Abstract
This study presents molecular characteristics of livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA) from pigs and swine workers in central Thailand. Sixty-three MRSA isolates were recovered from pigs (n = 60) and humans (n = 3). Two major LA-MRSA lineages, including sequence type (ST) 398 and clonal complex 9 (ST9 and ST4576, a novel single-locus variant of ST9), were identified. ST398 had spa type t034 (n = 55). ST9 and ST4576 had t337 (n = 8) and carried staphylococcal cassette chromosome mec (SCCmec) IX only. MRSA-ST398-t034 contained various SCCmec, including SCCmec V (n = 42), a novel SCCmec composite island (n = 12), and a nontypeable SCCmec (n = 1). All isolates were multidrug resistant and carried common resistance genes found in LA-MRSA. This is the first report of the presence of swine MRSA ST398 and multidrug resistance gene cfr in MRSA ST9 in Thailand. With identical molecular characteristics, pigs could be a source of MRSA ST398 spread to humans. A minor variation of genetic features and resistance gene carriage in both lineages represented a heterogeneous population and evolution of the endemic clones. A monitoring program and farm management, with prudent antimicrobial uses, should be implemented to reduce spreading. Strict hygiene and personal protection are also necessary to prevent transfer of LA-MRSA to humans.
Introduction
Methicillin-resistant Staphylococcus aureus (MRSA) is one of the major antibiotic-resistant and opportunistic bacteria causing serious infections, with limited antimicrobial treatment options. 1 Antimicrobial uses in livestock production were associated with the emergence of livestock-associated MRSA (LA-MRSA) clones spreading worldwide. 2 Pigs are recognized as a reservoir of LA-MRSA that can be transferred to humans, especially to swine workers and their household members. 3 In addition to resistance to β-lactams mediated by mecA, LA-MRSA can usually mediate multidrug resistance by accumulating mobile genetic elements encoding multiple resistance mechanisms by possessing uncommon resistance genes, which are not found in human MRSA.
LA-MRSA belongs to specific sequence types (ST) such as ST398, ST9, and ST49.4,5 In pig production, LA-MRSA-ST398 is endemic in Europe and North America, but less widespread in Asia and has so far not been detected in livestock in Thailand. In Asia, MRSA-ST9 is the most frequent lineage, which has been detected in China, Hong Kong, Taiwan, Malaysia, and Thailand. 6 Previous studies showed that pigs and swine workers in the northern and northeastern parts of Thailand carried MRSA-ST9.7–10 This clone has also been associated with infections in human in the community. 11 MRSA-ST398 colonizing human and canine hosts was already reported in Thailand,12,13 but its presence in livestock is still unknown.
As the central part of Thailand is a main region for swine production, we screened pigs and swine workers for the presence of MRSA in central Thailand and characterized the MRSA isolates using genotyping methods and determined their antimicrobial resistance phenotypes and genes.
Materials and Methods
Sources of isolates
From May 2015 to April 2017, 467 pig samples, including 80, 156, and 161 from individual sows, nursery pigs (3–8 weeks), and fattening pigs (>8 weeks), respectively, and 70 from suckling piglet litters (aged <3 weeks old, pooled 3 piglets per litter) were obtained by nasal swabbing from 27 farms located in central Thailand. The sampling protocol was approved by the Chulalongkorn University Animal Care and Use Committee (CU-ACUC) with the Animal Use protocol number 1631051. Thirty-eight samples from volunteered swine workers were obtained by nasal swabbing from 16 farms at the same period. The protocol was approved by the Ethics Review Committee for Research Involving Human Research Subjects, Health Sciences Group, Chulalongkorn University (No. 198.1/59).
Swabs preserved in Stuart's transport medium (Difco, Bordeaux, France) at 4°C were sent for culture within 24 hr. The Chulalongkorn University Faculty of Veterinary Science Biosafety Committee (CU-VET-BC) certified biosafety of the study providing the Biosafety Use protocol number IBC1631037.
