Knowledge of associations between fungal hosts and their bacterial associates has steadily grown in recent years as the number and diversity of examinations have increased, but current knowledge is predominantly limited to a small number of fungal taxa and bacterial partners. Here, we screened for potential bacterial associates in over 700 phylogenetically diverse fungal isolates, representing 366 genera, or a tenfold increase compared with previously examined fungal genera, including isolates from several previously unexplored phyla. Both a 16 S rDNA-based exploration of fungal isolates from four distinct culture collections spanning North America, South America and Europe, and a bioinformatic screen for bacterial-specific sequences within fungal genome sequencing projects, revealed that a surprisingly diverse array of bacterial associates are frequently found in otherwise axenic fungal cultures. We demonstrate that bacterial associations with diverse fungal hosts appear to be the rule, rather than the exception, and deserve increased consideration in microbiome studies and in examinations of microbial interactions. Robinson et al. explore bacterial associations in over 700 phylogenetically diverse fungal isolates with 16 S rRNA sequencing of fungal collections from across the globe and bioinformatic screening of publicly available fungal genome sequencing projects. This study reveals that bacterial associations with fungal hosts are widespread, and represents a vast increase in the representation of explored fungal-bacterial relationships. Fungi have been primarily considered as one of the microbial components of the host-associated microbiome in numerous animal and plant studies4 and are often overlooked in human studies5. However, the existence of a fungal bacteriome, consisting of bacteria found both within and in close association with cells of a fungal host, is an emerging concept6.Microbiome research permeates all domains of biology and the concept of the holobiont  is changing the perception of individual biological eukaryotic units7. While community-level studies of environmental co-occurrence between bacteria and fungi are important and have revealed interesting patterns10, such community-level correlations do not reveal specific fungal-bacterial associations. For example, some bacteria and fungi that co-occur under similar environmental conditions, but never actually interact with each other, could be highlighted in a co-occurrence study, but likely be excluded when examined for BFI specifically. Examining bacterial associates of fungal hosts that have been isolated and maintained in otherwise axenic culture removes any uncertainty about the fungal partner, while providing greater confidence that the bacteria form a tight and long-term association with the fungal host. Focusing on fungal isolates has the aforementioned benefits, but distinguishing between true biological associations and other factors resulting in the detection of a bacterial signature (e.g. co-isolation or contamination) can still be difficult without detailed and time-intensive investigations of each putative association. The current catalog of known associations between bacteria and fungal isolates is largely the result of specific examinations of endohyphal bacterial associates of mycorrhizae and plant-associated fungal endophytes, and is focused primarily on a small number of fungal genera from either the early diverging fungal phylum Mucoromycota13 or the large and highly diverse subkingdom Dikarya (Ascomycota and Basidiomycota)17. Despite the growing number of examples of specific bacterial associations with isolated fungal hosts, a broader perspective of both intra and extracellular bacterial associates among the larger diversity of fungal hosts is necessary in order to understand the potential evolutionary and ecological consequences of these interkingdom interactions.The field studying bacterial–fungal interactions (BFI) is complex and dynamic, with research ranging from specific bacterial–fungal interactions to larger scale community analyses18 deliberately sampling the widest possible range of fungal phylogenetic diversity. Stringent quality control standards and procedures were employed for both screens, to increase confidence in the accuracy of the observed results. This work provides a considerably more comprehensive exploration of the fungal bacteriome by examining over 700 fungal isolates, including 366 fungal genera  and multiple representatives from six out of the eight recognized fungal phyla. Putative bacterial associations were found to be both common and complex, with an unexpected diversity of bacteria detected across all examined fungal lineages. This raises a multitude of questions: how frequently do fungi serve as hosts for specialized bacteria, or as potential substrates for bacteria with broad ecological niches? To what extent are bacteria transient or more persistent in their occurrence with fungi? Considering both transient co-occurrences and more persistent associations, what are the potential roles and impacts of the bacteriome on fungi, including impacts on interactions with other microscopic (e.g. protists) or macroscopic  organisms?To address this knowledge gap and gain a more comprehensive view of the diversity of bacterial–fungal associations, we employed two complementary approaches to identify signals of potential bacterial associates among a phylogenetically broad range of fungi. We analyzed 16S ribosomal RNA (rRNA) gene amplicon sequences obtained from total DNA extractions of distinct fungal isolates belonging to four different fungal culture collections from diverse environments within North America, South America, and Europe (hereafter referred to as 16S-CC screen). We also searched for bacterial-specific sequences (hereafter referred to as BSS screen) within publicly available fungal genome sequencing projects from the Joint Genome Institute (JGI) Mycocosm portal,Two complementary methods were used to explore the diversity of constituents of the fungal bacteriome: an amplicon community profiling survey of four independent and predominantly soil-derived culture collections from three geographical origins , and a scan of fungal genome sequencing projects for bacterial genomic signatures.19, were less common in our collections. Our 16S-CC screen included five genera from the Mucoromycota, all of which have previously described bacterial associates 24. This study also represents the first examination of bacterial associates in the Zoopagomycota  rDNA region was used to characterize the diversity of 294 cultivable fungal isolates from four culture collections  were previously described associates of fungi, but in all cases, they associated with different fungal genera than those observed in this screen , with an average of 34 OTUs per isolate, indicating that it was typical for diverse bacteria to co-exist within the examined fungal isolates, many of which have been propagated for years , Massilia , Streptococcus , Brevundimonas , and Sphingomonas  were the most frequently detected bacterial OTUs and were also found in isolates from all four examined fungal phyla. These bacterial genera have been found to be common soil inhabitants and, with the exception of Brevundimonas, these common bacterial genera from the 16S-CC screen were also previously described as associating with fungi29. While 38 previously described bacterial–fungal associations were corroborated using this screen, we report a more than 100-fold increase with an additional 4818 putative genus-level bacterial–fungal associations , Gammaproteobacteria (258), Alphaproteobacteria (256), Actinobacteria (247), and Bacilli (219) were detected most frequently across all examined fungal isolates and OTUs assigned to these five lineages were also responsible for a large  proportion of all 7830 detected bacterial–fungal associations 32. We detected 89 bacterial genera not previously described as associates of Mortierella, although 27 of these have been previously found as associates of other fungi . To our knowledge, bacterial associates have not been previously reported for isolates of Ilyonectria, increasing the need to further validate their associations with bacteria. Bacterial cells were visible in all seven examined fungal cultures  techniques targeting the 16S rRNA to visualize the presence and localization of bacterial associates for seven diverse fungal isolates examined in the 16S-CC screen. Given the large number of fungal isolates examined overall, it would have been impractical to attempt FISH staining on every single isolate. Instead, fungal isolates were selected due to their filamentous growth characteristics , which are ideal for imaging, and because these isolates represent fungal genera with the highest number of isolates in the 16S-CC screen  with bacterial ASVs representing 32 distinct genera . The bacterial genus Lacunisphaera was selected as one of our targets to validate our findings with a novel putative associate, as members of this bacterial genus have not previously been described as associates of any fungi, and the relative abundance of Lacunisphaera ASVs detected in this Aspergillus isolate were quite low . A target with a lower relative abundance was selected to aid in gauging the sensitivity of the probes, and to increase confidence that these potential bacterial associates are yet present, despite their low relative abundance.To demonstrate the utility of the 16S ASV data to generate unique FISH probes specific to certain bacterial taxonomic groups, we selected an tes Fig. . To ensuLacunisphaera 16S rRNA in the Aspergillus isolate using fluorescence microscopy is shown in Fig. Lacunisphaera shape and size38, together with mono- and diplococci-like structures (white arrows). DNA staining using DAPI also displayed similar structures correlating with the 16S signal, which were distinct from the Aspergillus nuclei indicated by the white asterisks. 