antibiotics Article A New Variant of the aadE-sat4-aphA-3 Gene Cluster Found in a Conjugative Plasmid from a MDR Campylobacter jejuni Isolate Pedro Guirado 1 , Elisenda Miró 2 , Yaidelis Iglesias-Torrens 2,3 , Ferran Navarro 2,3 , Susana Campoy 3 , Tyler Scott Alioto 4,5 , Jessica Gómez-Garrido 4 , Cristina Madrid 1, * and Carlos Balsalobre 1, * 1 Department of Genetics, Microbiology and Statistics, School of Biology, University of Barcelona, Av. Diagonal, 643, 08028 Barcelona, Spain; pedro.guirado.frias@gmail.com 2 Hospital de la Santa Creu i Sant Pau, Institut d’Investigació Biomèdica Sant Pau (IIB Sant Pau), Sant Quintí 89, 08041 Barcelona, Spain; emiro@santpau.cat (E.M.); yiglesiast@santpau.cat (Y.I.-T.); fnavarror@santpau.cat (F.N.) 3 Departament de Genètica i Microbiologia, Universitat Autònoma de Barcelona, 08193 Cerdanyola del Vallés, Spain; susana.campoy@uab.cat 4 CNAG-CRG, Center for Genomic Regulation (CRG), Barcelona Institute of Science and Technology (BIST), Baldiri i Reixac 4, 08028 Barcelona, Spain; tyler.alioto@cnag.crg.eu (T.S.A.); jessica.gomez@cnag.crg.eu (J.G.-G.) 5 Universitat Pompeu Fabra (UPF), 08002 Barcelona, Spain * Correspondence: cmadrid@ub.edu (C.M.); cbalsalobre@ub.edu (C.B.); Tel.: +34-934-039-382 (C.M.); +34-934-034-622 (C.B.) Abstract: Campylobacter jejuni is a foodborne pathogen causing bacterial gastroenteritis, with the highest incidence reported in Europe. The prevalence of antibiotic resistance in C. jejuni, as well as in many other bacterial pathogens, has increased over the last few years. In this report, we describe the  presence of a plasmid in a multi-drug-resistant C. jejuni strain isolated from a gastroenteritis patient.  Mating experiments demonstrated the transference of this genetic element (pCjH01) among C. jejuni Citation: Guirado, P.; Miró, E.; Iglesias-Torrens, Y.; Navarro, F.; by plasmid conjugation. The pCjH01 plasmid was sequenced and assembled, revealing high similarity Campoy, S.; Alioto, T.S.; (97% identity) with pTet, a described tetracycline resistance encoding plasmid. pCjH01 (47.7 kb) Gómez-Garrido, J.; Madrid, C.; is a mosaic plasmid composed of a pTet backbone that has acquired two discrete DNA regions. Balsalobre, C. A New Variant of the Remarkably, one of the acquired sequences carried an undescribed variant of the aadE-sat4-aphA-3 aadE-sat4-aphA-3 Gene Cluster Found gene cluster, providing resistance to at least kanamycin and gentamycin. Aside from the antibiotic in a Conjugative Plasmid from a resistance genes, the cluster also carries genes coding for putative regulators, such as a sigma factor MDR Campylobacter jejuni Isolate. of the RNA polymerase and an antisigma factor. Homology searches suggest that Campylobacter Antibiotics 2022, 11, 466. https:// exchanges genetic material with distant G-positive bacterial genera. doi.org/10.3390/antibiotics11040466 Academic Editor: Sara M. Soto Keywords: Campylobacter; conjugative plasmid; antibiotic resistance; aminoglycoside Received: 28 February 2022 Accepted: 28 March 2022 Published: 30 March 2022 1. Introduction Publisher’s Note: MDPI stays neutral Campylobacter jejuni is a worldwide foodborne pathogen. It is the zoonotic agent with regard to jurisdictional claims in with the highest reported incidence in Europe, with 220,682 confirmed human cases and published maps and institutional affil- 47 reported deaths in 2019 [1]. C. jejuni causes campylobacteriosis, a self-limiting gastroen- iations. teritis that can sporadically cause systemic infection and trigger neurological disorders, such as Guillain–Barré syndrome [2]. C. jejuni is a motile, Gram-negative, microaerophilic epsilon proteobacteria that are characterized as being thermophilic with optimal growth in Copyright: © 2022 by the authors. a temperature range between 30 and 45 ◦ C, approximately. It is commonly found in the Licensee MDPI, Basel, Switzerland. gastrointestinal tract of birds, including poultry species. In fact, the most common transmis- This article is an open access article sion pathway to humans is the consumption of undercooked chicken meat. Nevertheless, distributed under the terms and its presence has also been detected in the gastrointestinal tracts of mammals, insects, water, conditions of the Creative Commons soil, etc. [3]. Attribution (CC BY) license (https:// Over the last few years, an increase in the prevalence of antibiotic resistance has creativecommons.org/licenses/by/ been noticed in many bacterial pathogens, including Campylobacter. A European Food 4.0/). Safety Authority report from 2018/2019 indicated that 61.5, 47.2, 1.5, 0.8, and 0.3% of the Antibiotics 2022, 11, 466. https://doi.org/10.3390/antibiotics11040466 https://www.mdpi.com/journal/antibiotics Antibiotics 2022, 11, 466 2 of 14 C. jejuni isolated from humans are resistant to ciprofloxacin, tetracycline, erythromycin, amoxicillin/clavulanic acid, and gentamicin, respectively [4]. The appearance of multidrug resistance (MDR) among Campylobacter species can jeopardize the treatment of prolonged and severe cases of campylobacteriosis. The spread of antibiotic resistance among bacteria is often mediated by plasmids that act as vehicles for antibiotic