, these threeoxidants also allowed conjugation of the non-chlorinated catecholmethyl esters 6 with pyridine derivatives (Table S4). When the selected catechol methylester mimics were redox inactive, such as compounds 7–10, neither FeCl3, I2,nor Ag2O were able to mediate their coupling with the pyridines(Table S4). In addition, the oxidationof model compound 11 was also tested by electrochemistry(in a nondivided cell) and found to also allow for the formation ofthe identical region-specific conjugate 20  as produced using FeCl3, I2, and Ag2O. With respect to the reactionefficiency, FeCl3, I2, and Ag2O allallowed for bonding the catechol moiety with 3- and 4-methylpyridines 12 and 13, while lower yields were observed with2-methylpyridines 14, and no product observed with 2,6-dimethylpyridine 15. Lower yields experienced with compound 14 and lack of reactivity with 15 can be rationally explainedbased on obstructed access to the nitrogen atom in these substrates.The fact that identical products formed with a variety of oxidantspoints out the fact that the Fe(III)-mediated conjugation followsthe same mechanistic pathway. In a way, the Fe-mediated coupling mostclosely resembles the one described for I2, even thoughin both cases the formation of the respective quinone could not bedirectly confirmed in either case. It therefore appears that in thepresence of an excess amount of FeCl3 the reaction equilibriumis pushed to catechol oxidation with the transient formation of thehighly reactive quinone, after which the pyridine nitrogen acts asa nucleophile by attacking the most electrophilic C-6 of the putativequinone in a Michael-type addition reaction trichloride method to produce 18a  or through the iodine-mediated methodto give compound 18b. It was postulated that dechlorinationof the 18 conjugate would introduce an additional aromatichydrogen into the catechol moiety of the conjugate to allow for thefacile distinction between two adjacent aromatic doublets with a large 3J-coupling constant (in the case of a dechlorinatedC6-N conjugate) or with a small 4J-couplingconstant . To achieve this dechlorination,reductive hydrogenation of 18 over a palladium catalystwas used,60 which also mediated the reduction of thepyridinium salts to the corresponding piperidine derivatives . Using this method, 18b (generatedusing iodine) was dechlorinated to the catechol-methylpiperidine product(18f) with the proton NMR of 18f revealingtwo aromatic doublets with large 3J-couplingconstants (3J = 8.5 Hz) at 6.71 and 6.87ppm, which, together with other NMR data  further consolidated that the C-Nconjugation occurs specifically at C6 of the catechol. When this procedurewas applied to the iron generated 18a, the respectivedechlorinated product 18e was obtained, with the correspondingproton NMR analysis mirroring those of 18f  and confirming that the conjugationproducts were identical. Collectively, these data validate that thechlorocatechol–pyridine linkage occurs between C6 of the catecholand the pyridine nitrogen atom. Unfortunately we were not able toextend this approach to ClDaf-Pyr:Ga 4:Gallium, as itshydrogenation led to uncontrolled degradation of the conjugate.To gain additional evidence on the C6-Nlinkage between the chlorocatechol and pyridine moieties of the generatedpyridinium conjugates, we decided to conduct further investigationon the model conjugate ith refs −63)  was synthesized from 5-carboxytetramethylrhodamineand 3-picolylamine. 3-Pyridyl-TAMRA 21 was then conjugatedto ClDaf 2 in the presence of 10 equiv of FeCl3, allowing for the successful formation of the iron bound ClDaf-3-pyridyl-TAMRAconjugate 22 .To evaluate the biological “Trojan horse”potential of siderophore conjugates generated using the here uncoveredcatechol–pyridine conjugation, it was decided to commence withthe attachment of a fluorescent cargo molecule to ClDaf ugate 22 4A. Follo22 conjugatecould be taken up by bacteria, confocal microscopy on D. fulvum was used to follow the compound uptake. The results showed thatin the presence of ClDaf-3-pyridyl-TAMRA 22 bacteriareadily became fluorescent , while this effect was less pronouncedin the presence of equimolar concentrations of the nonconjugated 3-pyridyl-TAMRA 21 , and the corresponding fluorescence uptake wasfound to be significantly decreased , in line with the before described constitutiveand non-iron regulated expression of the ClDaf BGC . Finally, uptake of ClDaf-3-pyridyl-TAMRA 22 and 3-pyridyl-TAMRA 21 was also evaluatedin other bacterial species, with data revealing ClDaf-3-pyridyl-TAMRA 22 uptake by all evaluated Dactylosporangium sp. and Streptomyces coelicolor, while no significantuptake of either fluorophore was observed for S. aureus and P. aeruginosa . Together these data support the hypothesis that ClDaf-3-pyridyl-TAMRA 22 can be actively taken up as a “Trojan horse fluorophore”by a number of D. fulvum and other closely relatedbacteria.To identify whether theClDaf-3-pyridyl-TAMRA TAMRA 21 4B. To suecreased 4B. As anTAMRA 22 4C. DIP m22 conjugates, it was questionedwhether biomimetic conjugates could allow for antibiotic uptake andsubsequent antibiotic activity. Initial studies with ClDaf-Pyr 4 (up to 50 μM) found it to be inactive against other Dactylosporangium strains, though neither of these bacteriawere susceptible to Pyr 1. To elucidate if antibacterialactivity could be attained, it was thus decided to evaluate the potentialof coupling other antibiotics to ClDaf 2. With D. fulvum being a Gram-positive bacterium (without an outercell membrane), a β-lactam antibiotic was first selected (23) to probe siderophore–antibiotic conjugate penetrationacross the well wall to the peptidoglycan layer (where the targetpenicillin-binding proteins are located) without having to cross amembrane. Second, a rifamycin (25) was selected, as ittargets the cytosol-located DNA dependent RNA polymerase (RNAP) andthus requires translocation across the bacterial membrane. To allowfor biomimetic conjugation to ClDaf 2, 3-pyridyl moietieswere introduced into these antibiotics to give 3-pyridyl-penicillin 23, a penicillin G analogue where the 3-pyridine group replacedthe phenyl group, and 3-pyridyl-rifampicin 25, a rifampicinanalogue with a 3-pyridine extension on the rifampicin piperazinefragment (23 and 3-pyridyl-rifampicin 25 maintained antibiotic activity on D. fulvum (27) was commercially obtained, but it failed to show appreciableantibiotic activity against D. fulvum.Following evidence for the uptake of fluorescentClDaf-3-pyridyl-TAMRA fragment 