MRSA isolation and identification
Swabs were inoculated in 5 mL of Müller–Hinton broth (Difco) with 6.5% NaCl and 4 μg/mL cefoxitin (FOX). The first inoculum of 0.5 mL was transferred into 4.5 mL of phenol red mannitol broth with 3.5 μg/mL cefoxitin before plating onto tryptic soy agar with 5% sheep blood. Each step required incubation at 32°C for 48 hr. Hemolytic Staphylococcus colonies were selected and primarily identified as S. aureus by gram-positive cocci staining and positive catalase and coagulase production. The presence of a 359-bp nuc fragment amplified by PCR identified S. aureus. 14 Bacterial DNA was isolated using the NucleoSpin® Tissue DNA Extraction Kit (Macherey-Nagel, Düren, Germany). The cefoxitin disk diffusion test and mecA PCR were used for methicillin resistance detection and confirmation. 15
Antimicrobial susceptibility
Minimal inhibitory concentrations (MICs) of 19 antimicrobials, including FOX, penicillin (PEN), tetracycline (TET), gentamicin (GEN), kanamycin (KAN), streptomycin (STR), erythromycin (ERY), clindamycin (CLI), ciprofloxacin (CIP), chloramphenicol (CHL), rifampicin (RIF), tiamulin (TIA), sulfamethoxazole (SMX), trimethoprim (TMP), mupirocin (MUP), fusidic acid (FUS), dalfopristin/quinupristin (SYN), linezolid (LZD), and vancomycin (VAN), were determined in Müller–Hinton by broth microdilution assay using EUST customized plate (Trek Diagnostic Systems Ltd., East Grinstead, United Kingdom) following guidelines of the European Committee on Antimicrobial Susceptibility Testing (www.EUCAST.org). Inducible clindamycin (iCLI) resistance was detected by D-zone test. Resistance to FOX (>4 mg/L), PEN (>0.125 mg/L), TET (>2 mg/L), GEN (>1 mg/L), ERY (>2 mg/L), CLI (>0.5 mg/L), CIP (>1 mg/L), CHL (>8 mg/L), RIF (>0.5 mg/L), TMP (>4 mg/L), FUS (>1 mg/L), SYN (>2 mg/L), LZD (>4 mg/L), and VAN (>2 mg/L) was interpreted according to clinical breakpoints recommended by EUCAST. 16 Resistance breakpoints for KAN, STR, TIA, and MUP were referred to epidemiological MIC cutoff values from EUCAST for KAN (>8 mg/L), STR (>16 mg/L), TIA (>2 mg/L), SMX (>128 mg/L), and MUP (>1 mg/L). 5
Molecular typing
All MRSA isolates were characterized by staphylococcal cassette chromosome mec (SCCmec) typing, pulsed-field gel electrophoresis (PFGE), and spa typing. SCCmec were typed by two multiplex PCR panels classifying ccr complex and mec complex. 17 DNA fingerprint analysis was performed using Cfr9I-macrorestricted PFGE. Briefly, bacterial cells embedded in 0.9% SeaKem Gold agarose (Lonza, Rockland, ME) were lysed by lysostaphin, lysozyme, and detergents before chromosomal DNA digestion by 50U of Cfr9I (Thermo Fisher Scientific, Inc., Waltham, MA). 18 Macrorestriction fragments were separated using CHEF-DRIII PFGE (Bio-Rad, Hercules, CA) with a switch time of 5–40 sec and a voltage of 6 V cm−1 for 21 hr. A dendrogram was constructed to illustrate genetic relatedness by Gene Directory software (Syngene, Cambridge, United Kingdom), using Dice coefficient with unweighted pair group method using arithmetic averages and position tolerance at 1.0%, and more than 80% band similarity was clustered in the same group (Fig. 1). 19 The polymorphic X region of the spa gene was amplified and sequenced for spa typing (http://spaserver.ridom.de).20,21 Representative isolates from different host and farm origins exhibiting different SCCmec types, spa types, and DNA fingerprint patterns were selected for multilocus sequence typing (MLST) analysis. 22 Seven housekeeping genes were sequenced for MLST as described.*
A dendrogram constructed by Cfr9I-macrorestricted pulsed-field gel electrophoresis using unweighted pair group method using arithmetic averages, 1% position tolerance, and 80% similarity grouped in the same cluster and molecular typing illustrating genetic relatedness of methicillin-resistant Staphylococcus aureus isolated from pigs and swine workers in central Thailand. Color images are available online.