resistance genes and can be mobilized by conjugation [5]. It should be noted that tetracycline resistance in Campylobacter, which was detected in 47.2% of the human isolates from the European Union in 2018/2019, is commonly encoded in plasmids [6]. Several plasmids carrying tetracycline resistance in C. jejuni have been characterized and their transfer by plasmid conjugation has been experi- mentally demonstrated. In a recent report, we described that the conjugation of several tetracycline resistance carrying plasmids was promoted at temperatures characteristic of the avian intestinal tract [7]. The role of plasmid conjugation in the transfer of resistance to antibiotics other than tetracycline has been poorly characterized in Campylobacter. In this report, we describe a C. jejuni plasmid, pCjH01, isolated from a gastroenteritis patient in Spain that confers resistance to tetracycline, kanamycin, and gentamycin. The sequence of the pCjH01 plasmid reveals high similarity with the well-described tetracycline-resistance- encoding plasmid, pTet [8,9], but carries an undescribed variant of the aadE-sat4-aphA-3 gene cluster providing resistance to kanamycin and gentamycin. 2. Results and Discussion 2.1. C. jejuni Strain H01 Carries a Conjugative Plasmid Providing Resistance to Tetracycline, Kanamycin, and Gentamicin C. jejuni strain H01 belongs to a C. jejuni collection characterized in terms of the popu- lation structure, antimicrobial resistance, and presence of virulence-associated genes [10,11]. H01, isolated from a human patient suffering from gastroenteritis, belongs to the singleton ST-441 sequence type, which has been isolated from diverse niches, including humans, chickens, dogs, and farm environments [12]. The H01 isolate displays the most common pro- file of prevalence of virulence genes in the mentioned collection—being cdtA+ , cdtB+ , cdtC+ , cadF+ , ciaB+ , htrA+ , hcp− , wlan− , and cgtB− —whereas it has a distinct antibiotic resistance profile among the 150 isolates of the collection. The H01 isolate is a multi-drug-resistant (MDR) strain, being resistant to tetracycline (TET), nalidixic acid (NAL), ciprofloxacin (CIP), kanamycin (K), and gentamicin (G), and sensitive to ampicillin, amoxicillin/clavulanic acid, imipenem, streptomycin, chloramphenicol, and fosfomycin. In Campylobacter, resistances to TET, K, and G, all of which coincide in H01, are often encoded in plasmid-borne genes [6,13,14]. The tet(O) gene is the most common genetic determinant involved in resistance to TET, and the aph(30 )-IIIa gene is the one involved in K and G resistances [15,16]. The presence of these genes in the genome of the H01 strain, including both chromosomal and extrachromosomal elements, was confirmed by PCR and sequencing using specific primers, as described in Supplementary Table S1 (data not shown). To determine whether these genes were located in a plasmid, S1 nuclease digestion of the genomic DNA was carried out, followed by pulsed field gel electrophoresis (PFGE) and the hybridization with specific probes for tet(O) and aph(30 )-IIIa. As a control, the genomic DNA of the strain 81-176 carrying the tet(O)-harboring plasmid pTet was also analyzed. Figure 1 shows that the S1 nuclease linearizes both plasmids, showing a band between 48.5 and 97 kb. The pCjH01 and pTet plasmids hybridize with the tet(O) probe, whereas only pCjH01 hybridizes with the aph(30 )-IIIa probe. These results clearly indicate that the resistance to TET, K, and G in H01 is conferred by the presence of a plasmid, from this point referred to as pCjH01. Antibiotics 2022, 11, 466 3 of 15 Antibiotics 2022, 11, 466 3 of 14 that the resistance to TET, K, and G in H01 is conferred by the presence of a plasmid, from this point referred to as pCjH01. Figure1.1.The Figure TheHS01 HS01isolate isolatecarries carriesaaMDR MDRplasmid, plasmid,pCjH01. pCjH01.PFGE-S1 PFGE-S1hybridization hybridizationwith withspecific specific probes for tet(O) and aphA-3 (aph(3′)-IIIa). 0 Genomic DNA from the C. jejuni 81-176 strain probes for tet(O) and aphA-3 (aph(3 )-IIIa). Genomic DNA from the C. jejuni 81-176 strain carrying carrying the pTet plasmid and the MDR H01 isolate was S1 digested, and linearized plasmids were separated the pTet plasmid and the MDR H01 isolate was S1 digested, and linearized plasmids were separated by PFGE. The DNA was visualized by ethidium bromide (EtBr) staining and the presence of the by PFGE. The DNA was visualized by ethidium bromide (EtBr) staining and the presence of the tet(O) and aphA-3 genes was detected by hybridization using specific probes, as indicated. Lane 1, tet(O) and aphA-3 molecular genes was mass marker (CHEFdetected DNA by hybridization Size using specific Standard, 48.5–1000 probes,Ladder, kb, Lambda as indicated. LaneThe Bio-Rad). 