5.Both 3. fulvum 1. In add23 and 3-pyridyl-rifampicin 25 were conjugated with ClDaf 2 using the ferriciron approach, to give the expected ClDaf-3-pyridyl-penicillin 24 and ClDaf-3-pyridyl-rifampicin 26 conjugates,respectively . As a note, HRMS confirmedthe mass and expected formula of ClDaf-3-pyridyl penicillin 24; its low stability in D2O resulted in temporalhydrolysis of the 24, which was confirmed by the correspondingNMR analysis. Even if 3-pyridyl norfloxacin 27 was inactiveagainst D. fulvum, ClDaf-3-pyridyl-norfloxacin 28 was prepared to demonstrate the translatability of theiron-based protocol  and 3-pyridyl-rifampicin 25 (MIC = 2.5 μM). Interestingly, ClDaf-3-pyridyl-penicillin 24 was found to retain antibiotic activity against D. fulvum (MIC = 2.5 μM)  were also susceptibleto ClDaf-3-pyridyl-penicillin 24 (D. fulvum susceptibilityto ClDaf-3-pyridyl-penicillin 24 (Table S5).The antibiotic activities of the 3-pyridyl antibiotics  2.5 μM) 1, suggesillin 24 1. Finall26 was encouragingly foundto elicit antibiotic activity against D. fulvum  was confirmedto be cross-resistant to 3-pyridyl-rifampicin 25 butalso to ClDaf-3-pyridyl-rifampicin 26 , in accordance with the constitutiveexpression of the ClDaf BGC and minimal impact of DIP on ClDaf-3-pyridyl-TAMRA 22:iron uptake . Using analyticalUHPLC-MS, ClDaf-3-pyridyl-rifampicin 26:iron was foundto be stable in D. fulvum culture filtrate (spentmedia), with more than 90% recovery measured following 2 days of incubation and without detectable releaseof 3-pyridyl-rifampicin (similar results were observed with incubationin fresh GYM media), suggesting that no conjugate decoupling occurredin the bacterial culture filtrate. These data were in line with thehighly rifamycin-susceptible bacterium, S. aureus, being 400 fold less susceptible to ClDaf-3-pyridyl-rifampicin 26 compared to 3-pyridyl-rifampicin 25. Togetherthese data suggest entry of ClDaf-3-pyridyl-rifampicin 26 into the D. fulvum cytosol.ClDaf-3-pyridyl-rifampicin cies, 26 1. To conpicin 26 1, confir26 mayoccur directly by the antibiotic conjugate itself or alternativelymay require an intracellular decoupling process for rifamycin releaseand subsequent RNAP inhibition. To this end, in vitro biochemical inhibition studies were performed to determine directinhibition of these compounds on commercially available E.coli and S. aureus RNAP assays. As expected,both rifampicin and 3-pyridyl-rifampicin 25 showed potentinhibition of both RNA-polymerases  for iron assimilation. Tothis extent, the discovery and characterization of the ClDaf-pyr 4 conjugate presents a novel insight into the design of siderophore–antibioticconjugates using catechol-containing siderophores.Few natural sideromycins have been discovered thus far, but theyhave served as invaluable blueprints for the rational design of customsiderophores–antibiotic conjugates. These natural systems haveprovided crucial insight into how nature has selected conjugatesthat hijack siderophore uptake systems for bacterial entry and antibioticactivity. Unfortunately, the restricted number of natural siderophore–antibioticconjugates has hindered the capacity to copy and translate their naturallyselected chemical properties for the synthesis of custom siderophore–antibioticconjugates. Moreover, these natural molecules are constituted by antibioticscoupled to hydroxamate-based siderophores, such as ferrichrome, ferrioxamine,and danoxamine, and therefore, they provide limited insight into howantibiotics can be coupled on to catecholate-containing siderophores.This is particularly important because antibiotic penetration intoGram-negative bacteria is a major hurdle, and many of these bacteriause catechol-containing siderophores  is intriguing and suggests thatsuch conjugates can potentially be formed in environments rich inferric iron. Interestingly, the critical role of environmental ironwas previously demonstrated for catechol-based reactions such as formussel adhesion.77Among theknown natural conjugates of siderophores and antibiotics,ClDaf-Pyr Enterobacterales, Acinetobacter baumannii, provide evidence that this conjugate is taken up preferentiallyover the unconjugated 3-pyridyl-TAMRA 21 and that thisuptake can be decreased through competition with unconjugated ClDaf 2 or increased by genetically preventing the bacteria fromproducing competing ClDaf 2. Similarly, the conjugatedantibiotics were found to have antibiotic activity suggesting thatthey reached their respective targets. Siderophore transporters areoften encoded by in cluster genes , and multiple attempts were made to link these transporterswith the uptake of the ClDaf conjugates. Unfortunately, using routinegenetic approaches that commonly work for D. fulvum , to disrupt dafT3 or dafT4 was not successful, neither by single cross over inactivation  nor by double cross over gene removal (onlysingle cross over achieved). The reason for this inability to knockoutthe transporters suggests that they play a critical role in bacterialviability or may be related to the high local GC content (75–80%).Further work is therefore required to pinpoint the exact mechanismof ClDaf-cargo uptake.The biological studies presented here go a long way towardcharacterizingthe uptake of catechol-pyridine-based siderophore antibiotic conjugates.Uptake studies with conjugated fluorophores , biochemicaldata showed that this inhibition could occur directly, and that decouplingwas not required. At this point, it is difficult to unambiguouslyestablish the existence of an intracellular decoupling mechanism,and further work with other conjugates is needed to address this question.In addition to the uptake of ClDaf conjugates,it was of interestto find out whether an intracellular decoupling mechanism existedto allow for antibiotic release. The design of ClDaf-3-pyridyl-rifampicin Together, this work has brought to light a novel “natural”mechanism by which catechol-containing siderophores can be coupledto antibiotics through a previously unexploited manner. This mechanismof this siderophore-antibiotic coupling was shown not to be restrictedto ClDaf-Pyr, and could be translate to other pyridine-based bioactiveor fluorescent molecules. Experiments with the model substrates suggestthat such conjugation can also be achieved with other catechol-containingsiderophores, as the main requirement is the need for a catechol group.Further work is now in progress with the focus on expanding this researchto target pathogenic bacteria for which antibiotic R&D is an ongoingpriority.