3D projections of the figure are provided in Supplementary Movie Our approach produced a pool of 11 FISH probe sequences specific to this bacterial genus, to increase the likelihood of detection even at relatively low abundance relative to other bacterial associates. Visualization of the 18  from the JGI Mycocosm portal39, revealed that bacterial signatures were detected in the majority  of the examined fungal genome projects  technologies were examined to maximize the probability of capturing BSS. Analysis performed with the sensitive and highly specific classification algorithm GOTTCHA2ty 323/40, or 79% While this BSS screen focuses specifically on the unique genomic fraction of any given bacterial genus (unique signatures), we found that on average, >55% of the unique signatures per genus were recovered, representing an average of over 241.5 kb of bacterial genomic signal at an average depth of coverage of 54 fold. The breadth and depth of unique (genus-specific) genome coverage that was captured for any given bacterial genus suggests a substantial relative number of bacterial cells associated with the fungal host, which also implies an active functional relationship with the fungal host. A graphical representation of the number and percentage of bacterial reads identified in each fungal genome sequencing project, as well as the proportion of each bacterial phylum detected in each project are presented in Supplementary Fig. Genomic signatures of putative bacterial associates were detected in representatives from every examined fungal phylum. The average number (3) and range 1–20) of different bacterial genera identified per fungal isolate were substantially lower than what was observed in the 16S-CC screen , Stenotrophomonas (72), Cutibacterium (72), Rhodococcus (45), and Methylobacterium (35) as the most frequently detected genera in this screen. Four of these most frequently detected genera have been previously described as fungal associates 28, but in different fungal genera than those observed here. These results further support the notion that some bacterial groups form diverse and unspecific associations with multiple fungal hosts.In terms of the taxonomic diversity of bacterial genera found associated with fungi, a smaller number of genera were discovered using the BSS screen relative to the 16S-CC screen, but the overall taxonomic distribution was proportionally similar Fig. . The mosIn total, more than half (111 out of 203) of the bacterial genera detected in this BSS screen had also been identified in the 16S-CC screen, while 85 genera were detected only in this screen Fig. . These 8Morchella, Penicillium, Suillus, and Trichoderma) of the 17 fungal genera detected using this network analysis had previous descriptions of bacterial associates40, while 13 of the 19 bacterial genera  were known associates of fungal isolates29. Many of these associations were also detected in several fungal isolates, in either or both of our screens. Independent observations of specific associations between the same fungal and bacterial genera, particularly when utilizing diverse screening methods and isolates, suggests that these associations could be beneficial or essential for one or both participants.The frequency and diversity of putative bacterial associations detected in the two complementary large-scale screens resulted in a complex network of possible bacterial–fungal associations that make overarching statements and interpretations challenging Fig. . Phylum-Aspergillus) to examine in detail whether closely related isolates shared similar profiles of bacterial associations. In an attempt to minimize method specific variance, this analysis was limited to bacterial partners of Aspergillus that were detected in both screens. Our examination of 51 Aspergillus isolates, including 23 described species from the BSS screen, revealed that while some general patterns of association were present, closely related Aspergillus isolates can have distinct profiles of bacterial associates  that were detected at high levels (see Methods section) by GOTTCHA2 in four or more taxonomically diverse fungal datasets to evaluate phylogenetic relationships between fungal associates of the same speciesUnlike the 16S-CC screen, where sequencing data was limited to a fragment of the 16S region, the fungal genome sequencing projects examined in the BSS screen often captured much larger fractions of bacterial genomes, which in some cases, allowed for more comprehensive genome-wide evolutionary comparisons. We selected three bacterial species , Melanomma pulvis-pyrius , Mrakia frigida , and Phycomyces blakesleeanus . Whole genome alignments and phylogenetic analyses of these fungal dataset-derived sequences  indicated that three of the B. cereus strains putatively associating with these fungi are closely related via a recent common ancestor (with average of 1136 SNPs in the core genome), while one strain associating with M. pulvis-pyrius appears to be more distantly related  , while the B. cereus strains from the two most closely related fungal hosts, L. macrosporus and M. pulvis-pyrius, appear more distantly related. These results indicate that diverse fungi may harbor phylogenetically related bacteria, and that multiple lineages within a bacterial species may be able to form such cross-kingdom associations.We examined es) Fig. . InteresE. coli and A. johnsonii sequences found in different fungal projects  and A. johnsonii . A total of 84,954 (E. coli) and 930 (A. johnsonii) SNPs were utilized to infer phylogenetic trees. Fungal-derived strains from these two bacterial species appear to be closely related, with an average of only 5197 (E. coli) and 17 (A. johnsonii) SNPs detected within the core genome across the analyzed strains. While the hosts of both bacterial taxa are phylogenetically diverse, the four examined fungal hosts of A. johnsonii, occupy similar ecological niches , while the examined six hosts of E. coli lack this similarity. Two of these A. johnsonii fungal hosts are well characterized pathogens of woody plants  and the other two are white-rot saprotrophs of woody plants (Panellus stipticus and Scytinostroma sp.). These overlaps in the niche and trophic mode of the fungal hosts could be one possible explanation for the phylogenetic similarity found between their potential bacterial associates.A phylogenetically less diverse series of potential associations were found using the same phylogenetic approach with cts Fig. , c. ThesThe results presented in this study demonstrate for the first time that multiple potential bacterial associates are common in a large diversity of fungal isolates across all examined phyla and suggest the fungal bacteriome can be quite complex. Potential bacterial associates were detected in the vast majority (617 or 88%) of the 702 fungal isolates examined across both the 16S-CC and BSS screens, including representatives from three phyla previously unexamined for the presence of bacterial associates . Quality control standards and procedures were employed throughout our investigations to increase confidence in the accuracy of the presented results and aid in interpretation. We consistently describe bacteria detected in our screens as ‘potential’ bacterial associates given that the nature of each interaction is not yet explored in depth. However, it is important to consider that bacteria capable of persisting alongside a presumably axenic fungal isolate in culture, without obvious signs of parasitism, regardless of their origin, should be considered at minimum a potential associate.41.Multiple representatives from both early diverging, and higher lineages of fungi were examined in this work, suggesting bacterial associations proliferate all branches of fungal evolution. The presence of potential bacterial associates in all examined lineages of early diverging fungi also suggests associations with bacteria arose early in the evolutionary history of fungi, which may be expected given that members of these kingdoms co-dominate most terrestrial environments and frequently overlap in environmental nichesOur results indicate a remarkable, and previously undescribed, diversity of bacterial lineages that appear capable of associating closely with fungi. In our 16S amplicon screen of diverse fungi belonging to multiple culture collections, at least a single putative bacterial associate was detected in every examined isolate. This detection rate was unexpected given that results from previously published surveys usually contain several isolates lacking any potential bacterial associates. However, differences in the applied methodology of these previous surveys such as the use of primers targeting specific taxonomic groups or the treatment of fungal tissue to eliminate or reduce external bacterial associates, compared with the more sensitive methods applied here, could explain the increased discovery rate, as our methods are designed to generally detect any potential bacterial associates.A total of 638 bacterial genera were detected across both screens, including representatives from 17 bacterial phyla that have not previously been described as having associations with fungi. Members of the Proteobacteria, Actinobacteria and Firmicutes were most frequently detected in both our results as well as previous examinations, suggesting that bacteria from these phyla are the most prevalent associates of fungal isolates. The average number of detected bacterial associates per fungal isolate from both screens demonstrates that it is common for fungi to harbor multiple bacterial associates, implying that the fungal bacteriome can be quite complex.Mortierella isolates are promising, with at least two different patterns emerging consisting of distinct sets of bacterial taxa  was used to extract DNA from the LANL isolates, while the Zymo Quick-DNA Fungal/Bacterial Miniprep Kit  was used for Neuchâtel, Houston, and São Paulo isolates. The internal transcribed spacer (ITS) rDNA regions were amplified from the DNA extracts using either ITS5F  or ITS1-F  as forward primer and ITS4 (5′-TCCTCCGCTTATTGATATGC-3′) as reverse primer and the Phusion High-Fidelity DNA polymerase . Amplified products were then submitted for cleanup and Sanger sequencing . Ambiguous peripheral bases were trimmed using 4Peaks  and forward and reverse reads were merged using AliView v1.2442.Fungal culture collections from Los Alamos National Laboratory , University of Neuchâtel , University of Houston , and University of São Paulo  were examined in this screen (Supplementary Data 43 and NCBI BLAST (https://blast.ncbi.nlm.nih.gov/Blast.cg) databases. The UNITE database is a curated database that is accepted as a reliable resource for ITS fungal classification44, however is less complete than the more encompassing BLAST database. The criteria for identification of fungal taxa required an Expect (E) value of 0.0 and a minimum of 95% sequence identity to an unambiguous top hit (for genus level) in UNITE. When our ITS sequences had matches to multiple closely related fungal UNITE genera with scores that passed our cutoffs (22 out of 294 isolates), the final taxonomic classification was then based on additional BLAST alignments conducted with the NCBI ITS RefSeq database, using the same classification thresholds mentioned above for the original UNITE hits. In addition, a small number of fungal isolates (10) did not meet the identity cutoffs listed, however all but one (AODJ.161.70 which had a best match at 85%) of these isolates aligned to a UNITE database reference with at least 92% sequence identity. Following published guidelines45 to ensure confident taxonomic classification, the ITS sequences for these 10 isolates were aligned to authenticated and/or published sequences from both the NCBI ITS RefSeq and the complete nucleotide (nt/nr) databases. Top matches were then scrutinized by comparing to other sequences with identical taxonomic classification and authenticated with closely related organisms