Resistance gene detection
Gram-positive resistance genes were screened in representative isolates by a customized DNA microarray tube version AMR+ve-5 (Alere Technologies GmbH, Jena, Germany).23,24 PCR was used to illustrate the presence of specific resistance genes found from screening by microarray in all MRSA isolates, including blaZ, mecA, tet(K), tet(L), tet(M), aac(6′)-aph(2")-Ia, ant(6)-Ia, ant(4′)-Ia, str, spc, spw, erm(A), erm(B), lsa(E), vga(A), cfr, catpC221, fexA, and dfrG (Supplementary Table S1).
Results
MRSA isolation
A total of 63 MRSA isolates, including 60 swine isolates from 12.6% (59/467) pig samples and 3 human isolates from 8% (3/38) swine workers, were recovered. Two swine isolates were collected from one pig sample because of distinct hemolytic colonies. Thirty-seven percent (10/27) of farms (Farm 1–10) had MRSA-positive pigs, and three MRSA-positive swine workers were from three farms (Farm 1, 3, and 11). Two MRSA-positive swine workers worked in two swine MRSA-positive farms (Farm 1 and 3), and another was from a swine MRSA-negative farm (Farm 11). Table 1 summarizes the MRSA isolates from different sources.
Isolation of Methicillin-Resistant Staphylococcus aureus from Pigs and Swine Workers
Farms 1 and 2 were farrow-to-finish holdings. Farms 3 and 5 were farrow-to-wean holdings. Farms 4, 6, 7, 8, 9, 10, and 11 were wean-to-finish holdings.
Three piglets pooled per litter.
Two MRSA isolates were derived from one nursery pig in Farm 8.
MRSA, methicillin-resistant Staphylococcus aureus.
Molecular characteristics
Three SCCmec types were identified, including SCCmec V (n = 42), IX (n = 8), and a novel SCCmec composite island (CI), consisting of ccrA1B1, ccrC, and class C mec complex (n = 12), while SCCmec of one isolate was nontypeable (NT) showing negative amplification of ccr and mec complexes. Two spa types were identified, including t034 (n = 55) and t337 (n = 8). Chromosomal restriction analysis by PFGE generated five clusters (A to E) (Fig. 1). Identical band patterns were consistent with the identical SCCmec, spa, and antimicrobial resistance profile for the isolates from the same farm.
The 16 representative isolates from each positive farm from distinct origins (pig or human) presenting identical genotype (SCCmec/spa/PFGE pattern) and antimicrobial resistance profile belonged to three STs, including ST398, ST9, and ST4576. The ST4576 was a new single-locus variant of ST9 by a substitution mutation (C335T) of glpF, resulting in allelic profile 3-3-625-1-1-1-10, which belonged to clonal complex (CC)9. The ST9 and ST4576 isolates harboring SCCmec IX had spa t337 (ST9-IX-t337 and ST4576-IX-t337). The ST398 isolates containing spa t034 were associated with SCCmec V (ST398-V-t034) and SCCmec CI (ST398-CI-t034). NT SCCmec was harbored by one ST398 with t034 (ST398-NT-t034).
Antimicrobial resistance phenotypes and genes
Antimicrobial resistance phenotypes and genes are illustrated in Table 2. All MRSA isolates exhibited resistance to PEN, FOX, CLI, TET, STR, CIP, TIA, and TMP. Additional resistance phenotypes were also found, including resistance to ERY (n = 60), GEN and KAN (n = 21), CHL (n = 8), SYN (n = 61), and SMX (n = 5).
Molecular Characteristics and Antimicrobial Resistance Phenotypes and Genes of Methicillin-Resistant Staphylococcus aureus Isolated from Pigs and Swine Workers in Central Thailand
Antimicrobial resistance phenotype was determined by microbroth dilution. Abbreviation of antimicrobial agents are as follows: CIP, ciprofloxacin; CHL, chloramphenicol; CLI, clindamycin; ERY, erythromycin; FOX, cefoxitin; GEN, gentamicin; KAN, kanamycin; LZD, linezolid; PEN, penicillin; SMX, sulfamethoxazole; STR, streptomycin; SYN, Synercid or dalfopristin/quinupristin; TET, tetracycline; TIA, tiamulin; TMP, trimethoprim.