1, molecular mass size of some of marker (CHEF the bands DNA Size in kilobases Standard,on48.5–1000 is indicated kb, of the left side Lambda Ladder, the EtBr panel.Bio-Rad). Lanes 2 andThe3, size of some of S1 digested the bands genomic DNAin kilobases is indicated from 81-176 and H01,on the left side of the EtBr panel. Lanes 2 and 3, S1 respectively. digested genomic DNA from 81-176 and H01, respectively. Conjugation is a widespread mechanism for the transfer of plasmids among bacteria, Conjugation including is [17]. C. jejuni a widespread mechanism To determine whetherfor the the transferplasmid putative of plasmids among present bacteria, in strain H01 including C. jejuni [17]. To determine whether the putative plasmid present encodes a functional conjugative apparatus, conjugation experiments were performed. in strain H01 encodes a functional Mating assays conjugative were conducted at apparatus, 42 °C for 4 conjugation h using A3S,experiments a streptomycinwere performed. (S)-resistant C. Mating assays were conducted at 42 ◦ C for 4 h using A3S, a streptomycin (S)-resistant jejuni strain, as a recipient strain [7]. Transconjugants were selected in the presence of S C. jejuni and TET.strain, as aDNA Plasmid recipient fromstrain [7]. Transconjugants H01 and were in TETRR transconjugants selected A3S wasin the presence isolated, of S digested and TET. Plasmid DNA from H01 and TET transconjugants in A3S was isolated, with BglII, and the restriction pattern characterized by electrophoresis. As it can be seen digested with BglII, and the restriction pattern characterized by electrophoresis. As it can be seen in Figure 2A, the digestion of the plasmid preparation from the H01 isolate displayed a in Figure 2A, the digestion of the plasmid preparation from the H01 isolate displayed a specific restriction band profile, which was identical to the profile derived from the plas- specific restriction band profile, which was identical to theRprofile derived from the plasmid mid preparation of oneR TETR transconjugantR (TetR and S colonies). The selected RTETR and R colonies). preparation of one TET transconjugant (Tet and S The selected TET and SR SR colonies were confirmed as transconjugants by the PCR genotyping of the chromoso- colonies were confirmed as transconjugants by the PCR genotyping of the chromosomal mal wlaN gene, coding for a galactosyltransferase, since the A3S recipient strain, but not wlaN gene, coding for a galactosyltransferase, since the A3S recipient strain, but not the the H01 donor strain, carries the wlaN gene (Figure 2B). Hence, transconjugant clones were H01 donor strain, carries Rthe wlaN gene (Figure 2B). Hence, transconjugant clones were characterized by being S , wlaN+, and carrying the pCjH01 plasmid (TETR). The selected characterized by being SR , wlaN+ , and carrying the pCjH01 plasmid (TETR ). The selected transconjugants were also resistant to K and G, clearly indicating that the resistance to the transconjugants were also resistant to K and G, clearly indicating that the resistance to the three antibiotics was encoded in plasmid pCjH01. Conjugation rate for pCjH01 at 42 °C three antibiotics was encoded in plasmid pCjH01. Conjugation rate for pCjH01 at 42 ◦ C wasestimated was estimatedtoto5.5 5.5×× 10 10−−77(Figure (Figure2C). 2C). In a previous report, we showed that the conjugation of several C. jejuni plasmids was thermodependent, being higher at 42 than at 37 ◦ C, the temperatures of the avian and human intestinal tracts, respectively [7]. Mating experiments with pCjH01 were also performed at 37 ◦ C and similar conjugation rates to those at 42 ◦ C were detected, indicating that pCjH01 conjugation is not temperature-dependent under the conditions tested. 2.2. pCjH01 Is Derived from the pTet Plasmid and Carries a New Variant of the aadE-sat4-aphA-3 Gene Cluster The plasmid pCjH01 was sequenced and assembled (Figure 3A) (GenBank accession number ON007234). It had a size of 47,758 bp and a GC content of 29.6 %. An in silico BglII restriction map was generated and, despite some deviation in the size estimation after electrophoretic analysis, the theoretical and experimental generated fragments coincided (Figure 2A), clearly confirming the presence of a single plasmid in strain H01. The annota- tion revealed 58 putative open reading frames (ORFs), covering approximately 94% of the plasmid (Table 1, Figure 3A). A BLAST alignment indicated that the pCjH01 plasmid had a high degree of identity with plasmids from C. jejuni and C. coli, including the pTet plasmid Antibiotics 2022, 11, 466 4 of 14 that has been previously characterized. The main known feature of the pTet plasmid is the presence of a canonical tet(O) gene that confers tetracycline resistance. The pCjH01 plasmid harbors a nearly identical tet(O) gene with only 11 nucleotide differences as compared with the tet(O) encoded in pTet, including a silent substitution in codon 21 and missense Antibiotics 2022, 11, 466 alterations in nine codons causing the following amino acid substitutions: P33L, K38E, 4 of 15 Q142R, C295Y, L346I, E363G, K366T, G371C, and C595Y. Figure2.2.The Figure ThepCjH01 pCjH01plasmid plasmidfrom fromthe theH01H01 isolate isolate is is a conjugative a conjugative plasmid plasmid that that carries carries resistance resistance to to tetracycline, kanamycin, and gentamycin. (A) BglII restriction profile of DNA tetracycline, kanamycin, and gentamycin. (A) BglII restriction profile of DNA plasmid from recipient plasmid from re- cipient (R), donor(R), donor (D), (D), and transconjugant and transconjugant (T) strains.