to increase confidence. Because we cultured all isolates, growth morphology was also considered and no morphologies contradicted our classification analyses.Classification of the ITS amplicons was performed using comparisons to the UNITE 8.346 and 907R 46 were used to amplify the V1-V5 region of 16 S rRNA followed by modified versions of primers 341F47 (5′-CCTACGGGNGGCWGCAG-3′) and 806R48 (5′-GGACTACHVGGGTATCTAATCC-3′) to further amplify the V3-V4 region prior to sample barcoding. Samples were sequenced on Illumina MiSeq (2 × 300 bp paired end reads). No-template controls and controls for reagent contamination during DNA extraction and library preparation were also included in the sequencing runs for the samples from each culture collection. Blank DNA extractions (controls) were processed in an identical manner to the fungal samples using the reagents from the same lot for both extraction kits . These extraction controls were amplified in a manner consistent with the fungal samples and included in the Sanger sequencing submissions. Additionally, all work was conducted in sterilized biological safety cabinets, including separate units in separate labs designated for culturing and molecular work, following strict sterile techniques  to limit potential contamination.Signatures of potentially associating bacteria were examined via amplicon sequencing of the V3-V4 region of 16 S rDNA from the fungal DNA extractions described in the section above. Nested PCR was performed to enhance the bacterial signal. Primers 27F 49 with DADA250 for error modeling and the generation of merged, denoised, chimera-free sequences to produce amplified sequence variants (ASVs). Representative sequences for these ASVs and the ASV table generated by QIIME2 are available in supplemental information . Separate controls were used for each culture collection to try and minimize generalizing contaminants across each collection, as there was not a case where a contaminant ASV or taxa was found in all control samples . Non-bacterial  ASVs were identified and were excluded from analyses, given our sole interest in bacterial associates. In an effort to reduce redundancy and increase efficiency of downstream analyses given the large number of bacterial ASVs identified (6594), ASVs with identical taxonomic classifications were clustered into 705 bacterial OTUs based on taxonomic identity rather than sequence identity . The methods and techniques utilized in this research, such as clustering ASVs into OTUs based on taxonomic identity and the use of DADA2 for error modeling and removal, were selected specifically to eliminate concerns about the artificial inflation of taxonomic richness, by ensuring the removal of spurious sequences and eliminating concerns about erroneous OTU splitting. A total of 22 taxa identified by SILVA were absent from the NCBI taxonomy database when comparing taxonomic classifications and were therefore excluded. OTUs that were not classified at the genus level were also excluded from downstream analyses to simplify taxonomic comparisons.Raw sequences from all four culture collections were de-multiplexed and were analyzed together as a single pooled group 16S-CC) using QIIME2S-CC usinhttps://github.com/glarue/jgi-query). The SRAdb51 package for R was used to identify the genomic SRA run IDs for each screened fungal isolate using the associated NCBI BioProject number and reads were downloaded using the NCBI SRA Toolkit  sequence length shorter than 50 bp, (2) sequences with average quality scores <15, (3) sequences with more than one consecutive ambiguous base (‘N’), or (4) sequences with a fraction of mono- or di-nucleotide repeats exceeding 65% of the sequence.Fungal genome assemblies were downloaded from JGI’s Mycocosm data portal using the jgi-query script . The default parameters and settings were used for all GOTTCHA2 analyses.In order to enrich bacterial sequences in these fungal projects and reduce computational burden and false positive assignments, fungal sequence data was removed by mapping all reads to the respective fungal genome assembly . Reads were mapped using the BWA-MEMA. johnsonii, B. cereus, and E. coli in multiple fungal hosts from fungal-filtered reads obtained during genome sequencing. Four or more of these fungal projects for each bacterial species were selected for phylogenetic analysis based on the quality of the GOTTCHA2 results (linear coverage and depth of coverage of unique signatures within each project) for the particular bacterial species in question. Once identified, the fungal-filtered reads from these projects were analyzed using PhaME55 together with several complete reference genomes or reference genome assemblies  from NCBI RefSeq. Briefly, PhaME automatically selects the most closely related reference genome or assembly to use for alignment of the raw read datasets during the determination of the conserved (core) genome among all input sequence datasets. From this core genome alignment, phylogenetic trees were generated using RAxML and 1000 bootstrap replicates were run for each analysis.GOTTCHA2 identified Aspergillus isolate (LANL.1351.96) into 32 distinct taxonomic groups. OligoMiner56 was used to design probes for each group following these steps: (1) repetitive sequences, homopolymeric runs and ambiguous bases were masked from all sequences; (2) candidate probes were identified based on provided criteria such as length, melting temperature, and GC content  multiplexable checking was performed for qualified probes using ThermonucleotideBLAST.The genus lineage level (SILVA designation D_5) was used to separate the 16S ASVs detected in the ate LANL.51.96 intate LANL.51.96 intCircular growth media disks were prepared by casting 4% gellan gum or phytagel  supplemented with 1X potato dextrose broth (Cat: EW-14200-28. Cole-Parmer) in between two standard microscope slides spaced by two No. 1.5 coverslips. Growth disks were then transferred to a microscope slide fitted with a 65μL Gene Frame . Growth slides were placed in a 100 mm disposable petri dish along with a cap from a 15 mL conical tube filled with water to control humidity. Fungal mycelia from isolates grown for at least 1 week on potato dextrose agar were transferred to the growth media pads and incubated for 3–5 days at 25 °C. It is worthy to note that the different genera of fungi exhibited variable growth phenotypes, and sample preparation for imaging was standardized as best as possible to prioritize thin imaging depths across hyphae.−1 lysozyme , 5 mg mL−1 β-Glucanase from Trichoderma longibrachiatum , and 500 μg mL−1 chitinase from Streptomyces griseus  for 1 h at 37 °C. The samples were washed three times with PBS and subsequently dehydrated with a series of ethanol  treatments at 50%, 75%, 100%, 75%, 50% and rinsed with PBS for 3 min each at room temperature. Samples stored at −20 °C in 100% ethanol were found to have no change in quality for at least 3 days.To prepare samples for FISH, fungal samples were fixed with 4% paraformaldehyde  in PBS  buffer at 4 °C overnight directly on the growth disks in the Gene Frames. Samples were washed three times with PBS and cell walls were lysed with a cocktail of 5 mg  mL59. Universal eukaryote staining was achieved using the 516 region of the 18 S rRNA  (Integrated DNA Technologies)58. Probes were hybridized for 4 h at 37 °C and then washed three times for 20 min with Stellaris Wash Buffer A at 37 °C. Nuclear staining was performed by incubating 1 μM DAPI  in Stellaris Wash Buffer A and for 10 min at room temperature. Excess DAPI was rinsed away with Stellaris Wash Buffer B for 5 min at room temperature. Samples were then rinsed with 2 X SSC buffer  and lastly placed under a No. 1.5 coverslip for imaging.For double-labeled oligonucleotide FISH, Stellaris RNA FISH buffers  were used for subsequent hybridization and washing steps. Samples were preconditioned with Stellaris Wash Buffer A supplemented with 30% formamide  for 5 min and replaced by Stellaris Hybridization Buffer supplemented with 30% formamide containing a final 125 nM concentration of each probe. To target endobacteria, a probe pool was generated based on EUB338 targeting the 16S rRNA: EUB338-I (5′-GCT GCC TCC CGT AGG AGT-3′), EUB338-II (5′-GCA GCC ACC CGT AGG TGT-3′), and EUB338-III (5′-GCT GCC ACC CGT AGG TGT-3′) with Cy3 dyes flanking the 5’ and 3’ ends (Integrated DNA Technologies)36, samples were preconditioned with Probe Hybridization Buffer (Molecular Instruments) for 20 min at room temperature. A pool of target-initiator sequences was proprietarily designed by Molecular Instruments based on the EUB338 target sequence (5′-ACA CUG GAA CUG AGA CAC GGU CCA GAC UCC UAC GGG AGG CAG CAG UGG GGA A′-3′). 1.2 pmol of target probes and 125 nM of EUK516 (5′-ACC AGA CTT GCC CTC C-3′-Quasar 570) (Biosearch Technologies) in Probe Hybridization Buffer was incubated with the samples overnight at 37 °C. Probes were washed 5X with Probe Wash Buffer (Molecular Instruments) for 5 min each and then preconditioned with Amplification Buffer (Molecular Instruments) for 30 min at room temperature. The buffer was replaced with 18 pmol of each HCR amplification probes (Molecular Instruments): B4-Alexa Fluor 647 or B1-Alexa Fluor 488 in Amplification Buffer and incubated overnight at room temperature. Probes were washed 4X with 5X SSCT ) for 5 min each at room temperature. In all, 1 μM DAPI in 5X SSCT was then added to the sample and incubated for 10 min at room temperature and rinsed with 2X SSC three times. A No. 1.5 coverslip was placed on the sample and immediately imaged. Fluorescence microscope images were collected using a Zeiss Axio Observer outfitted with a mercury short-arc lamp, a 1.3 N.A. 100X oil-immersion objective lens, and filters for DAPI, FITC, CY3, and CY5.If direct hybridization of fluorescent probes was insufficient to provide adequate signal, a fluorescence amplification technique was employed. For hybridization chain reaction FISH (HCR-FISH)Aspergillus was performed following the protocol outlined above for double-labeled oligonucleotide FISH. Here, Cy3-labeled probes of the probes above were purchased from Biosearch Technologies  to identify research and review articles containing previous descriptions of bacterial–fungal associations. Artificial associations, such as forced associations between bacteria and fungi not isolated from the same environment or associations involving genetically engineered strains, were excluded, as well as co-occurrence studies involving multiple fungi. Our findings are summarized in Supplementary Data Further information on research design is available in the Transparent Peer Review FileSupplementary FiguresDescription of Additional Supplementary FilesSupplementary Movie 1Supplementary Data 1Supplementary Data 2Supplementary Data 3Supplementary Data 4Supplementary Data 5Supplementary Data 6Supplementary Data 7Supplementary Data 8Supplementary Data 9Supplementary Data 10Supplementary Data 11Supplementary Data 12Supplementary Data 13Supplementary Data 14Reporting Summary