Antimicrobial resistance genes and mechanisms are as follows: mecA, penicillin-binding protein; blaZ, β-lactamase; tet(K) and tet(L), tetracycline efflux protein; tet(M), ribosomal protective protein; ant(4′)-Ia and ant(6)-Ia, aminoglycoside nucleotidyl transferase; aac(6′)-Ie, aminoglycoside acetyltransferase; aph(2"), aminoglycoside phosphotransferase; erm(A), erm(B) and erm(C), erythromycin resistance methylase; dfrG, dihydrofolate reductase; lnu(B), lincosamide nucleotidyltransferase; vga(A) and lsa(E), ABC transporter; fexA, florfenicol exporter; cfr, ribosomal RNA methyltransferase; spc and spw, spectinomycin adenyltransferase; catpc221, chloramphenicol acetyltransferase; str, streptomycin adenyltransferase.
SCCmec was identified by two multiplex PCR panels. CI was a novel SCCmec CI consisting of ccrA1B1, ccrC, and class C mec complex and NT was an NT SCCmec presenting negative amplification of ccr complex and mec complex.
The isolates exhibited intermediate resistance to dalfopristin/quinupristin.
ABC, ATP-binding cassette; CI, composite island; NT, nontypeable; PFGE, pulsed-field gel electrophoresis; SCCmec, staphylococcal cassette chromosome mec.
All the isolates had mecA, blaZ, and dfrG. tet genes were detected, including tet(M) (n = 63), tet(K) (n = 55), and tet(L) (n = 5). Only 3 isolates carried just tet(M), and 60 isolates carried 2 tet genes, namely tet(M)+tet(L) (n = 5) and tet(M)+tet(K) (n = 55). Genes encoding aminoglycoside-modifying enzymes were found, including ant(4′)-Ia (n = 60), ant(6)-Ia (n = 55), and bifunctional aac(6′)-aph(2")-Ia (n = 21). Sixty isolates carried an erythromycin resistance methylase gene, including either erm(B) (n = 5) or erm(C) (n = 34) alone, or a combination of erm(A)+erm(C) (n = 8) and erm(A)+erm(B) (n = 13).
The same 55 isolates contained lnu(B) and lsa(E), and spw. spc (n = 21), vga(A) (n = 8), and cfr (n = 5) were detected. Two genes encoding phenicol resistance mechanisms were detected, including fexA (n = 5) and catpC221 (n = 3). Also, the cfr gene conferring resistance to LZD was detected in the same fexA-positive isolates. Resistance to VAN, RIF, FUS, and MUP and iCLI resistance were not detected in any isolates.
Clonal distribution
MRSA-ST398 was recovered in nine swine farms in central Thailand. ST398-V-t034-C (ST-SCCmec-spa-PFGE cluster) was the major characteristic found in seven farms (Farm 3, 4, 5, 7, 8, 9, and 10) and contained mecA, blaZ, tet(K), tet(M), ant(4′)-Ia, ant(6)-Ia, dfrG, erm(C), lnu(B), and lsa(E). The isolates from Farm 3 and Farm 4 also contained erm(A) and spc. Farm 3 supplying piglets to Farm 4, and Farm 5 supplying piglets to Farm 7, 8, and 9 shared the strains presenting each identical feature. MRSA from sows and piglets from the same herd also exhibits the same characteristics.
Swine and human MRSA-ST398-CI-t034 was isolated from Farm 1 (PFGE cluster E) and Farm 11 (PFGE cluster D). The MRSA-ST398-NT-t034-E isolate was found in a pig from Farm 1 only. MRSA CC9 (ST9 and ST4576) carrying SCCmec IX isolates was isolated from pigs in three farms (Farm 2, 6, and 8). Only ST9 isolates from Farm 2 expressed ERY, SMX, and LZD resistance and additionally carried cfr, fexA, ant(4′)-Ia, erm(B), and tet(L). Human MRSA-ST398-V-t034-C and ST398-CI-t034-E were identical to those from swine from the same holding.