(T) strains. The R strainThewasR strain A3S, andwasthe A3S, and the D strain wasD H01. strainLane was H01. Lane M, molecular mass marker (1 Kb DNA Ladder, Nippon Genetics, M, molecular mass marker (1 Kb DNA Ladder, Nippon Genetics, Düren, Germany)—the size of some Düren, Germany)—the size of some of the bands in kilobases is indicated on the left side of the panel. On the right side of of the bands in kilobases is indicated on the left side of the panel. On the right side of the panel, the the panel, the theoretical size in base pairs of the bands derived from the deduced BglII restriction theoretical size in base pairs of the bands derived from the deduced BglII restriction map from the map from the pCjH01 sequence obtained is indicated. The three lowest mass bands were better de- pCjH01 sequence tected when obtained the gel is indicated. (Supplementary was overexposed The three lowestFiguremass bands were S1). (B) better detected Genotyping of the when R, D, the and gel was overexposed T strains, as in A, by the(Supplementary PCR amplification Figure S1).wlaN of the (B) Genotyping gene. Lane M, ofmolecular the R, D, and massTmarker strains,(Gene- as in A, by the Ruler 100PCR amplification bp DNA of the wlaN gene. Lane Ladder, ThermoScientific, M, molecular Waltham, MA, USA)—themass marker size of(GeneRuler some of the100 bp bands in kilobases DNA Ladder,isThermoScientific, indicated on the left side ofMA, Waltham, the panel. USA)—thePCR size detection of the of some housekeeping of the gene gltA bands in kilobases is was usedon indicated as the a control. (C)ofConjugation left side the panel. PCR frequency detectionof pCjH01 at 37 and 42 °C of the housekeeping using gene donor gltA was and usedrecip- as a ient cells control. (C)asConjugation in A. Both cultures frequency andofmating pCjH01mixtures at 37 andwere incubated 42 ◦ C using donorat the andindicated recipienttemperatures. cells as in A. Significance was tested by an impaired two-tailed t-test. Statistical significance Both cultures and mating mixtures were incubated at the indicated temperatures. Significance (p-value) is indi- was cated, n.s. indicates no significance. tested by an impaired two-tailed t-test. Statistical significance (p-value) is indicated, n.s. indicates no significance. In a previous report, we showed that the conjugation of several C. jejuni plasmids was Athermodependent, comparison of the being higher pCjH01 andatpTet 42 than at 37 °C, plasmid the temperatures sequences (Figure 3A)ofrevealed the avianthat and human intestinal tracts, respectively [7]. Mating experiments with pCjH01 both plasmids are related by shared homology over most of the sequence: 89% of pCjH01 were also per- formed aligns toat 37 °C pTet and with similar 97% conjugation identity rates (with 5.8% to those of pTet at 42 °C and unaligned), wereincludes detected, indicating most genes that pCjH01 related conjugation to plasmid is not temperature-dependent mobilization. Clear differences among underthethe conditions two plasmidstested. were also detected, specifically within three distinct sequence regions (Figure 3B): region I (R1), 2.2. pCjH01 where Is Derived the pCjH01 from carries plasmid the pTetaPlasmid and Carries large sequence a New tract (4011Variant bp) thatof the aadE-sat4- replaces a 1564 bp aphA-3 Gene Cluster tract of the pTet plasmid; region II (R2), where the pCjH01 plasmid carries a sequence tract of 1163 bpplasmid The that replaces pCjH01 a 749 wasbpsequenced sequence of pTet; and and region assembled III (R3), (Figure 3A)where, (GenBankas compared accession to the pTet number plasmid, itItseems ON007234). had a that size aof288 bp deletion 47,758 bp and aoccurs. GC content of 29.6 %. An in silico BglII restriction map was generated and, despite some deviation in the size estimation after electrophoretic analysis, the theoretical and experimental generated fragments coincided (Figure 2A), clearly confirming the presence of a single plasmid in strain H01. The anno- tation revealed 58 putative open reading frames (ORFs), covering approximately 94% of the plasmid (Table 1, Figure 3A). A BLAST alignment indicated that the pCjH01 plasmid Antibiotics 2022, 11, 466 5 of 14 Antibiotics 2022, 11, 466 7 of 15 Figure 3. The pCjH01 plasmid has high similarity with the pTet plasmid. (A) Comparison between Figure 3. The pCjH01 plasmid has high similarity with the pTet plasmid. (A) Comparison between the pCjH01, pTet, and pGB19 plasmids. The regions I (RI), II (RII), and III (RIII) are indicated. The the pCjH01, pTet, and pGB19 plasmids. The regions I (RI), II (RII), and III (RIII) are indicated. The three plasmids were aligned by blastn to an inclusive artificial “pangenome” sequence constructed three fromplasmids wereand both pCjH01 aligned pTet by blastnregions to show to an inclusive artificialor“pangenome” that are present sequence absent from each plasmidconstructed with from both respect topCjH01 and The the others. pTetlevel to show regionspercent of BLAST that areidentity presentisorindicated absent from each plasmid by color accordingwith respect to the tolegend. The annotated the others. The levelORFs correspond of BLAST percenttoidentity the