An estimated 1% of endovascular aneurysm repair (EVAR) devices become infected, carrying a high mortality rate. Surgical explantation is recommended and prognosis is guarded. This retrospective cohort analysis focuses on the role of outpatient parenteral antimicrobial therapy (OPAT) in the management of aortic vascular graft infections following EVAR.Patients who received OPAT for aortic graft infections (AGI) following EVAR from 2014 to 2018 inclusive were identified using the OPAT database. Clinical, microbiological and radiological data were collected. Survivors were followed up for a median of 36 months (range 25–60) after first presentation with infection. Outcomes were assessed.Eleven cases with 20 OPAT episodes were identified: 10/11 male, median age 76 (IQR 71–81). Median time to presentation was 7 months (range 0–81 months) after EVAR. OPAT lead to a 55% reduction in length of hospital stay. One patient had graft explantation; four others had temporising measures. Eight of 11 were alive a median of 36 months after presentation with infection, having had a median of 2 re-treatments on OPAT (range 1–3). Seven of the eight survivors were on continuous suppressive oral antimicrobials; three were also intermittently on intravenous antibiotics for flares of infection. Patient/ infection outcomes were cure (1/11), improved (7/11), failure (3/11).AGI following EVAR usually presents in the first year after graft deployment. OPAT has an important peri-operative role in patients suitable for curative surgery. OPAT followed by oral suppressive antimicrobial therapy can be a feasible long-term treatment for non-curative management of AGI. Survival in our cohort was longer than expected, and OPAT was feasible despite the complexity of these infections. OPAT can avoid multiple and lengthy hospital admissions and maximise time at home and quality of life in this cohort with life-limiting infection. Aortic graft infection (AGI) can follow open aortic aneurysmal repair or endovascular aneurysm repair . An estiThis study was performed in a tertiary referral hospital with a vascular surgery department, an infectious diseases (ID) department and an OPAT programme all on the same site. The majority of the OPAT programme in our hospital is run by the ID department. Joint decisions are made by the vascular surgeons and ID physicians regarding suitability of patients with AGI for OPAT. Once accepted onto the OPAT programme, patients are reviewed by face-to-face weekly visits with an ID physician and OPAT clinical nurse specialist in the ID department. There is a direct telephone line for virtual review of any issues falling outside of weekly visits, and extra in-person review can be organised as needed. Though there is no joint ID/ vascular clinic, review by the vascular surgeons can often be organised on the same or following day if necessary. Standard weekly laboratory testing includes full blood count, renal and liver profiles and C-reactive protein. Once patients complete the course of OPAT and are switched to oral suppressive treatment, the frequency of clinical review is decreased. While on the OPAT programme patients can call the OPAT nurse specialist directly. Once discharged from the OPAT programme patients are followed up in the general ID clinic, which can also be contacted directly if issues arise between visits. Once discharged from care, patients can still phone the ID clinic to be seen if any new clinical issue arises.The hospital Outpatient Parenteral Antimicrobial Therapy (OPAT) database was used to identify all patients who received at least one course of OPAT for infected EVAR over a 5-year period from January 2014–December 2018. Only AGIs following EVAR were included (referred to here as AGI for brevity). Patient medical and electronic records were accessed to collect data. Management of Aortic Graft Infection Collaboration (MAGIC) case definitions were applied to stratify infections as early or late, suspected or definite . AGI wasEleven patients, with a total of 20 OPAT episodes, were identified. Eight patients (72%) met MAGIC criteria for definite infection, and 3 (27%) for suspected infection . Ten of Patients presented with infection a median of 7 months range 0–81 months) after EVAR. Symptoms at presentation with infection included fever in 6/11(55%), anorexia in 6/11(55%), abdominal pain in 5/11 (45%), back pain in 3/11(27%) and bleeding in 2/11(18%) (Table  months aPseudomonas aeruginosa (2/11) and Candida albicans (2/11) and Salmonella species (1/11) (Tables Causative organisms were identified in 7/11 (64%) cases, 4 of whom had polymicrobial infections. Three patients 27%) had positive blood cultures, one had positive vertebral biopsy, three a positive culture from sac aspirate and two a positive sinus culture. Five patients had Gram-negative organisms isolated; 3/5 had suspected aorto-enteric fistulae. Organisms isolated included staphylococci and streptococci species(6/11), anaerobes (4/11), enterococci (2/11), 7% had poTwenty OPAT episodes were analysed, with patients having a median of two range 1–3) episodes. Most patients (10/11) received more than one intravenous antimicrobial agent at some stage during the course of their treatment, including their inpatient stay. Six patients received dual IV antimicrobial therapy on OPAT. All patients received oral antibiotics for a minimum duration of 6 months after OPAT and some received concomitant oral antibiotics during their OPAT treatment. Antibiotics received on OPAT can be found in Table –3 episodThree patients received more than two OPAT courses to treat clinical flares of infection. Patient 9 had recurrent abscess formation at the site of axillobifemoral graft; this patient had multiple temporising measures (sac drainage) and three OPAT episodes, before eventually having elective graft explantation. The patient had a stormy post-operative course with a long admission to intensive care but was alive and on oral antimicrobials 25 months after diagnosis of AGI and 15 months after surgical explantation of the graft. Patient 8 successfully completed 12 weeks of OPAT, and later presented with fatigue and rising inflammatory markers while on oral suppression therapy; the patient was switched back to intravenous antibiotics until symptoms and markers improved. This was done on two occasions and on one occasion was achieved without a hospital admission. Patient 4 had increasing abdominal pain and fevers on oral suppressive therapy and was admitted for switch to intravenous (IV) antibiotics and repeat surgical opinion. The decision was made that the patient was still not for surgical intervention and he was discharged within a week via OPAT. At the time of writing, he was on oral suppressive therapy, clinically stable, but with deteriorating radiological appearances.A total of five adverse events occurred in three patients during OPAT; all were antimicrobial-related. One patient presented with a daptomycin-induced eosinophilic pneumonitis for which they required oxygen therapy and antibiotics were changed to vancomycin (Patient 9). He made a full recovery from the pneumonitis. A second patient was admitted with transfusion-requiring anaemia during therapy with linezolid and caspofungin (Patient 8). Three other adverse events occurred which did not require re-admission; piperacillin/tazobactam-induced skin rash, cefuroxime-induced skin rash, and asymptomatic eosinophilia on daptomycin.Four patients had to be re-admitted for infection-related reasons at least once while on OPAT; patient 9 was admitted 3 times ; patient 8 was admitted twice (once for failure of therapy and once for complications of antibiotics). Patient 4 was re-admitted for failure of therapy and a repeat surgical opinion and patient 7 was re-admitted electively for repeat imaging Tables  and 2.Eight (72%) patients were alive at a median of 36 months of follow up (range 25–64 months) Table . OPAT ouThe median number of days on OPAT was 46.5 (range 14–91 days). Patients were on IV antibiotics for a total of 1560 days; 696 in hospital and 864 on OPAT. OPAT facilitated a saving of 864 total hospital days, representing a 55% reduction in length of stay.Our patients had similar demographics to those in previously published literature on aortic vascular endograft infections, insofar as they were mostly male, older and with multiple co-morbidities, and these latter factors correlated with mortality , 9, 10. Expert consensus favours surgical removal of an infected graft where possible , 8. One However, this small selective cohort cannot be used to compare surgical versus medical therapy for AGI, but only to describe outcomes in those who survive and are considered candidates for outpatient management. There is an inherent bias in the selection of patients for analysis from an OPAT registry, as those who presented in extremis or died during the first presentation of infection are not included. By definition, the patients in our cohort were well enough for hospital discharge at least once with OPAT, skewing overall survival as compared with cohorts including all patients who present with AGI. In a series including all presenting AGI patients, 17/22 (77%) presented with an urgent surgical indication (rupture or bleeding), as compared with only two of our patients presenting with bleeding and none with rupture [To the best of our knowledge there is no other published data on OPAT outcomes for AGI. As with other complex infections, OPAT can deliver significant savings in inpatient bed-days and healthcare costs . This stOPAT has multiple roles in the setting of AGI, and both patients who undergo surgery and those who do not may benefit. OPAT may be used as a temporizing measure in those awaiting radical surgery, as induction therapy in those AGIs for medical management, and for treatment of clinical ‘flares’ in patients otherwise well-maintained on oral suppressive therapy. In each of these instances, OPAT can avoid multiple and lengthy hospital admission, reduce costs and maximise time at home and quality of life in this cohort with life-limiting infection. Importantly, we have also demonstrated that long-term outpatient medical management  of patients with AGI for whom cure is not intended is feasible. No deaths attributable to vascular graft infection occurred during the follow-up period, but the prognosis and unpredictability of AGI warrants early setting of treatment goals by a multi-disciplinary team comprised of vascular surgeons, infections specialists and, where appropriate, palliative care practitioners. Patients with early AGI and frailty who are not considered candidates for curative surgery should be counselled about their prognosis and consider criteria forre-admission and preparedness for potential vascular catastrophes in the community if appropriate. OPAT followed by oral suppressive therapy can be a suitable long-term non-curative treatment option for certain patients with AGI.