Nuevo Amanecer-II intervention, tested through a randomized controlled trial, reduced anxiety and improved stress management skills among Spanish-speaking Latina breast cancer survivors. This paper describes the implementation and equity evaluation outcomes of the Nuevo Amanecer-II intervention delivered in three California rural communities.The 10-week Using implementation and equity frameworks, concurrent convergent mixed methods were applied to evaluate implementation  and equity (shared power and capacity building) outcomes. Quantitative data were collected using tracking forms, fidelity rating forms, and program evaluation surveys; qualitative data were collected using semi-structured in-depth interviews. Respondents included community-based organization (CBO) administrators, recruiters, compañeras (interventionists), and program participants.Of 76 women randomized to the intervention, 65 (86%) completed at least 7 of 10 sessions. Participants’ knowledge (85% correct of 7 questions) and skills mastery were high (85% able to correctly perform 14 skills). Mean fidelity ratings across compañeras ranged from 3.8 (modeled skills) to 5.0 (used supportive/caring communication); 1–5 scale. The program was rated as very good/excellent by 90% of participants. Participants and compañeras suggested including family members; compañeras suggested expanding content on managing thoughts and mood and healthy living and having access to participant’s survivorship care plan to tailor breast cancer information. CBOs adopted the program because it aligned with their priority populations and mission. Building on CBOs’ knowledge, resources, and infrastructure, implementation success was due to shared power, learning, responsibility, and co-ownership, resulting in a co-created tailored program for community and organizational contexts. Building intervention capacity prior to implementation, providing funding, and ongoing technical support to CBOs were vital for fidelity and enhancement of recruiter and compañera professional skills. Two of three CBOs created plans for program sustainability beyond the clinical trial; all administrators discussed the need for new funding sources to sustain the program as delivered.Building on community assets and using equitable participatory research processes were central to the successful implementation of a peer-delivered psychosocial intervention in three rural communities among Spanish-speaking Latinas with breast cancer.The online version contains supplementary material available at 10.1186/s13690-023-01207-y. Psychosocial interventions for women with breast cancer can effectively decrease psychological distress and improve quality of life , 2. ThesDespite efforts to provide supportive services to women with breast cancer, very few have been offered to rural Latinas with breast cancer –8, repreNuevo Amanecer (NA) program into Nuevo Amanecer-II (NA-II), after demonstrating the efficacy of NA among urban Latinas living with newly diagnosed breast cancer [NA for these rural settings are described elsewhere [NA-II was evaluated with a 6-month randomized controlled trial (RCT), and was found to reduce anxiety and improve stress management skills [NA-II within three rural communities using the Proctor implementation Outcomes Framework [To fill this gap, we designed a stress reduction intervention specifically for rural-dwelling Spanish-speaking breast cancer survivors. We partnered with three CBOs in rural settings to translate/adapt our first-generation t cancer . The adalsewhere . The efft skills . Brieflyramework  and the ramework . ResultsNA-II was a partnership between the University of California San Francisco (UCSF) (academic partner), Círculo de Vida Cancer Support and Resource Center (lead community partner/compañera (interventionist) supervisor), and three CBOs that implemented the program in rural California communities. The lead community partner is a bilingual-bicultural clinical psychologist, Co-Principal Investigator on the study, and Executive Director of a San-Francisco-based CBO providing cancer support services to Latinos. Partnering CBOs serving as implementation sites included: WomenCARE , Kaweah Delta Health Care District , and Cancer Resource Center of the Desert . Sites are described in detail elsewhere [lsewhere .NA-II was guided by the Transcreation Framework [NA-II partners were engaged in all research phases . Monthly partnership meetings included all study staff . The partnership shared responsibility for and ownership of intervention and data collection activities, while emphasizing CBO’s strengths and resources and building capacity [Implementation of ramework  and CBPRramework . NA-II pcapacity .NA-II in the community, explained the study, and enrolled eligible women . Two individuals (Latina breast cancer survivors at least three years post-diagnosis with no recurrence) were identified by each CBO to deliver the intervention (compañeras). Recruiters and compañeras were trained by the academic and lead community partners . The lead community partner supervised the compañeras in the field.CBOs received funds for study implementation  and controlled their budget. Three types of CBO personnel participated in the study: administrators, recruiters, and compañeras (interventionists). One administrator from each CBO was actively involved throughout the study, serving as key decision maker regarding program design and implementation. Input was secured throughout from all study staff. Two CBO staff members or volunteers were identified by each administrator to be recruiters. Recruiters promoted NA-II was a 10-week structured program delivered in Spanish by a trained compañera in the woman’s home or alternate site chosen by participants. Structured weekly modules provided training in cognitive-behavioral coping skills to manage stress and emotions and emotional support from the compañera . The program is described in detail elsewhere [lsewhere . SessionWe used a concurrent convergent mixed-methods design with qualitative and quantitative data collected from multiple perspectives . Multiple data types were collected concurrently, analyzed separately, and then integrated and converged to conduct a comprehensive equity-informed implementation process evaluation . The evaCBO administrators, recruiters, and compañeras were contacted for a semi-structured interview about program implementation. Compañeras completed a structured program tracking form for all intervention group participants (end-users) after each weekly session. The compañera supervisor completed a structured fidelity rating form for observed program sessions. All participants completing the program  were invited to complete a structured program evaluation survey. We randomly selected 10 participants who completed the program evaluation survey for a semi-structured interview about their experiences.Five types (sources) of program evaluation data were collected. Data were managed using a secure REDCap  data sysRecruiters used a paper tracking form to document recruitment and retention for each potential participant (name obtained through outreach). The form included name, contact information, study ID, study eligibility questions, and a check list of study enrollment requirements , with places to record dates/times of phone calls/contacts, recruitment disposition , and reasons why participants did not enroll . The academic team entered all RCT tracking forms into the REDCap data system. Similar tracking forms were used for women enrolled in the study that included a check list of 3-month study requirements: 3-month survey, program evaluation survey (if assigned to intervention group) and 6-month survey. The disposition  and reasons why participants did not complete the surveys  or reasons that intervention group women discontinued the study at any point  were documented. The academic team used tracking forms to assess retention rates.The compañera supervisor made site visits to CBOs to directly observe intervention sessions (1–2 intervention sessions per compañera). Using structured rating scales , the supervisor rated compliance with six program components  and the extent to which compañeras encouraged participants to practice the seven skills being taught.Compañeras completed structured program tracking forms after each session and recorded program attendance and logistics, reasons why participants missed a session, and several aspects of program uptake  for that week (yes or no), whether the participant reported difficulty in doing the goal (yes or no) and type of difficulty (open-ended), whether participants were able to answer correctly a few questions about a session’s material (correct or incorrect), and whether they were able to demonstrate skills covered in the prior session (yes or no).A few weeks after completing the program, a structured program evaluation survey was administered by telephone by a bilingual-bicultural research associate to participants who completed at least 7 of 10 sessions. The interview lasted about 10-minutes and women received $10.After the RCT, all CBO administrators, recruiters, compañeras, and a subsample of participants were invited to semi-structured interviews via telephone to debrief them about their experiences in implementing the program and participating in the study. Interviews with administrators were conducted in English (by informants’ choice) by a trained bilingual-bicultural interviewer and lasted 60-minutes. Interviews with recruiters and compañeras were conducted in Spanish (by informants’ choice) by a trained bilingual-bicultural interviewer and lasted 90-minutes. Administrators, recruiters, and compañeras each received $50. Participant semi-structured interviews were conducted in Spanish via telephone by a trained bilingual-bicultural interviewer; the interview lasted 30-minutes, and each participant received $25.Semi-structured interviews were audio-recorded and transcribed verbatim in English or Spanish