combined annotation is indicated of allaccording by color three plasmids. to the (B) Rep- The legend. resentationORFs annotated of regions I, II, and correspond III incombined to the both pCjH01 and pTet. annotation of Green all threearrows and black plasmids. lines indicate of (B) Representation the sequences present in both plasmids, whereas orange arrows and red lines indicate the sequences regions I, II, and III in both pCjH01 and pTet. Green arrows and black lines indicate the sequences only present in pCjH01. present in both plasmids, whereas orange arrows and red lines indicate the sequences only present in pCjH01. A comparison of the pCjH01 and pTet plasmid sequences (Figure 3A) revealed that both plasmids are related by shared homology over most of the sequence: 89% of pCjH01 The large pCjH01 specific sequence (region I), from position 3292 to 7302, had a higher aligns to pTet with 97% identity (with 5.8% of pTet unaligned), and includes most genes GC content (39.6%) than the plasmid on whole (29.6%), suggesting that this sequence was related to plasmid mobilization. Clear differences among the two plasmids were also de- acquired in a later event. Homology searches revealed that part of region I (1–858 and tected, specifically within three distinct sequence regions (Figure 3B): region I (R1), where 1793–4011) the pCjH01was foundcarries plasmid in several Campylobacter a large sequences sequence tract (4011 bp)located in bothathe that replaces chromosome 1564 bp tract and of the pTet plasmid; region II (R2), where the pCjH01 plasmid carries a sequence tract of is a plasmids, such as the plasmid pGB19 (Figure 3A, Table S2). pGB19 (CP071593.1) plasmid 1163 bp from C. jejuniastrain that replaces 749 bpGB19 associated sequence with of pTet; andgastrointestinal infections region III (R3), where, linked to the as compared onset of Guillain–Barré syndrome [18]. Interestingly, to the pTet plasmid, it seems that a 288 bp deletion occurs.significant similarity was also found with sequences from distant microorganisms, such as Mycoplasma bovirhinis (AP018135.1) and Streptococcus pyogenes (CP010449.1) (Table S2). Antibiotics 2022, 11, 466 6 of 14 Table 1. ORFs annotated in the plasmid pCjH01. The pCjH01-specific ORFs, as compared to pTet, are highlighted (grey background). Gene (ORF) AA(MW) Putative Function * tetO 639 (72.5) Tetracycline resistance ribosomal protection protein cpp2 57 (6.7) Conjugal transfer protein g1828 221 (25.5) Relaxase/mobilization nuclease-domain-containing protein cpp3 190 (22.1) Hypothetical protein aadE 206 (24.3) Aminoglycoside 6 adenyltransferase. sat4 113 (13.4) Streptothricin acetyltransferase g1778 209 (23.9) Aminoglycoside phosphotransferase aphA-3 264 (31) Aminoglycoside O phosphotransferase APH(30 )IIIa g1780 221 (25.1) Anti sigma factor (Zinc-finger domain containing protein) g1781 150 (17.1) Sigma 70 family RNA polymerase sigma factor cpp6 58 (7.0) Hypothetical protein cpp7 96 (11.9) Hypothetical protein cpp8 117 (13.4) Hypothetical protein cpp9 170 (19.6) Hypothetical protein cpp10 185 (22.1) Hypothetical protein cpp11 88 (10.6) Hypothetical protein cpp12 186 (21.5) ParA hypothetical protein cpp13 88 (10.2) Hypothetical protein cpp14 1932 (224.3) DEAD/DEAH box like helicase g1790 314 (37.3) HTH transcriptional regulator, XRE family cpp17 462 (54.0) Relaxase/mobilization domain containing protein cpp18 183 (21.1) Hypothetical protein cpp19 93 (11.5) Hypothetical protein cpp20 203 (23.9) NTPase. Hypothetical protein cpp21 217 (26.0) Hypothetical protein traC 408 (47.1) DNA primase, TraC family cpp23 87 (9.7) EexN family lipoprotein. IncN-type entry exclusion virB6 32 (3.6) Truncated VirB6 protein g1799 85 (10.2) Hypothetical protein g1832 61 (7.4) Hypothetical protein cpp24 72 (8.1) Type-II toxin-antitoxin system HicB family antitoxin cpp25 67 (7.9) Type-II toxin-antitoxin system HicA family toxin cpp26 597 (69.0) AAA family ATPase cpp27 204 (26.6) Recombinase family protein vapD 125 (15.0) Virulence associated protein cpp29 107 (12.6) Hypothetical protein virB2 87 (9.2) TrbC/VirB2 family protein. Conjugal transfer protein TraC virB4 992 (106.3) VirB4 family type IV secretion/conjugal transfer ATPase cpp32 188 (21.8) Rha family transcriptional regulator/phage regulatory protein cpp33 221 (25.6) Hypothetical protein ssb1 141 (15.8) single-stranded DNA-binding protein cpp35 91 (10.7) Hypothetical protein Antibiotics 2022, 11, 466 7 of 14 Table 1. Cont. Gene (ORF) AA(MW) Putative Function * virB5 323 (37.2) Type IV secretion system protein virB6 332 (35.5) Type IV secretion system protein. TrbL/VirB6 family protein virB7 56 (6.3) Hypothetical protein virB8 220 (25) Type IV secretion system protein virB9 295 (34.1) P-type conjugative transfer protein VirB9 Type IV secretion system protein VirB10. TrbI/VirB10 family virB10 391 (43.1) protein virB11 330 (37.6) P-type DNA transfer ATPase VirB11 Type IV secretory system conjugative DNA transfer family virD4 603 (63.7) protein cpp44 145 (16.8) cag pathogenicity island protein cpp45 254 (29.4) Hypothetical protein cpp46 265 (30.6) Hypothetical protein g1831 30 (3.9) Hypothetical protein cpp47 206 (23.8) Hypothetical protein topB 730 (84) DNA