Most individuals treated for heroin use disorder receive opioid agonist treatment (OAT)(methadone or buprenorphine). However, OAT is associated with high attrition and persistent, occasional heroin use. There is some evidence for the effectiveness of contingency management (CM), a behavioural intervention involving modest financial incentives, in encouraging drug abstinence when applied adjunctively with OAT. UK drug services have a minimal track record of applying CM and limited resources to implement it. We assessed a CM intervention pragmatically adapted for ease of implementation in UK drug services to promote heroin abstinence among individuals receiving OAT.Cluster randomised controlled trial.552 adults with heroin use disorder (target 660) enrolled from 34 clusters (drug treatment clinics) in England between November 2012 and October 2015.Clusters were randomly allocated 1:1:1 to OAT plus 12× weekly appointments with: (1) CM targeted at opiate abstinence at appointments (CM Abstinence); (2) CM targeted at on-time attendance at appointments (CM Attendance); or (3) no CM . Modifications included monitoring behaviour weekly and fixed incentives schedule.Primary outcome: heroin abstinence measured by heroin-free urines (weeks 9–12). Secondary outcomes: heroin abstinence 12 weeks after discontinuation of CM (weeks 21–24); attendance; self-reported drug use, physical and mental health.CM Attendance was superior to TAU in encouraging heroin abstinence. Odds of a heroin-negative urine in weeks 9–12 was statistically significantly greater in CM Attendance compared with TAU . CM Abstinence was not superior to TAU  or CM Attendance  . Reductions in heroin use were not sustained at 21–24 weeks. No differences between groups in self-reported heroin use.A pragmatically adapted CM intervention for routine use in UK drug services was moderately effective in encouraging heroin abstinence compared with no CM only when targeted at attendance. CM targeted at abstinence was not effective.ISRCTN 01591254. To our knowledge, this is the first clinical trial to examine the effectiveness of contingency management (CM) in promoting heroin abstinence among those in opioid agonist treatment in the UK.This study is a large cluster randomised trial conducted at 34 drug treatment services in England.CM, as developed in the USA, was pragmatically adapted for ease of implementation in resource-poor UK substance use treatment settings.Adaptions included using drug service staff to deliver all aspects of the CM intervention ; staff were trained, supported by a CM Handbook and regular supervision.Illicit drug use was monitored through urine drug screens once a week rather than more frequently using a fixed rather than escalating schedule.Opioid agonist treatment (OAT) (methadone and buprenorphine) is recognised globally as a clinicallyCM is a behavioural intervention based on the principles of operant conditioning,Evidence, primarily from US trials, shows that CM is an effective adjunct to substance use treatment in encouraging abstinence from drug use, including in treating drug use regardless of treatment setting, and for treating drug use  in opiate addiction treatment.UK guidance, based on the evidence at the time,We employed a pragmatic cluster randomised controlled trial design described elsewhere.Within each cluster, participants received the same allocated condition, thus minimising the risk of contamination between intervention and control arms.The trial tested the following research hypotheses:CM (positive reinforcement targeted at treatment attendance) will increase abstinence from street heroin when compared with TAU in which no positive reinforcement is offered.CM (positive reinforcement targeted at the provision of opiate-negative urine samples at treatment appointments) will increase abstinence from street heroin when compared with TAU in which no positive reinforcement is offered.Differences in the type of CM schedules will be associated with differences in heroin abstinence.Having both CM interventions allowed us to assess whether any benefit (abstinence) derives from a direct effect of CM on abstinence as the behavioural target, or is a general benefit resulting from CM-stimulated improved attendance at clinical appointments (and possibly consequent improvements in treatment retention).Findings from parallel economic and process evaluations are to be reported separately.Eligible patients were aged 18 and above; seeking a new episode of OAT; regular users of street heroin , and all (minimum 1) urine drug screens (UDS) in previous month positive for opiates); meeting International Classification of Diseases 10th Revision criteria for opiate dependence; willing to receive 12-week CM intervention; at liberty to participate in the study for 24 weeks; and willing and able to provide informed consent. We excluded patients if they could not read English and required an interpreter to understand a brief oral description of the study to ensure they would be able to understand the CM intervention provided; were pregnant or breast feeding ; and/or were referred through the criminal justice pathway and were receiving a community sentence on condition of attending drug treatment as they would be subject to additional contingencies which might influence their behaviour.Each cluster (drug clinic) was randomly assigned to OAT plus 12× weekly clinical appointments with either (1) CM Abstinence: positive reinforcement contingent on opiate abstinence monitored through UDS undertaken at each weekly appointment; (2) CM Attendance: positive reinforcement contingent on attendance on time; or (3) no CM .Randomisation was undertaken independently by the King’s Clinical Trials Unit. Clusters were assigned to treatments using random permuted blocks within type of service provider strata (NHS or non-NHS) using a block length of 3 in a 1:1:1 allocation ratio. Laboratory personnel who completed the urinalysis and the statistician analysing primary outcome data were all blinded to treatment allocation.OAT was delivered in line with existing service protocols at all clinics and included weekly clinical appointments between 15 and 50 min with a named drug worker. Attending the service to obtain a prescription did not constitute attendance. The CM interventions (described below) were delivered adjunctively.In our trial, CM, as developed in the USA, was pragmatically adapted for use in resource-poor UK drug treatment services to ease future implementation. Key CM principles were retained; targeting a clearly defined behaviour, regular monitoring of that behaviour and providing an immediate reinforcer ensured that a clear contingent relationship was made between the positive behaviour and the reinforcer. The reinforcer was withheld if the target behaviour was not achieved. Adaptions included: (A) training drug workers to deliver CM ; (B) monitoring illicit drug use through weekly UDS (rather than more frequently) to fit routine UK practice of taking UDS once a week at the start of treatment and less frequently thereafter; and (C) using a fixed rather than escalating schedule, as drug service staff had previously reported the use of escalating schedules to be too difficult.CM Abstinence’: Reinforcement was conditional on attendance at weekly appointments and (A) during weeks 1–4 (priming weeks) providing a UDS whatever the result and (B) during weeks 5–12 providing an opiate-negative UDS. UDS were undertaken using a drug integrated cup test to detect opioids. This test is able to detect opioids up to 1–3 days after use. While unable to confirm opioid abstinence over the week period, the test nevertheless provided clinically significant evidence of the participants’ ability to abstain from using opioids over this briefer period. Instant UDS tests were completed at the start of each appointment. Staff provided the reinforcer immediately the target behaviour was achieved. Priming ensured participants gained experience of the reinforcer, while also addressing practitioner concerns about the difficulty in achieving abstinence during contemporaneous titration up to a stable dose.‘CM Attendance’: Eligibility to receive the reinforcement was conditional solely on on-time attendance at the appointment (within 15 min of the scheduled appointment time).‘Control: Participants received TAU (OAT plus 12× weekly appointments) with no CM.The CM interventions ceased after week 12, at which point the frequency of appointments reverted to usual care for each service .All staff providing clinical appointments received training on trial procedures. At CM sites, staff received bespoke 1-day training on the principles and practice of CM . Training was supported by a CM Handbook designed for UK services but which drew on international evidence and practice models.CM was delivered in the first 5–10 min of the appointment and audio recorded. A CM adherence measure was developed from measures available at the timeEvidence suggests training alone is unlikely to change health professional behaviour unless supported by effective supervision.Consenting participants completed a research interview (and were reimbursed £20 for time and travel) before enrolment into the trial and again at 12 and 24 weeks after enrolment. All participants were asked to provide (research) UDS weekly between weeks 9–12 and 21–24. Weekly ‘urine collection clinics’ were established by the research team at each clinic (participants were reimbursed for travel) to enable participants to provide UDS outside of appointments. Participants’ attendance at appointments, receipt of CM vouchers and provision of urines for CM and research purposes were recorded.The primary outcome measure of heroin abstinence was a once-weekly binary positive/negative heroin UDS collected at weeks 9–12 for laboratory drug testing.Secondary outcome measures included retention in treatment, self-reported illicit drug use in the last 30 days,The Adult Service Use Schedule adapted for drug users,et alMore detail is provided in the protocol.Statistical analysis was carried out using Stata V.14/15 according to the