by a professional transcription service. Transcriptions were de-identified and were analyzed in their original language to prevent nuances from getting ‘lost in translation’ .Implementation outcomes include: feasibility, fidelity, acceptability, adoption, appropriateness, and sustainability . Shared Feasibility is defined as the extent to which a program can be successfully used or carried out within a given setting [ setting . We focuFidelity is the degree to which a program was implemented as described in the original protocol [NA-II, fidelity was operationalized separately for participants (adherence to program) and compañeras (adherence to program delivery). For participants, fidelity was operationalized in terms of 1) participants’ adherence to and uptake of the program protocol as noted on tracking forms by compañeras. For compañeras, fidelity was operationalized in terms of (1) compañeras’ adherence to the program delivery protocol, and (2) the quality of program delivery, based on supervisor ratings during directly observed sessions.protocol . For NA-Acceptability reflects participants’ and compañeras’ perceptions of whether the program was agreeable, palatable, or satisfactory [sfactory . To asseAdoption is defined as the intention, initial decision, or action to employ an evidence-based program by CBO administrators as part of the real-world implementation efforts in their settings [NA-II and its relevance to their site and community needs.settings . We askeAppropriateness reflects perceptions of the fit or practicability of the program and research methods [ methods . ApproprSustainability is defined as the extent to which a newly implemented program is maintained or institutionalized within a CBO’s ongoing operations [erations . SustainShared power reflects the perceptions of individuals engaged in the partnership including leadership, dynamics, communication, decision-making, resources, governance mechanisms, efficiency, and partnership challenges [allenges , 28. RelCapacity building reflects perceptions of personal growth  or how their organization was enhanced  as a result of the partnership [Nuevo Amanecer program benefit your organization?” and “What were the changes in your community or organization as a result of this study?”tnership , 28. RelWe first analyzed quantitative and qualitative data separately then converged qualitative and quantitative findings.Feasibility data from the RCT and compañera program tracking forms were summarized in terms of frequencies and percentages for the recruitment rate (participants enrolled/invited/ineligible), retention (completed 6-month survey), and program dose (completed at least 7 program sessions/assigned to intervention group).The compañera supervisor’s fidelity rating forms were summarized across compañeras using means and standard deviations. For the seven skills, we report the frequency of how much compañeras encouraged participants to practice skills.Acceptability outcomes from the structured program evaluation survey were summarized in terms of frequencies and percentages. Semi-structured interviews with participants and compañeras were analyzed using a deductive thematic approach  using DeFor adoption, appropriateness, sustainability, shared power, and capacity building outcomes, the semi-structured interviews with CBO administrators, recruiters, and compañeras were analyzed using the similar methods  using DeWe conducted semi-structured in-depth interviews with all three CBO administrators, four of five recruiters (one was unavailable), and five of six compañeras (one was unavailable), and nine of 10 participants sampled (one was unavailable). Sixty of 65 participants completed the program evaluation survey (5 were unable to be reached).CBOs reached their enrollment goal of 50 women each (two enrolled 50 women and one enrolled 53 women). Across sites, 231 women were invited to participate, and 24 were ineligible. Of 207 eligible, 54 refused for a recruitment rate of 74% (207/231). We randomly assigned the final sample of 153 women to the intervention (n = 76) or control group (n = 77). Six-month retention was 92% overall (140/153), 88% for the intervention group, and 95% for the control group.≥ 7 sessions were no longer needing a support program because they had completed active treatment, treatment side effects, and travel.Of 76 women randomized to the intervention group, 65 (86%) completed at least 7 of 10 sessions . Primary reasons women did not complete Across sessions, participants’ knowledge (> 85% correct on seven questions) and uptake of skills mastery were high ; Table Mean fidelity ratings across the 6 compañeras ranged from 3.8 (modeled skills presented) to 5.0 (used supportive/caring communication) on the 1–5 rating scale (Table The compañera supervisor (direct observer) rated highly the compañeras’ ability to deliver the program as designed, and this was supported further by compañeras’ ratings of participants’ ability to learn new information, master skills, and complete most weekly goals.≥ 7 sessions, 60 (92%) completed the program evaluation survey.Of 65 intervention group participants who completed Regarding program timing, 55% would have preferred to have started the program when they were diagnosed, a quarter when they were undergoing treatment, and 18% after Table . No one Qualitative results from participants and compañeras confirmed quantitative results Table  with theThe program was rated as very good/excellent by most participants (90%). The DVD was rated as very good/excellent by 77% and poor/fair/good by 17%. Few respondents (7%) lacked a DVD player. Most respondents (97%) rated compañeras’ skills as very good/excellent. Suggestions from qualitative participant and compañera interviews included having alternative ways of playing the videos. Participants related strengths of the program as having personal contact with a breast cancer survivor.Almost all participants (98%) rated how much the program helped them cope with breast cancer as quite a bit/very much. The highest ratings pertained to communicating with doctors (92% quite/very useful) and family members (94%), managing thoughts and mood (95%), managing stress (97%), healthy eating (98%), and setting goals (93%). Participants and compañeras related strengths of the program as stress management skills, communication skills with doctors and family, learning to cope with thoughts and mood, and goal setting. The lowest ratings were for cancer information (77%) and survivorship care planning (70%). Participants found the breast cancer treatment video less applicable to their post-treatment circumstances.Participants and compañeras suggested including family members. Compañeras suggested expanding content on managing thoughts and mood and healthy living and having access to participant’s survivorship care plan to tailor breast cancer information.Ease of understanding the manual (88% reported being quite/very easy) and program convenience (88% reported being very convenient) received high participant ratings. About 3/4 (77%) of participants reported currently practicing the skills often. From the qualitative results, participants and compañeras said the program was easy to use but found some content repetitive. Participants found the program convenient because sessions were delivered in participants’ homes or CBO sites. Compañeras identified challenges with scheduling sessions and travel distances (mileage was reimbursed). Compañeras’ mean weekly round trip travel time was 37 min (SD = 24.4) and mean round trip travel distance was 29 miles (SD = 24.5).NA-II . Sub-themes included availability of CBO internal resources  for program implementation, and pre-existing relationships with external organizations  to obtain program referrals or additional resources .Administrators highlighted how they capitalized on their program fit their community setting because it utilized community health workers, home visits, and one-on-one delivery given common barriers in rural areas . Administrators and compañeras stressed that elicitation and incorporation of their input on program and training materials prior to implementation was a major strength.We identified two overarching themes  on contextual factors that supported/hindered appropriateness Table . Respondinput on research methods so they were more appropriate and easier to use. Administrators and recruiters discussed that having the flexibility with locale to conduct program sessions or enrollment at the CBO office or women’s homes made it practical.Recruiters commented on how the academic partner made adaptations to the research methods  based on their staffing, with three sub-themes , affected appropriateness for their setting. Administrators talked positively about their ability to hire community members to deliver the program or enroll women. They also expressed challenges overseeing staff, e.g., role confusion or lack of communication.Administrators discussed how staff turnover was a limiting factor. One organization lost one of their compañeras and had to pause recruitment until the position was filled. Another organization had difficulty identifying a suitable, bilingual recruiter.Lastly, need for new funding sources to sustain the program as delivered. Two administrators talked about potential funding sources . All administrators discussed how they would incorporate aspects of program into current CBO services such as delivering skills training within their support groups. They discussed keeping the program but altering the delivery mode to a group setting or individual sessions at the CBO (no home visits).Administrators identified two sustainability related themes Table . All admpartnership structural dynamics  that supported program implementation and indirect staff . All three administrators spoke highly of the partnership collaboration and the high-quality communication that provided opportunity for information exchange between CBO and academic partners. They