Discussion
This study demonstrated the distribution of LA-MRSA ST398 and CC9 in swine herds in Thailand. MRSA ST398 and ST9 are the major LA-MRSA clones disseminating worldwide and in Asia, respectively. Swine MRSA-ST398 was first detected as a major lineage in this study, while MRSA-ST9 has been previously reported.8,10 MRSA-ST398 is the predominant LA-MRSA disseminating in many parts of the world. For over a decade, MRSA-ST398 has been recognized as the major LA-MRSA clone in Europe and North America, and can be found only in some Asian countries, including South Korea and Singapore.25,26 spa t034 was commonly associated with MRSA-ST398 strains from Thailand and other Asian countries.25,27 Despite t034 being one of the predominant spa types found in MRSA CC398 in Europe, other types such as t011, t108, and t889 can be found.28–32
MRSA-ST9-t337 was also found in central Thailand, as well as in some northern and northeastern provinces.9,10 Asian MRSA-ST9, from pigs, contains various associated spa types in the mainland of China and Hong Kong (t899), Taiwan (t337, t1430), and Malaysia (t4358),33–36 while ST9-t4794 has been detected in Europe. 28 In our study, spa types t034 and t337 were specific to MRSA-ST398 and MRSA-ST9, respectively, which represented two, major endemic LA-MRSA clones spreading in Thai swine herds in central Thailand.
SCCmec V and SCCmec CI were identified in MRSA-ST398-t034. The SCCmec V is a common type in MRSA CC39837 and has been detected in MRSA-ST398 from canine and human sources in Thailand. 12 The novel SCCmec CI classified by the presence of two ccr sets (ccrA1B1 and ccrC) was found in MRSA-ST398-t034 from both pigs and humans. These ccr components possibly illustrated a hybrid form between SCCmec V and SCCmec IX, both commonly found in MRSA from Thailand. The SCCmec IX was identified in the MRSA-ST398 strain from a Thai swine practitioner and in MRSA-ST9 from swine and workers.10,13 It has been suggested that type V and IX SCCmec and the CI could evolve into endemic strains in Thailand. Therefore, structures of the new CI and the NT SCCmec need to be determined by whole-genome sequencing in the future. The presence of MRSA-ST9-IX-t337 in central Thailand was consistent with previous reports, and ST4576-IX-t337 belonging to CC9 was first identified in our study. It has also been speculated that the endemic Thai MRSA-ST9 evolved by the specific acquisition of SCCmec IX.10,11 The present findings of heterogeneous characteristics by STs and various SCCmec and spa types could infer continuous evolution of LA-MRSA in Thai swine herds.
All MRSA exhibited multidrug resistance phenotypes and carried multiple resistance genes. Most of the resistance genes are found in staphylococci and gram-positive species, conferring resistance to the common, available antimicrobial classes used in human and veterinary medicine. Resistance to some drugs could be mediated by more than a single mechanism or gene such as tetracycline, erythromycin, and clindamycin, reflecting a high accumulation of acquired resistance determinants. Dihydrofolate reductase gene dfrG was found in both CC9 and CC398 from pigs and swine workers, but MRSA-ST398 from canine and veterinarians from our previous study contained dfrA. 12 Such a difference possibly means distinct events of gene acquisition and different spreading of the ST398 subpopulation among pigs, dogs, and humans in Thailand.
Resistance gene carriage and phenotypes were mostly specific to molecular characteristics and origins of the isolates. Bifunctional aac(6′)-aph(2")-Ia was carried in the MRSA CC9 and MRSA-ST398-SCCmec CI, but not in MRSA-ST398-SCCmec V. The CC9 isolates exhibited CHL resistance mediated by different resistance genes from different farms. Those from Farm 2 carried fexA and cfr, genetically linked on Tn558, 38 whereas other CC9 isolates had the plasmid-borne catpC221. The presence of cfr is of high concern as a public health threat due to the cross-resistance activity to oxazolidinones, which are last-resort antibiotics used for the treatment of MRSA infections.
All CC9 and ST398 isolates harbored vga(A) and lsa(E), respectively, mediating lincosamide/pleuromutilin/streptogramin A resistance. The colocalization of lsa(E) and lnu(B) was first detected on the multidrug resistance plasmid, pV7037, which carried ant(6)-Ia, aac(6′)-aph(2")-Ia, and erm(B) of MRSA-ST9-t899. 39 These genes were concurrently detected in MRSA-ST398-SCCmec CI and ST398-SCCmec V. The difference in resistance of gene carriage in the MRSA-ST398 lineage in this study supported different and continuous genetic alterations for the evolution of mobile elements in LA-MRSA strains.