intention-to-treat principle. Variables were summarised using mean/SD, median/quartiles or frequencies/proportions as appropriate.Refusal and non-attendance UDS were coded as positive (positive assumption) but results missing for other reasons  were initially left missing. This positive assumption is commonly used in clinical practice and in studies of CM and was used in our previous trial.These responses were the dependent variables in a mixed effects logistic regression model (9–12 and 21–24 weeks in separate models), with trial arm, the clinic type stratification factor and week dummy variables as fixed effects, and random intercepts for participants nested within clinic. Results are presented both from fitting a model fitted to data where (1) the positive assumption has been applied, but no further imputation has taken place, (2) further missing data were imputed (100 data sets) and estimates obtained using Rubin’s rules. The imputation model included clinic, trial arm, NHS stratification factor, gender, heroin use at 12 and 24 weeks, and baseline measures including frequency of heroin use, AUDIT score, OTI social functioning score anxiety and depression, SF-36 physical and mental component scores and delay discounting, in addition to the four binary weekly urine sample measures. Moderation by type of opioid replacement (methadone or buprenorphine), NHS clinic versus non-NHS and the Charlson Index of multiple drug use was tested using the non-imputed data by adding treatment by moderator interaction terms each separately to the model described.Differences in secondary continuous outcomes between the groups were estimated using linear mixed effects models with the 12 and 24-week outcomes as dependent variables, the same fixed and random effects as for the primary outcome, plus time point, treatment by time interaction and baseline values of the outcome where applicable as predictors. For the self-report binary drug use variables, marginal logistic generalised estimating equation (GEE) regression models were fitted ,Retention in the 12-week treatment programme was coded as weeks attended up to the point of continuous future non-attendance. Retention was compared between the groups using: (1) Fisher’s exact test for retention in the trial overall, (2) a logistic GEE model for binary retention in opioid treatment at 12 weeks (with exchangeable correlation structure and 1000 repetition bootstrap SEs), and (3) Kaplan-Meier and Cox regression analyses for time until continuous treatment non-attendance, with trial group and NHS stratification factor as predictors and clinic as a clustering variable.A Service User Research Advisory Group (SURAG) was established before the trial commenced, comprising 14 service users with direct experience of receiving OAT. The group met once every 6 months to provide advice on the design, conduct and progress of the research. The SURAG helped significantly to make the trial procedures acceptable and aided implementation.Between September 2012 and September 2015, we randomly allocated 34 clinics (clusters) (of 61 considered) to three treatments . Of the 10.1136/bmjopen-2020-046371.supp1Supplementary dataRetention in the trial was high and comparable across trial arms. Eight participants actively withdrew from the trial (CM Abstinence=4 (2%); CM Attendance=1 (1%); TAU=3 (2%), p=0.50).Study participants were broadly representative of patients entering OAT in England . They weAll participants (552) self-reported using heroin in the month prior to interview . Three-quarters reported using crack  while 23% used benzodiazepines (n=129). Half drank alcohol  and nearly all used tobacco  .There was reasonable balance across the three trial arms in terms of baseline sociodemographic, drug use, health and treatment experience variables. However, there were more women in the CM Attendance group, fewer white individuals in the CM Abstinence group and a smaller proportion with insecure housing status in the CM Attendance group.Of the 2208 scheduled UDS (552 participants × 4 urines for weeks 9–12), 601 (27%) were not obtained due to non-compliance  and were imputed as positive (positive assumption), with the greatest number of these being in the TAU group. Additionally, 454 (21%) were treated as missing, including: 260 (12%) not obtained due to unable to contact, 77 (3%) not obtained due to clinic error, 15 (1%) unable to provide a sample, 55 (3%) in prison (47) and in hospital (8), and for 10 (0.5%) the reason was missing. The percentage of UDS results missing by week and trial arm are shown in Results from the analyses with and without imputation were similar , so resu2(2)=1.89; p=0.389); NHS or non-NHS service (χ2(2)=4.96; p=0.084)) or by other or multiple drug use (Charlson Index of multiple drug use (χ2(2)=4.93; p=0.085)).There was no moderation of trial intervention outcome by medication or treatment setting  down to 45% (week 5) with a steady decline thereafter to 33% attending at week 12. Attendance in the CM Attendance group remained consistent with a slight decline from 59% (week 1) to 51% (week 12). Most attendance was on time (within 15 min) .The proportions attending all of the 12-week appointments differed across the groups, with a higher proportion attending in CM Attendance as compared with CM Abstinence and TAU groups (56% vs 39% and 30%). Participants in the CM Attendance group had statistically significantly greater odds of full attendance compared with those in TAU . There were no statistically significant differences between CM Abstinence and TAU .Participants in the CM Attendance group attended longer than those in the CM Abstinence group or TAU group. Participants in the CM Abstinence group had statistically significantly greater risk of dropping out of the appointments before week 12 compared with those in TAU group  and in CM Attendance group  .Self-reported heroin use reduced in all groups from nearly 100%  to 74% (291) at week 12, while the median number of days used reduced from 28  to 4 . Self-reported use of crack and benzodiazepines reduced in the trial population from 76% to 54% and 23% to 16%, respectively, at 12 weeks. Self-reported alcohol and tobacco use remained similar . There were no significant differences in these outcomes between groups at 12 weeks .At 24-week follow-up, we found no difference in self-reported illicit drug use between groups. However, there was weak evidence of increased odds of alcohol use in the last 30 days in CM Attendance as compared with CM Abstinence , and evidence of fewer days using tobacco in the last 30 days in TAU as compared with CM Abstinence  see . Mixed eThere were no differences between the groups in clinical outcomes at week 12 or 24 ; CM Attendance=44.15%, SD=17.07 (range 16.7%–68.2%)).There were 25 serious adverse events in 22 participants: CM Abstinence=10 in 9 participants; CM Attendance=8 in 7 participants; TAU=7 in 6 participants. The number of deaths was 3 . None were related to the trial intervention.We found the adapted CM intervention targeted at attendance was moderately effective in encouraging heroin abstinence compared with TAU. CM Abstinence was not more effective than TAU or CM Attendance.The UK’s clinical guidance,When our adapted CM intervention was targeted at attendance only, it was superior to TAU in encouraging heroin abstinence. It has been argued that clinical benefit arises from regular attendance at clinical appointments and overall retention in treatment.The CM interventions we evaluated retained the main principles of CM, but modifications were made to ensure ease of implementation in UK settings with limited treatment budgets.First, in the UK, resources are limited. Patients receiving OAT attend their service and receive UDS once a week at the start of treatment and less frequently thereafter. Previous studies exploring views of CM among UK drug treatment staff and patients found that staff viewed the US frequency of urine testing to be too resource heavy for the UK settings and service users felt strongly that such a regime would act as a disincentive.Second, while the CM evidence base demonstrates the efficacy of escalating value incentives,Third, it was critical that the CM schedule was acceptable to participants and supported by staff. Our SURAG argued that having to provide opiate-negative UDS would be challenging during titration and staff felt monitoring at this point may be undermining. In response, we introduced an initial 4-week priming phase in the CM Abstinence arm, during which participants received reinforcement for on-time provision of a UDS, irrespective of the result. The use of priming in CM ensures that patients experience receipt of the reinforcer early in treatment.Fourth, vouchers were chosen as reinforcers as there is good evidence for their effectivenessAdherence to CM as set out in the training manual was somewhat better in the CM Attendance group than CM Abstinence group but not significantly so. Movement of trained staff and supervisors away from participating services may well have impacted on adherence. Further exploration of the relationship between adherence and outcomes and the factors which may affect this  will help to clarify this.Although CM has a strong evidence base in achieving behaviour change, there is weaker evidence for behaviours sustained once the incentives are removed. Previous studies have found differing results with only a small treatment effect following discontinuation of incentives.While there is a growing body of evidence for CM, it has not been widely implemented in UK drug treatment services. Similarly, the actual adoption and implementation of CM programmes in US community-based treatment programmes (including those where most research on CM has been undertaken) is rare,Adapting CM for ease of implementation in resource-poor settings may have led to a loss of some of its potential effectiveness. Therefore, future research should consider how best to implement CM in routine practice both in UK and international contexts and should consider different models of delivery (including the use of mobile telephone technology).