valued the monthly conference calls to address issues, troubleshoot, and brainstorm solutions. There was resounding agreement among the administrators about the professionalism of the academic and lead community partner and the sense of mutual respect. Administrators spoke about the academic team’s reputation working with community as a strength of the partnership. One administrator said that they joined the partnership because a colleague from another organization (not involved in the project) recommended the principal investigators and program.We identified five themes regarding shared power Table . Administraining and technical support, three sub-themes emerged . Compañeras and recruiters spoke highly of the training provided and that they felt prepared for their duties. Compañeras would have liked training on additional breast cancer related content . An administrator suggested that compañeras and recruiters be cross trained on each other’s roles to address turnover. Administrators at times were unsure how to best support recruiters and compañeras given the roles of the academic and lead community partner.We identified three overarching themes related to capacity building, training/ongoing technical support, individual-level capacity, and CBO-level capacity Table . With reprovision of ongoing technical support to compañeras and recruiters by the academic team and lead supervisor. Compañeras, appreciated receiving positive reinforcement and feedback on program delivery via fidelity checks. However, they mentioned that fidelity check visits were difficult to coordinate given that they required alignment across supervisor, compañera, and participant schedules. Conducting fidelity checks required the supervisor to travel from 100 to 600 miles to study sites. In addition to the monthly meetings of all staff, there was a sentiment that compañeras needed more support to process their interactions with participants.Informants reported that a key determinant of success was role modeling had on enhancing their program delivery or recruitment capacity.Recruiters discussed that the academic team was readily accessible to answer questions. Two administrators discussed how well the academic team supported their recruiters. Another administrator talked about how the academic team assisted them in developing institutional review board approved procedures to recruit potential participants using hospital records, providing template letters and post cards. Lastly, compañeras and recruiters spoke highly of the value that enhanced capacity to deliver the NA-II program and their self-application of program skills to their personal lives. Several compañeras wished they had had access to this type of program when they were undergoing their diagnosis. Recruiters reported increased research skills capacity and increased knowledge of the psychosocial impact of breast cancer.Compañeras reported their individual-level enhancing their CBO’s program capacity. They saw the cognitive behavioral coping skills as being transferable to individuals with other cancer types/other genders. Administrators indicated that because the organization had limited staff development funds, staff were able to obtain skills through the study that they otherwise would not have. In addition, it enhanced their CBO’s research skills capacity to participate in other research projects with other researchers. Lastly, administrators discussed that their participation enhanced their CBO’s reputation in the community through media coverage or town halls of their CBO’s participation in NA-II.At the CBO-level, administrators talked about Distilled from the data, we summarize best practices for how investigators can equitably engage community organizations to implement behavioral interventions Table . HighligNA-II, a peer delivered stress management program designed for Spanish-speaking Latinas with breast cancer in partnership with CBOs serving three rural California communities. A broad implementation evaluation framework was applied to explore various implementation process outcomes, supplemented with an equity evaluation framework that went beyond traditional implementation science outcomes.This study used an innovative, comprehensive, mixed methods approach to evaluate the implementation processes of NA-II design, which was guided by the Transcreation Framework for Community-Engaged Behavioral Interventions to Reduce Health Disparities [Our choice of this broad approach was based on the innovative parities . The TraWe illustrate here how we tailored our implementation evaluation to this unique approach. Our traditional implementation evaluation framework included feasibility, fidelity, acceptability, adoption, appropriateness, and sustainability. However, we defined these in somewhat unique ways. For example, fidelity was examined in terms of participants’ uptake of program components  as well as in terms of community-based interventionists’ ability to deliver the program per protocol (unique definition). We selected equity outcomes to evaluate the extent to which we succeeded in sharing power and building capacity. In another example, we evaluated shared power by exploring their sense of the partnership dynamics such as governance and communication.NA-II aligned with the CBOs’ priority populations and missions. Understanding local community contexts for implementing psychosocial interventions helps address large differences that can exist across rural communities and relative to the original program test sites [Implementing psychosocial interventions in rural areas face considerable challenges over and above those in urban areas. Because of the paucity of services in rural areas, strategies require extensive collaboration between institutions to augment conventional delivery systems . The thrst sites . ContextEquity success was due largely to use of strategies of shared responsibility and learning and co-ownership (shared power), resulting in a co-created, co-tailored program for Spanish-speaking Latinas with breast cancer and the community and organizational context. In this study, it was important that all partnership members share responsibility and ownership of intervention and data collection processes  for a suChallenges to community involvement typically relate to communication, inclusiveness, and trust issues, which can affect implementation  and CBPRNA-II was due in part to the long history of the academic and lead community partners’ use of CBPR principles to test community-based psychosocial interventions among Latina breast cancer survivors. Even though the three rural CBOs were new partners, they recognized the reputations and the prior partnership of the academic and lead community partner with other CBOs to improve psychosocial health among Latinos with cancer [Successful implementation of h cancer , 40–43. NA-II intervention reduced anxiety and improved stress management skills [NA-II; thus more funding to provide technical assistance to achieve these plans would have been extremely helpful.CBOs and staff participating in our study may not be representative of other rural organizations. Results therefore may not generalize to other culturally and linguistically diverse communities and populations. While the t skills , parts ot skills . Sample Nuevo Amanecer-II offered the opportunity for individual and organizational capacity building, an equitable partnership, and an acceptable and effective psychosocial intervention designed for a vulnerable population. The inclusion of both implementation and equity outcomes from multiple community perspectives offers a comprehensive evaluation to better inform community implementation of peer-based programs designed to address populations and settings that have experienced limited access to health preserving resources.Applying an equity-focused approach to co-creation, implementation, and evaluation of Below is the link to the electronic supplementary material.Supplementary Material 1


Engaging young people in sport and physical activity (PA) requires awareness of their preferences and priorities. This study provided less active young people (14–17 years of age) with an opportunity to articulate their perspective on an ideal sport program.The features of a sport program that I would really like to participate in are…. Twenty-four participants then sorted these statements into groups based on similarity of meaning, and 37 participants rated each statement on 5 point Likert scales for its importance  in a program and how common  it is in the programs they participate in. We analysed sorting data using multi-dimensional scaling and hierarchical cluster analysis, and rating data using descriptive statistics.We used online concept mapping, involving qualitative data collection and quantitative data analysis. Forty-eight less active young people in regional Victoria, Australia brainstormed 65 relevant, unique and single-focus statements in response to the prompt: access and convenience ; social connection ; inclusion and belonging ; confidence ; design and structure ; skill development and goals ; cost and time ; age and gender ; and specific features —as the most appropriate representation of the participants’ sorting data. The two single statements—both in the inclusion and belonging cluster—rated most important were a sport program where you are accepted (4.57) and in which you do not feel judged (4.51).We identified a map with the following nine clusters (in order of relative mean importance ratings)—Young people want affordable, accessible, convenient, safe, welcoming, socially engaging and inclusive sport and PA programs. They want programs to be well organised, delivered by experienced coaches with flexible content that builds their confidence, fitness and skills through challenging and fun activities. Youth sport and PA program designers can use this data to inform program development and delivery planning.Victorian Health Promotion Foundation .