topoisomerase III cpp50 473 (57) Hypothetical protein cpp51 59 (7.1) Hypothetical protein * The predicted amino acids were used to search for putative function using the NCBI BLAST service with default blastp parameters. The pCjH01-specific region I sequence carried six putative ORFs (Table 1, Figure 3B). Four of them (g1776 to g1779), transcribed in the same orientation, encoded proteins puta- tively involved in antibiotic resistance, consistent with the antibiotic resistance phenotype associated with the acquisition of the pCjH01 plasmid. The other two ORFs (g1780–g1781), transcribed in the opposite direction with respect to the previous ORFs, encoded proteins presumably involved in gene regulation. The sequence from g1776 to g1779 showed a certain degree of similarity with the well- described aadE-sat4-aphA-3 cluster [19] (Table S3). Several variants of these gene clusters have been described within the chromosome of Campylobacter, as well as transposons, plasmids, and phages of both G-positive and G-negative bacteria [20–24]. The canonical aadE-sat4-aphA-3 gene cluster encodes a 60 -aminoglycoside-adenyltrasferase (also named AAD(60 )), a N-acetyltransferase, and a 30 -aminoglycoside-phosphotransferase which confer resistance to streptomycin, the atypical aminoglycoside streptothricin/nourseothricin, and to kanamycin/gentamicin, respectively. The g1776-g1779 gene cluster in pCjH01 exhibits a high degree of similarity with the aadE-sat4-aphA-3 cluster in plasmid pGB19 (Figure 4A), despite some important sequence rearrangements that will be further discussed below. The pGB19 aadE-sat4-aphA-3 cluster is widespread among Campylobacter isolates and many other bacteria, including G-positive bacteria (Table S4). The g1776-g1779 cluster in pCjH01, though, seems to have been derived from an aadE-sat4-aphA-3 that subsequently suffered drastic alterations in the DNA sequence: the pCjH01 cluster spanned 2630 bp in contrast to the 2047 bp of the pGB19 cluster. The homology among both clusters abruptly vanished within the sat4 ORF, from codon 75 of 181. A sequence of 935 bp with no homology in the DNA databases replaced a 352 bp sequence of pGB19 carrying the 30 of the sat4 ORF and 34 bp of the intergenic region between sat4 and aphA-3 from pGB19 (Figure 4B). The 935 bp insertion generated a putative chimeric ORF (g1777) carrying the first 72 codons of sat4 plus 39 extra codons. Moreover, the inserted sequence encoded an extra putative ORF (g1778) of 630 bp. The nucleotide sequence of g1778 only had resemblance with Antibiotics 2022, 11, 466 8 of 14 sequences obtained from uncultured bacteria (KU544893). Homology searches indicated that the g1778 predicted amino acid sequence had similarities with proteins from G-positive Antibiotics 2022, 11, 466 9 of 15 bacteria with phosphotransferase activity and involvement in aminoglycoside modification (Table S5). Figure4.4.The Figure Thenew newvariant variantofofthe theaadE-sat4-aphA-3 aadE-sat4-aphA-3genegenecluster clusterofofpCjH01. pCjH01.(A). (A).Comparison Comparisonbetween between aadE-sat4-aphA-3 gene clusters among the pCjH01, pGB19 (CP071593.1), and pN29710 (CP004067) aadE-sat4-aphA-3 gene clusters among the pCjH01, pGB19 (CP071593.1), and pN29710 (CP004067) plasmids. Homology at the protein level is indicated both with a color code according to the legend plasmids. Homology at the protein level is indicated both with a color code according to the legend and ribbons indicate the pairwise percent identity with respect to pGB19. (B) Brackets and dotted and ribbons lines indicate demarcate the pairwiseofpercent the substitution identity the 3′ sat4 withand sequence respect to pGB19. insertion (B)gene, of a new Brackets and g1778, in dotted pCjH01 lines demarcate the substitution of the 3 0 sat4 sequence and insertion of a new gene, g1778, in pCjH01 with respect to pGB19. with respect to pGB19. The two other ORFs present in pCjH01-specific region I encoded for a putative pro- teinThe two other sharing ORFs present high homology within an pCjH01-specific RNA polymerase region I encoded sigma70 for a putative subunit protein (ORF g1781) and sharing high homology with an RNA polymerase sigma70 subunit (ORF g1781) and a a protein carrying a zinc finger HC2 domain that was described in antisigma factors (ORF protein carrying a zinc finger HC2 domain that was described in antisigma factors (ORF g1780). There was no intergenic region between g1781 and g1780, suggesting that the two g1780). There was no intergenic region between g1781 and g1780, suggesting that the two ORFs were cotranscribed. The 1363 bp sequence from the end of the aphA-3 ORF to the ORFs were cotranscribed. The 1363 bp sequence from the end of the aphA-3 ORF to the end of region I, carrying both g1781 and g1780, has been found in many C. jejuni and C. end of region I, carrying both g1781 and g1780, has been found in many C. jejuni and C. coli coli isolates in both plasmid and chromosome locations, as well as in several G-positive isolates in both plasmid and chromosome locations, as well as in several G-positive bacteria, bacteria, such as Streptococcus, Staphylococcus, and Enterococcus (Figure 4A and Table