Platalea minor) strictly breeds in marine environments and is threatened by the rapid loss of coastal wetlands within its breeding range. Adults with chicks are thought to gradually switch feeding sites from freshwater wetlands to coastal mudflats as the chicks’ osmoregulatory system develops. We investigated age-dependent shifts in the diet of Black-faced Spoonbill chicks at four breeding colonies with varying freshwater habitat availability by examining stable isotopes  between the tip (grown at the age of 10 days) and middle (grown at the age of 22 days) portions of their primary feathers. The δ13C value of the middle portions was significantly higher than that of the tips, which suggested that the ratio of marine resources increased with the growth and development of chicks. A Bayesian isotope mixing model revealed that the diet proportion of marine prey in the early-chick rearing season was slightly higher than in the late-chick rearing season at three colonies in inshore areas, although this proportion was approximately 60% even in the early chick-rearing period. In contrast, isotopic values and reconstructed diet composition suggested that chicks in an offshore colony with limited freshwater wetlands relied more heavily on freshwater diets for both chick-rearing periods (>80%). Our results suggest that the shifts in feeding sites seen in previous studies might be related to the age-dependent dietary shift of chicks, highlighting the importance of freshwater wetlands for spoonbills on offshore islands without an inflow of freshwater in nearby intertidal mudflats. These findings emphasize the importance of freshwater prey and wetlands even for the endangered marine-breeding spoonbills, even though the negative impact of salt stress remains inconclusive.The endangered Black-faced Spoonbill ( Colonial nesting waterbirds are typical central place foragers, repeating trips between the breeding colony and remote foraging areas. Thus, the energetic costs for feeding chicks depend on the distribution and abundance of prey within their foraging range . In manyPlatalea minor) is an endangered wading bird distributed along with coastal areas of East Asia; it has the smallest range of the six Platalea species in the family Threskiornithidae [The Black-faced Spoonbill  in the same family Threskiornithidae, as well as Common Eiders (Somateria mollissima) and Laughing Gulls (Leucophaeus atricilla), have little osmoregulatory capacity [E. ruber) in saline habitats provide chicks with low-salinity diets by changing foraging habitats from salt marshes to inland freshwater wetlands after chick hatching [Waterbirds living in saline habitats can regulate excess salt consumed with prey through salt glands –21, whiccapacity –25. Prevcapacity , 26. In hatching –31. Thushatching –36. In ahatching , 18, and13C) provides insight into the relative importance of marine versus freshwater prey in the diets of consumers [13C value than those in freshwater because of differences in the carbon sources of primary production between the two ecosystems [15N) is a useful measure of trophic level because consumer tissues are enriched at successive trophic levels [Fulmarus glacialis) [The stable isotope ratio in consumer tissues can provide useful information on such topics because it reflects the stable isotope ratio of diet in a predictable manner –39. Manyonsumers , 41. Orgosystems , 43. Thec levels . Keratinc levels . Severalacialis) , Laughinacialis) , 47, andacialis) .13C and δ15N stable isotopes of the tip and middle portions of a primary feather from each chick, reflecting diet at different ages. In addition, prey availability for each breeding spoonbill may vary depending on hatching timing, which may influence foraging behavior. Therefore, we controlled for the possible effect of seasonal variation in foraging habitat characteristics by including estimated hatching dates for each chick as a covariate in statistical analysis. δ13C values were expected to be lower in the tip portions than the middle portions if adult spoonbills exhibited a foraging shift from freshwater to intertidal wetlands as their chicks grew. In addition, we compared stable isotope values between breeding colonies to investigate spatial variation in reliance on freshwater wetlands of breeding Black-faced Spoonbills among four breeding populations with varied foraging habitat availability.In this study, we sought to determine whether the observed change in foraging habitats of breeding Black-faced Spoonbills was related to an age-dependent shift in diets of chicks at four breeding colonies on the west coast of the Korean Peninsula. We compared the δThis study was conducted at four breeding colonies of Black-faced Spoonbills along the west coast of South Korea . The GujDuring the chick-rearing period, adult Black-faced Spoonbills forage within an average distance of 11 km around breeding colonies . To deteWe visited breeding colonies once or twice to collect primary feather samples from June to July 2013. Chicks were captured on or near their nests within the colony. A total of 34 Black-faced Spoonbill chicks were captured by hand or using hand-held nets: six individuals at Gujido, eight individuals at Suhaam, seven individuals at Namdongji, and 13 individuals at Chilsando . The cap2 feather vanes from the distal tip and the middle part (8 cm from the tip) of the 8th primary feather for stable isotope analysis. The estimated hatching dates of sampled chicks were between May 1 and June 15, and all were more than 25 days old  linked with a CN analyzer  at the National Instrumentation Center for Environmental Management (NICEM) at Seoul National University of the Republic of Korea.Feather samples were cleaned to remove oil or dirt that could affect the value of the stable isotope ratio by using both a chloroform:methanol 2:1 solution and detergent, as a previous study indicated that cleaning feathers with a solvent might not be sufficient to ensure accuracy and repeatability of isotope analysis . We washX represents δ13C and δ15N, R is the corresponding ratio 13C/12C and 15N/14N, and Rstandard is the ratio of the international references: Vienna Peedee Belemnite (VPDB) for carbon and air for nitrogen. The known measurement precision with the reference materials in NICEM was better than 0.2‰ for δ13C  and 0.2‰ for δ15N , respectively [Stable isotope ratios were expressed in δ-values and parts per thousand (‰), according to the following equation:ectively , 67.15N values were obtained for the distal portion (early chick-rearing period) in three individual chicks from Chilsando due to technical problems and sample loss during analysis. We replaced the missing data with mean δ15N values of a distal portion of feathers from chicks in the early chick-rearing period in Chilsando. We ran the model with Markov chain Monte Carlo sampling based on the following parameters: number of chains = 3; chain length = 300,000; and thin = 100. We examined model convergence with a Gelmin-Subin Heidelberger diagnostic test and trace plots. To include the main diet groups of the spoonbill, we used the isotopic values of potential prey including fish, crustaceans, and aquatic insect larvae collected from freshwater and intertidal habitats in South Korea reported in previous studies  compared to the other colonies and had virtually no freshwater wetlands within the normal foraging range due to its remoteness from the nearest coastline  and -17.42 ± 0.50‰ (n = 34), respectively  for the early chick-rearing period and 14.55 ± 0.26‰ (n = 34) for the late chick-rearing period.The mean δectively . The mea13C analysis, the full model was significantly different from the null model . We found that there was a significant interaction effect between breeding colony and hatching date  and removed other two-way interactions from the full model  were lower than those of the other three colonies including Suhaam (-16.4 ± 0.62‰), Namdongji (-17.3 ± 0.75‰), and Chilsando  over the chick-rearing period, while there was no evidence of change on other colonies .The interaction between breeding colony and hatching date indicated that the change in δ15N, the full model differed significantly from the null model . We found that there was a significant interaction between chick-rearing period and breeding colony  and breeding colony and hatching date , while the interaction between chick-rearing period and hatching date for δ15N was not significant  than those during the early chick-rearing period ; among the other three colonies, the δ15N values of chicks at Chilsando (16.3 ± 0.41‰) were substantially higher than those at Suhaam (14.8 ± 0.30‰) and Namdonji (14.9 ± 0.34‰). For the late chick-rearing season, δ15N values of chicks were lower at Gujido (11.8 ± 0.41‰) than at other breeding colonies, and there was no difference among the other colonies .We failed to find supportive evidence for the change in δThe Bayesian mixing model also showed a spatial difference in diet use between breeding colonies , which i13C values, which were used to examine diet proportions from freshwater and intertidal wetlands, were higher during the early chick-rearing period than during the late chick-rearing period. In addition, δ13C values for Suhaam increased along with hatching timing. These results indicate that, in Suhaam, the freshwater diet proportion