Also, MRSA-ST398 strains had spw, while only SCCmec CI-carrying strains carried physically linked erm(A) and spc. 40 The vga(A), lsa(E), lnu(B), cfr, fexA, spc, and spw are considered uncommon resistance genes for MRSA from human origins but widely abundantly detected in LA-MRSA. 41 Resistance to VAN, RIF, FUS, and MUP was absent and was rarely found in other studies. As demonstrated, MRSA CC9 and ST398 acquired from different sources showed heterogeneous accumulation of acquired resistant determinants being successful clones in animals.
Clonal distribution, endemic circulation, and pig-to-human transmission among these populations could be supported by their identical features. MRSA-ST9-IX-t337-A, carrying cfr and fexA, and MRSA-ST398-CI-t034-E could be recovered from two different holdings that present specific, subclonal lineages circulating in the farms. Also, identical characteristics of swine and human strains demonstrated that pigs could be the source of the human strain. MRSA-ST398-NT-t034-E shared a similar PFGE profile with the major strains (MRSA-ST398-CI-t034-E) from the same farm, presenting a minor change of lineage in the hypervariable region as SCCmec. Also, MRSA CC9 had minor variations presented as a single-locus mutation and acquisition of resistance genes.
Identical characteristics and resistance patterns of MRSA-ST398-V-t034-C supported transmission between farms having a history of supplying pigs. MRSA-ST398-CI-t034 also contained erm(A), erm(B), aac(6′)-aph(2")-Ia, and spc, which were not found in MRSA-ST398-V-t034, suggesting a divergent evolution in this clone mediated by genetic recombination and horizontal transfer. Only ST398 was recovered from swine workers in our population, but human colonization by MRSA-ST9 has also been demonstrated in Thailand. Both, associated with infection, have been increasingly reported worldwide as suspects in livestock-to-community spread. 11
A study demonstrated that up to 6 of 15 samples are MRSA positive in a swine holding in Samut Songkhram province of central Thailand. 3 The present study reported the isolation of MRSA from a larger scale of pig samples and swine herds in central Thailand, which promoted finding more diverse MRSA lineages and their distribution. However, epidemiological information in terms of prevalence is limited by the criteria of sampling and the number of farm workers who volunteer. Pigs in the swine industry are considered a reservoir for LA-MRSA.6,42,43 Isolation of identical LA-MRSA from swine workers indicated an occupational risk for MRSA colonization. High-density farming is identified as a risk for LA-MRSA colonization in humans.27,44 Several transmission routes can promote MRSA circulation in endemic farms, including direct and indirect contact, and airborne and environmental contamination.45–47 Strict personal hygiene, monitoring, and infection control programs are highly recommended to reduce the risk of MRSA colonization in people working with pigs.
This study provides more insights into molecular epidemiological information of LA-MRSA spreading in swine herds and workers in Asia. MRSA-ST398 was also found to be an endemic clone in Thailand and illustrated heterogeneous features in addition to MRSA-ST9. The presence of cfr is alarming as it confers resistance to the critically important linezolid used in human medicine. Monitoring of MRSA in products, livestock, the human population, and hospital settings should be implemented to have a global surveillance of MRSA. Personal protection and strict hygiene, farm management, and judicious antimicrobial use need to be promoted to reduce the emergence and spread to humans.
Footnotes
Acknowledgments
The authors thank Alexandra Collaud from University of Bern, Switzerland, and Jitrapa Yindee, Nitisit Dokkhan, Nuttaphop Thamkittikhun, Pawarut Narongpun, and Stayu Tantanasarn from Chulalongkorn University, Thailand, for technical assistance. We gratefully acknowledge Keith Jolley from the University of Oxford, United Kingdom, for assigning a new allelic number and sequence type and the cooperation of pig farmers for participating in this study.
Disclosure Statement
No competing financial interests exist.
Funding Information
This study was supported by the TRF Grant of New Scholar (No. MRG5980024) and TRF Senior Scholarship to Alongkorn Amonsin (No. RTA6080012) from the Thailand Research Fund and the Grant for Development of New Faculty Staff, Ratchadaphiseksomphot Endowment Fund (No. DNS 60-003-31-001-2) from Chulalongkorn University.
References
Supplementary Material
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