Remote patient monitoring (RPM) is being increasingly utilized as a type of telemedicine modality to improve access to quality health care, although there are documented challenges with this type of innovation. The goals of this study were to characterize clinic delivery strategies for an RPM program and to examine barriers and facilitators to program implementation in a variety of community clinic settings.Primary data were collected via individual and small group interviews and surveys of clinical staff from South Carolina primary care clinics participating in an RPM program for patients with diabetes mellitus type 2 in 2019. We used a parallel convergent mixed methods study design with six South Carolina primary care outpatient clinics currently participating in a diabetes remote monitoring program. Clinic staff participants completed surveys to define delivery strategies and experiences with the program in a variety of clinical settings. Interviews of clinic staff examined barriers and facilitators to program implementation guided by the Consolidated Framework for Implementation Research (CFIR). Quantitative survey data were summarized via descriptive statistics. Qualitative data from interviews were analyzed in a template analysis approach with primary themes identified and organized by two independent coders and guided by the CFIR. Quantitative and qualitative findings were then synthesized in a final step.RPM program delivery strategies varied across clinic, patient population, and program domains, largely affected by staffing, leadership buy-in, resources, patient needs, and inter-site communication. Barriers and facilitators to implementation were linked to similar factors that influenced delivery strategy.RPM programs were implemented in a variety of different clinic settings with program delivery tailored to fit within each clinic's workflow and meet patients' needs. By addressing the barriers identified in this study with focused training and support strategies, delivery processes can improve implementation of RPM programs and thus benefit patient outcomes in rural and community settings. For example, one study found that clinics serving low-income patients report greater patient-level RPM barriers, whereas clinics serving middle-income patients report greater system barriers including challenges with program scalability and reach.7 Implementation challenges impede widespread use and risk continued use of small-scale inefficient RPM programs. Community-based practices are especially vulnerable to implementation barriers and yet are uniquely poised to deliver health care to vulnerable populations.Despite the promise of RPM, barriers to implementation and scalability exist.8Partnerships between academic and community health centers may support RPM implementation. Such partnerships can capitalize on existing relationships between patients and their local primary care home with the resources and specialty staffing of a central RPM site. This type of arrangement has demonstrated success in statewide specialty consultation services.RPM intrinsically enables a patient–provider connection that transcends geographic and other barriers. To advance RPM program dissemination, a better understanding of barriers to implementation is needed. We report on implementation experiences from a diabetes RPM program that capitalizes on academic–community partnerships. The objectives of this mixed methods study were to (1) characterize clinic delivery strategies for an RPM program and (2) examine barriers and facilitators to program implementation in underserved and/or low-income community settings.9 with six South Carolina primary care outpatient clinics currently participating in the Technology-Assisted Case Management in Low-income Adults with Type 2 Diabetes (TACM-2) program. These 6 parent clinics comprise 15 individual sites that were actively utilizing the program at time of this study. Practice managers were contacted by email with information about the study and to request participation. This study was approved by the Medical University of South Carolina Institutional Review Board, and a waiver of written informed consent was granted. The Consolidated Criteria for Reporting Qualitative Research (COREQ) checklist guided qualitative data methods and results reporting.10We used a parallel convergent mixed methods study design11 TACM-2 utilizes a cellularly enabled remote monitoring device that transmits home glucose readings to a web-based secure server for provider review. Program organization, oversight, and monitoring is conducted centrally at an academic hospital, whereas local sites maintain responsibility for individual patient care and HgbA1c reporting to central data repository.The TACM-2 program was established in 2016 with the goal of improving chronic disease management in South Carolina. TACM-2 is a care delivery program focused on supporting diabetes and hypertension management, although in this study we focus on diabetes only because barriers to implementation are likely to vary by clinical focus. The program is based on the success of a pilot study, which demonstrated improved diabetes control among participants randomized to TACM compared with controls.12 This framework was developed based on existing implementation theories to guide a pragmatic approach to understanding implementation barriers, facilitators, and processes and has been widely used in chronic illness management research.13 The CFIR includes five main domains that can influence implementation, including individuals involved , inner setting factors , outer setting factors , intervention characteristics , and implementation processes .12The Consolidated Framework for Implementation Research (CFIR) was used to guide data collection tools and assessment of a comprehensive set of implementation factors.Basic program enrollment characteristics, including date of program initiation, number of enrolled patients, and mean HgbA1c values, were collected as part of program participation. Additional data were collected via surveys and interviews. For parent clinics with several locations, surveys were sent to all locations and responses were averaged across the sites to maintain anonymity and address missing data.A clinic representative completed an online survey assessing clinic type, staffing structure, and patient characteristics.Program champions were identified jointly by local clinic staff and academic medical center program staff as the main advocate for the program at the local level. Some criteria considered for champion identification included being a primary contact for the program, having hands-on experience delivering the program, and length of time with program.14 The percentage of clinics endorsing each barrier as moderate or major was calculated. Finally, we assessed perceptions concerning leadership and implementation culture using two 4-item validated scales (1 = strongly disagree to 5 = strongly agree).15 Due to small sample size and symmetric data, average scores (rather than median) were calculated, with higher scores indicating higher leadership and more positive culture.These champions from each clinic location completed an online survey assessing TACM-2 staffing roles and processes. This survey also assessed perceived barriers to carrying out the program  using a 12-item instrument developed for previous implementation studies.16Individual and small group interviews were conducted with the identified clinic program champion and all available team members responsible for TACM-2 delivery and/or clinic leadership. Interviews were completed in-person or via telephone by two female investigators (E.J. and K.R.S.) with doctoral training in qualitative methods, who did not know participants. Using a semi-structured guide informed by the CFIR, participants described their clinic's patient care priorities, previous care practices, and perceptions related to the TACM-2 program including barriers and facilitators to implementation. Interviews were audio-taped and lasted 25–50 min. Strategies to assure theme saturation included using clinics that varied in program implementation processes to ensure coverage of diverse experiences, using probes to explore responses in depth, and monitoring field notes to track emerging themes.17 A template analysis approach was used with an initial codebook guided by the CFIR also allowing new codes to be generated directly from the data.17–19 An iterative process was used with two independent coders. The codebook evolved over