of chicks might be higher during the early chick-rearing season than the late chick-rearing period, while early-breeding spoonbills might feed their chicks with more freshwater prey than spoonbills breeding later. These results are consistent with earlier observations conducted in the coastal areas around Suhaam, which showed that the number of spoonbills foraging in rice paddies decreased over the chick-rearing season in the coastal area around Suhaam [The most prominent finding was that the proportion of freshwater diets was high at Gujido, where no freshwater wetlands are available for foraging within the typical foraging range (11 km from the colony). On the other hand, at other colonies located near the coastline, including Suhaam, Namdongji, and Chilsando, adults fed their chicks with prey from intertidal mudflats and freshwater wetlands at a similar rate. This result is in agreement with those of previous reports, which showed that the adults at other offshore islands did not feed in nearby intertidal flats but from more distant freshwater wetlands instead, while those at inshore islands used both habitats. These results also corroborate the findings of previous reports showing that the diets of chicks in offshore colonies mainly originated from freshwater habitats , 18. Witd Suhaam , 36, 78.13C values and the estimated proportion of freshwater prey should have shown lower salt intake or exclusive use of freshwater prey during the early chick-growing period. However, the range of δ13C demonstrated high overlap between the early and late chick-rearing periods, and some of the individuals at Suhaam, Namdongji, and Chilsando might have been fed with more marine prey than freshwater prey even during the early chick-rearing period. δ13C and δ15N Bayesian mixing models estimated that more than 50% of diets during the early chick-rearing period originated from intertidal mudflats at Suhaam, Namdongji, and Chilsando. In addition, the contribution of each prey type did not vary substantially by chick-rearing period. Comparing the estimated contribution of diets between chick-rearing periods may be limited because we estimated the average contribution of each prey type by colony and chick-rearing period without accounting for within-individual variation in δ13C and δ15N. In addition, we used potential prey sources in the model, not data collected at foraging sites of study populations, which might influence the uncertainty of the estimation. Nonetheless, δ13C values indicated that salt intake might not be substantially different between chick-rearing periods, suggesting a sufficient osmoregulatory capacity to consume marine diets among chicks during the early growing period.Our data could not conclusively prove or disprove the hypothesis presented in previous reports that adults may feed a larger freshwater diet proportion during the early chick-rearing period due to lower salt tolerance among young chicks. To definitively support the salt stress hypothesis with our stable isotope analysis, δ13C from our analysis indicate that the hypothesis of osmoregulatory system development should be investigated by further research. For the Laughing Gull, chicks exhibit lower salt tolerance than adults, and prey exceeding a certain level of salinity negatively affect chick growth rate [13C values of some individuals were close to marine invertebrates, which are osmo-conformers with body fluid concentrations similar to seawater [13C values of young spoonbills increased slightly with age, the shift in δ13C values regardless of breeding season and colony might partially reflect a change in the proportion of freshwater and marine diet during the chick-rearing period. Previous studies showed that the salt glands of chicks become functional around six days after hatching in many waterbirds [13C and δ15N values in the growing feathers gradually change along the shaft after the dietary shift [However, age-related changes and spatial variation in δwth rate . Stable wth rate . Howeverseawater . This reterbirds –82. Thusry shift . Therefory shift , 85, in 15N values due to changes in nitrogen use efficiency depending on nutrition status [13C values were negatively correlated with body condition [13C values accounted for individual variation concerning nutritional intake and body condition, showing a consistent shift in δ13C values during the chick-rearing period. Therefore, these results suggest that variation in δ13C values with the growth of chicks might not relate to physiological factors but instead to changes in diet composition.Physiological factors such as nutrition status and growth may influence the stable isotope ratio of chicks –88. Prevn status , 87. Forondition . HoweverThe proportion of freshwater prey of chicks in each colony also showed the possibility that young spoonbills might be more susceptible to salt intake than adults. All breeding populations used freshwater wetlands to a considerable degree, despite the availability of marine resources in intertidal mudflats located closer to colonies. The central place foraging theory predicts that a forager will favor large-sized or high-quality prey to maximize the net rate of energy delivery as foraging distance increases . In the 13C values at Suhaam showed that chicks hatched later were fed more marine prey. This seasonal change in the proportion of freshwater diets in Suhaam might be related to the decrease in prey availability at rice paddies over the breeding season due to mid-season drainage. On the other hand, there was no significant seasonal trend in δ13C values at Gujido, Namdongji, and Chilsando. Although there has been no detailed investigation of the foraging habitats around breeding colonies except for Suhaam, a few observations reported that spoonbills at Namdongji foraged in different types of freshwater wetlands such as reservoirs and lakes [δnd lakes . Therefo15N values, at Gujido was lower than those of other three populations, while those at Chilsando were higher than those at Suhaam and Namdongji only during the early chick-rearing period. These results were consistent with the high estimated diet proportion of freshwater invertebrates at the lowest trophic level at Gujido, while chicks at Chilsando were fed more freshwater and marine fish at higher trophic levels than youngs at Suhaam and Namdongji in the early chick-rearing period. Although δ15N values at Chilsando decreased with age between the early and late chick-growing periods, there was no significant and consistent change in δ15N values over time and the values varied among individuals at Gujido, Suhaam, and Namdongji  showed no inflow of freshwater to nearby intertidal habitats and showed a heavy reliance on freshwater prey. These findings emphasize that the availability of freshwater wetlands may be beneficial to Black-faced Spoonbills strictly breeding in marine environments, as shown in the case of White Ibises breeding in South Carolina [Our study found that δCarolina , 30.Freshwater marshes, river estuaries, and intertidal mudflats on the western Korean Peninsula, which are potential foraging habitats for Black-faced Spoonbills, have steeply declined over the last 50 years due to reclamation, construction of river barrages and seawalls, and coastal development , 94. AppOur findings regarding freshwater diet reliance in spoonbill chicks suggest that the ongoing loss of quantity and quality of coastal freshwater wetlands in the vicinity of existing breeding colonies may result in greater energy expenditure for both provisioning adults and growing chicks. This expenditure may negatively affect the reproductive output of spoonbills adapted to marine breeding habitats, such as spoonbills breeding in offshore colonies, due to the high freshwater diet requirements of their chicks. Protection of coastal freshwater wetlands appears to be as important as the preservation of intertidal mudflats in the conservation of endangered Black-faced Spoonbills. Consequently, retention and better management of natural freshwater wetlands and intertidal mudflats as well as sound agricultural practices in rice paddies could help to improve the breeding success of Black-faced Spoonbills.S1 Fig13C values for each individual between chick-rearing periods.(A) Gujido, (B) Suhaam, (C) Namdongji, and (D) Chilsando. Lines link the δ(TIF)Click here for additional data file.S2 Fig13C values.Hatching dates were adjusted for each breeding colony by subtracting the hatching dates of individuals from the earliest hatching dates. The solid line and grey shading represent the estimated mean and standard error of effect size for age in the linear-mixed effect model for δ(TIF)Click here for additional data file.S3 Fig15N values for each individual between chick-rearing periods.(A) Gujido, (B) Suhaam, (C) Namdongji, and (D) Chilsando. Lines link the δ(TIF)Click here for additional data file.S1 Table13C values in the early chick-rearing period at the Gujido colony.The model included individual ID as a random effect. The intercept in this model estimated δ(DOCX)Click here for additional data file.S2 Table15N values in the early chick-rearing period at the Gujido colony.The model included individual ID as a random effect. The intercept in this model estimated δ(DOCX)Click here for additional data file.S3 Table(XLSX)Click here for additional data file.