S6). such as Streptococcus, Staphylococcus, and Enterococcus (Figure 4A and Table S6). Sigma subunits are responsible for promoter recognition and transcription initiation. Sigma subunits are responsible for promoter recognition and transcription initiation. Their expression is often regulated at the protein level by repressor proteins, known as Their expression is often regulated at the protein level by repressor proteins, known as antisigma subunits [25]. The presence of genes coding for a specific sigma/antisigma sys- antisigma subunits [25]. The presence of genes coding for a specific sigma/antisigma tem ininthe system theplasmid plasmidpCjH01 pCjH01suggests suggeststhatthat some some plasmid genes will plasmid genes will require requirethethespecific specific sigmasubunit sigma subunitfor fortheir theircorrect correctexpression. expression. Considering Considering the the specific specificlocation locationofofthese these sigma/antisigma systems in pCjH01 and that pTet does not carry sigma/antisigma systems in pCjH01 and that pTet does not carry such genes, we suggestsuch genes, we suggest thatthese that theseproteins proteinsmaymaycontrol controlthetheexpression expressionofofgenes genesthat thatarearespecifically specificallypresent presentininthe the plasmid pCjH01, such as the genes involved in antibiotic resistance. plasmid pCjH01, such as the genes involved in antibiotic resistance. Homology searches Homology searches indicatedthat indicated thatthe thegenes genespresent presentininthe thepCjH01 pCjH01region regionI Iwere werefound foundininphylogenetically phylogenetically distantorganisms, distant organisms,including including firmicutes firmicutes (Table (Table S2).S2). Genes Genes acquired acquired fromfrom distant distant organ- organisms isms might not be properly expressed in the new host due to a lack of promoter might not be properly expressed in the new host due to a lack of promoter recognition by the recogni- tion by the transcriptional transcriptional machinery. The machinery. acquisitionThe ofacquisition of a sigma a sigma subunit mightsubunit allow the might allow the expression of expression of the newly acquired genes. One can also expect that the newly acquired genes. One can also expect that the sigma/antisigma system encoded the sigma/antisigma insystem encoded the pCjH01 in the is not pCjH01to restricted is controlling not restricted thetoexpression controllingofthe expression of plasmid- plasmid-encoded genes encoded genes and could be involved in crosstalk between the plasmid and the chromo- some if the sigma subunit from plasmid pCjH01 can recognize chromosomally encoded genes. Remarkably, genes highly similar to g1780 and g1781 have been found within a chromosomal multidrug resistance genomic island in the C. coli strain SH96 [26]. Sigma factors encoded in plasmids are rare. The plasmid pBS32 from B. subtilis encodes a sigma Antibiotics 2022, 11, 466 9 of 14 and could be involved in crosstalk between the plasmid and the chromosome if the sigma subunit from plasmid pCjH01 can recognize chromosomally encoded genes. Remarkably, genes highly similar to g1780 and g1781 have been found within a chromosomal multidrug resistance genomic island in the C. coli strain SH96 [26]. Sigma factors encoded in plasmids are rare. The plasmid pBS32 from B. subtilis encodes a sigma factor that is expressed under certain environmental stresses causing cellular death after DNA damage [27]. Interestingly, when using a longer sequence of pCjH01—spanning from the end of aphA-3 to the start of cpp7—including the pTet sequences where the gene cluster was inserted, only pGB19 showed 100% homology in a correlative sequence (Table S7). The identical relative location of the gene cluster in both plasmids suggests that they both derive from an ancestral pTet plasmid that acquired a gene cluster carrying the aadE-sat4-aphA-3 cassette and the sigma and antisigma subunits, most probably from G-positive bacteria. This ancestral plasmid seems to have evolved in different ways: (i) it has acquired sequences coding for antibiotic resistances (pCjH01), and (ii) it has suffered severe rearrangements with other circulating plasmids generating mosaic plasmids, such as pGB19, that contain part of the ancestral pTet in a distinct plasmid backbone. A total of 96% of pCjH01 aligns to pGB19 with 96.24% identity (10.5% of pGB19 is unaligned); 83% of pGB19 aligns to pTet with 95.9% identity (10.6% of pTet is unaligned). The pTet boundaries where the pCjH01-specific region I was inserted were the in- tergenic region between cpp3 and cpp4 and within the repA ORF, 146 bp upstream of the repA end. Therefore, the region I pCjH01-specific sequence replaced cpp4 and part of repA from pTet. The cpp4 ORF encodes a hypothetical protein that has domains that have been attributed to relaxase/mobilization nuclease domain-containing proteins [28]. RepA shares high homology with several proteins involved in replication. In previous reports, it has been shown that the repA gene can be a target for insertions [26]. A unique ORF, g1790, seems to be found in the pCjH01-specific region II (Figure 3A). Homology searches revealed that g1790 encoded for