time by refining code definitions, collapsing a set of codes, and adding several new codes, while maintaining an audit trail of the process. Coders read and reread each transcript, organizing primary themes and resolving discrepancies in group meetings.21 Results from each source were summarized and compared to identify similarities and differences. The final analysis step involved synthesis of quantitative and qualitative findings.Descriptive statistics were used to summarize quantitative survey data for each clinic. Transcriptions of interviews were analyzed using NVivo software .N = 20 participants overall; range number of participants 1–8) with each clinic represented (n = 6).Six clinics, with 15 unique practice sites participating in TACM-2, were invited to participate in qualitative and quantitative study elements. Five of six parent clinics completed champion and site surveys. We conducted 10 individual and small group interviews  to support patient monitoring. Some clinics only retrieved data to use as ancillary data during office appointments, whereas other clinics used data in real time or between visits to support additional follow-up. While some clinics were highly satisfied with program communication and receipt of supplies and data to monitor patients, others complained about delays. The majority of clinics reported that the program improved quality of care by providing increased communication and monitoring between the clinics and patients.N = 5). The following factors were considered a major or moderate barrier in only one clinic each: having designated staff to coordinate, other issues being higher priority and information technology. The top barrier was time, endorsed by three of five (60%) of clinics. Interviews identified unique themes representing barriers and facilitators to implementation .Limited feedback was provided regarding the complexity of the TACM-2 program and how this impacted implementation. One free clinic and one Federally Qualified Health Center (FQHC) described the TACM-2 program as simple, user-friendly, and easy to implement; yet, staff from a different clinic reported difficulties in troubleshooting the devices. Another FQHC site commented on the value of patient-directed technical support.Patient demand and clinic commitment to address uncontrolled diabetes represented key drivers of implementation. All clinics reported high demand for a program such as TACM-2. Due to high rates of diabetes, lack of insurance, and poverty in the rural and underserved communities, patients experience barriers to blood glucose monitoring that TACM-2 addresses, including expense of testing supplies and medications. One FQHC clinic considered the pros and cons of investing time and resources into programs, ultimately committing to TACM-2 due to high patient demand and promising benefits.In parallel to high patient demand, five clinics perceived that patients were grateful for TACM-2 as it supports diabetes management and provides supplies not otherwise accessible. Participants from these five clinics observed that through the program, patients learned to take ownership of their diabetes and how to identify abnormal glucose levels. Over time, patients valued being able to see their HgbA1c levels decrease.Although TACM-2 was perceived to meet patient demand and improve patient satisfaction, participants within all clinics still noted implementation barriers as some patients lacked transportation for enrollment and follow-up visits. Low educational attainment, low literacy levels, and language barriers were described as challenges to device training. In addition, some clinics perceived that access to technology  represented communication barriers that impacted program feasibility, representing a true digital divide.Several external factors, including diabetes management and reporting guidelines, were considered influential to TACM-2 delivery. Various guidelines were followed for quality initiatives and care goals, and two free clinics directly described how the program facilitated adherence to quality initiatives . The TACM-2 portal for data monitoring and reporting allowed clinics to efficiently aggregate data for reporting.There were mixed perspectives about partnerships between clinics and the academic medical center and their influence on program implementation. Five clinics reported timely and effective communication with the program during early implementation and start-up processes. These clinics appreciated the detailed easy-to-follow setup steps as well as ongoing support for troubleshooting, technical assistance, and reporting.Challenges establishing partnerships were defined by one of the clinics, typically during program setup and abated by identification of a clinic champion and clear delineation of clinic responsibilities. Suggestions to improve program–clinic partnerships included quarterly in-person training and monitoring, connections to other clinics implementing TACM-2 for networking, and more support and resources as patients' transition off the 12-month program.Teamwork and communication within the clinic were viewed as important to program delivery, but clinics varied in the ways they assembled teams around the program. Respondents commonly reported that it took time to establish implementation practices after trialing varied roles and strategies. Regardless of the approach taken by teams, all clinics highlighted the importance of communication for timely completion of program steps. Some successful clinic strategies described for improving communication and teamwork included consideration of varied models to identify best practices and holding routine team meetings to discuss program issues.Perceptions about program compatibility were important to implementation. Reflections included staff perceptions about the suitability of the program to address patient needs as well as consideration of the program's fit within clinic workflows. Generally, respondents emphasized that the program was a good match as it focused on meeting high-risk patients' needs, although a concern was raised about technological access and literacy.Two clinics appreciated that the program streamlined current workflows (facilitating review of data at appointments) and offered additional support (program oversight of patient data). In contrast, a different site highlighted inconsistency between program and clinic practices , which added work and was disruptive.While clear leadership for the program was not explicitly described as a driver of successful program implementation, all sites did identify a program leader and described their varied roles. Leadership structure and involvement varied among sites with most sites having one primary leader and a few sites having a more collaborative leadership team. The majority of leaders were on-site, with some holding administrative roles and others with clinical roles.Resources such as staffing, space, and supplies were considered important to the program and facilitated successful delivery. As described above, clinics had varied models of staffing to support and reinforce the program. Clinics frequently described the burden of program-related tasks in addition to other job responsibilities. Having a small number of staff and high rates of turnover were reported as common challenges to TACM-2 delivery in all free clinics and FQHCs, and retraining efforts drained clinic resources. A few clinics mentioned the need for materials and staff to communicate with Spanish-speaking patients. Suggestions to improve patient engagement from individual clinics included direct technical support, educational materials and support in Spanish, and patients' ability to log on and monitor blood sugar numbers themselves.Quantitative data showed that clinic, patient, and program delivery characteristics varied in TACM-2 clinics. Qualitative data offered additional insight into implementation barriers and facilitators. Survey data showed variability in resources available to support program delivery, and interview data highlighted that appropriate staffing models as well as infrastructure were supportive of the program. Both survey and interview data highlighted time as a key barrier to program implementation. Interviews also highlighted high staff turnover rates as a challenge, causing staff shortages, a need for increased training, and further increasing time demands. Qualitative data also described patient resource barriers including transportation as well as language and knowledge barriers hindering program understanding and the ability to carry out program demands at home.Surveys demonstrated positive perceptions of leadership in clinics, and interviews revealed that while all sites did identify a leader, there were different leadership styles among clinics that may have influenced implementation. Participants appeared to perceive the presence of a collaborative leadership team as facilitating implementation processes. Surveys revealed positive perceptions of the implementation climate for delivery of the program, but interviews highlighted variability in this area with only some clinics reporting positive communication and teamwork practices. Interviews additionally revealed that positive collaborations between the clinics and the academic center contributed to productive implementation climates. High patient demand, or high rates of diabetes in clinic communities, and clinic commitment to help patients were the main facilitators to implementation.22 However, there have been challenges to widespread dissemination of this multifaceted complex innovation.22 This CFIR-guided mixed methods study addressed this research–practice gap and utilized implementation science methodologies12 to examine program delivery strategies and common implementation barriers and facilitators to delivery of a diabetes RPM program in underserved community settings.RPM has been increasingly utilized in clinical practice for patients with diabetes and has demonstrated improved patient outcomes.Delivery strategies varied widely across clinics as they had diverse workflows, staffing and leadership models, and practical support to maintain the program. This variability highlights the need for RPM programs to be flexible and the feasibility of delivering programs in varied settings. Results also identified barriers and facilitators at the clinic, patient, and program levels.leadership buy-in emerged as important to program implementation. However, despite strong endorsement of leadership for the program, practical barriers had to be overcome to facilitate progress. Specifically, staffing was influential to program delivery, as has been documented in RPM studies of other chronic diseases.23 Free clinics generally relied on more volunteer staff, whereas academic clinics relied more heavily on trainees and resident physicians.At the clinic level, All sites struggled with the impact of staff turnover, which demanded additional time for training and programmatic inefficiencies. This could lead to lower program enrollment, less consistent monitoring, less fidelity in process measurement, and diversion of resources to accommodate repeated trainings. Importantly, staffing time increased to meet patient needs, including lower health literacy, lack of diabetes awareness, and non-English-speaking language preference. This may have stretched staffing, increased program-specific burnout, and/or limited available resources to engage other patients.multidisciplinary resources embedded within the clinics, including on-site laboratories, pharmacists, and clinical diabetic educators (CDEs) who provided ancillary support and education. By sharing program responsibilities with other staff members, multidisciplinary clinics may have faced less strain on staffing and time. Additionally, the added educational support and convenience of completing blood draws on-site may influence a patient's likelihood to remain engaged in the program. Further investigation into the impact of multidisciplinary resources on program engagement is important.Several clinics described 24Patient factors influenced program delivery as clinics tailored implementation to meet their needs. The free devices and monitoring were seen as a benefit for low-income and uninsured patients. As such, clinics serving low-income populations may be more likely to invest in the program, and patients may be more likely to engage. Contrarily, a large non-English-speaking population may be a barrier to implementation owing to greater staffing demands, as described above. Patients lacking stable housing, transportation, and insurance face barriers to program retention and follow-up data collection, a metric by which clinics were measured. A tailored implementation approach accounting for needs of the target population is important, as demonstrated by our work and that of others.23–26 In our study, most clinics communicated frequently with the central site and received timely supply shipments, thereby facilitating program implementation.Program factors, including local-central communication patterns and comfort with the data reporting system, impacted program delivery and were represented in reported barriers and facilitators to implementation. Program-level barriers to RPM implementation are well documented and include cost, poor integration with current workflows, data overload, and increased workload for providers.27 Our qualitative data demonstrate the importance of clear expectations and roles, with ambiguities threatening program success.For clinics well versed in online monitoring and data reporting, the program's online reporting system was viewed as beneficial, increasing clinics' abilities to achieve reliable convenient data exchange between patient and provider. Alternatively, some clinics found the program to be taxing on staff and disruptive to usual workflow. Collaboration between academic medical centers and community partners has been accomplished in other studies, as in ours, and has been emphasized as an important tool for disseminated care.28Results highlight the promise of RPM programs in community settings and potential support strategies needed at the clinic, patient, and program levels for implementing RPM programs. Similar to findings in a recent qualitative study of mobile health technologies, we found complex relationships of clinical staff with telehealth programs, specifically regarding inter-site collaboration, care delivery efficiency and flexibility, financial and technological barriers, training requirements, and patient needs and skills.A rigorous planning phase could support the assessment of clinic readiness and patient needs to guide tailored practices for staffing and workflows to set clinics up for success. Building clinic teams, assigning roles and responsibilities, establishing communication preferences, and providing training will also help teams prepare for implementation. Clear messaging in the planning phase can help define expectations to prepare to meet patient needs. More work is needed to guide development of protocols for ongoing training, technical assistance, and troubleshooting challenges to support programs over time.23 Third, the program under study is implemented within a single state, and thus, generalizability is limited. There are differences in state regulations related to RPM across states, including insurance payor variations, which were not assessed in this study.Despite the strengths including use of the CFIR to guide examination of a comprehensive set of implementation factors and our mixed methods approach, there are several limitations of this work. First, findings are subject to nonresponse bias as not all participants responded to all portions of the study. A strength, however, is that all sites were represented in interviews to allow comprehensive qualitative perspectives. Second, the perspective of patients is not captured and should be a focus of future studies, although work in the cardiovascular field suggests that patients appreciate the benefits of RPM.28 to improve patient outcomes. Future research is needed to expand the current study's findings to other geographic areas, which will allow descriptions of differences across state populations and with regard to differences in state regulations and payor variations. Future studies should also evaluate planning, training, team building, and implementation strategies to support program sustainability.Clinic, target population, and program elements impact RPM program delivery, but such programs can be adapted for community clinics with varied structures. Clinical staff perceive a number of barriers to program implementation that can be mitigated with training and support strategies, such as a structured planning to accommodate unique clinic staffing and clinic workflows, support for team-building within clinics, and development of tailored protocols for training, program delivery, and support. Despite implementation challenges, RPM programs can capitalize on clinical staff's motivation and commitment to help patients