a putative HTH transcriptional reg- ulator from the XRE family. The presence of genes coding for XRE-type transcriptional regulators has been described in several conjugative plasmids. For instance, the pLS20 plas- mid of Bacillus subtilis encodes an XRE regulator that represses plasmid conjugation [29,30], and the pRet42a plasmid from Rhizobium etli encodes an XRE-type protein that is required for conjugation from specific hosts [31]. These data suggest that the acquisition of this gene might provide differential regulation of plasmid conjugation affecting the optimal physiological and environmental conditions promoting pCjH01 conjugation and/or the range of specific hosts during conjugation. In a previous report, we showed that the conju- gation of pTet and pTet-related plasmids are affected by temperature, being promoted at temperatures higher than 42 ◦ C as compared to 37 ◦ C [7]. Interestingly, pCjH01, despite sharing with pTet most of the sequence, including genes involved in plasmid transfer, did not seem to be temperature-dependent. Whether the regulator encoded in g1790 is involved in the temperature regulation of conjugation remains to be elucidated. The acquisition of g1790 causes the deletion of the cpp16 ORF. Homology searches with the nucleotide sequence revealed that this gene was found in many Campylobacter isolates, located both in chromosomes and plasmids. Using the deduced amino acid sequence, it seemed to encode for a putative Gcn5-related N-acetyltransferase (GNAT). Acetyltransferases are enzymes involved in multiple bacterial processes, such as metabolism, cell wall modification, and antibiotic resistance. The role of this protein encoded in the pTet plasmid is uncharacterized. The last difference of note between the sequences of pCjH01 and pTet (region III) is the lack of a 288 bp sequence tract of pTet (from 5630 to 5917 bp, AY394561) in pCjH01 (Figure 3B). The deletion of this sequence causes alteration in two pTet ORFs: cpp7 (5346–5726, 126 amino acids) and cpp8 (5868–6266, 112 amino acids), with unknown functions. As a result, two new variants of the predicted cpp7 and cpp8 were generated (Figure 3B), which had no homology with any annotated protein. Antibiotics 2022, 11, 466 10 of 14 3. Conclusions The spread of MDR among pathogens is a public health concern of capital relevance. Campylobacter is among the bacterial pathogens causing high incidence of infections in humans, particularly in certain geographical areas such as the EU, North America, and Australia [32]. Understanding the mechanisms of antibiotic resistance and its mobilization among bacteria is of crucial importance to design strategies aiming to avoid the spreading of antibiotic resistances [33]. In this report, we describe pCjH01, a plasmid from a C. jejuni isolated from a patient with gastroenteritis that confers resistance to tetracycline, kanamycin, and gentamycin. pCjH01 has particular and distinctive characteristics. We demonstrate that pCjH01 is self-transmissible by conjugation and it seems to evolve from the very well-known pTet plasmid, characterized to transfer resistance to tetracycline, since it shares a high percentage of identity. The main distinctive features of pCjH01 are the gain of a gene cluster encoding for antibiotic resistance and a sigma/antisigma system, the gain of a gene encoding for a transcriptional regulator, and the loss of some ORFs. Homology searches using pCjH01-specific sequences revealed high similarity with sequences from G-positive bacteria, indicating that Campylobacter exchanges genetic material with distant bacterial species, such as Staphylococcus and Streptococcus. Evidence of gene exchange between Campylobacter and G-positive bacteria has been proposed earlier [14,24]. Whether the exchange occurs via natural transformation or conjugation remains unclear. The pCjH01 plasmid carries a new variant of the aadE-sat4-aphA-3 gene cluster. In pCjH01, the aadE and aphA-3 genes were intact, as compared to the aadE-sat4-aphA-3 gene cluster present in pGB19 and many other C. jejuni and C. coli isolates. In contrast, the downstream sequence of sat4 was substituted, generating a chimeric protein of smaller size (13.4 kDa as compared to the 21.3 kDa of sat4) with an unknown function. Moreover, the pCjH01 variant of the aadE-sat4-aphA-3 gene cluster carried an extra ORF encoding a putative phosphotransferase that may have been involved in antibiotic resistance. In many cases, the aadE-sat4-aphA-3 gene cluster was found together with two genes encoding for putative sigma and antisigma subunits, as exemplified by the plasmid pGB19 (Figure 4A). Homology searches using the genetic element including both the aadE-sat4-aphA-3 gene cluster and the sigma and antisigma genes indicated that several variants of the genetic environment existed when compared to pGB19, for instance, in the pN29710 plasmid and related (Figure 4B and Table S2). To our knowledge, the previously detected variations do not affect directly the aadE-sat4-aphA-3 gene cluster, but the genomic environment, whereas, in the pCjH01 plasmid, the aadE-sat4-aphA-3 gene cluster was altered by the insertion of a new gene. Further studies will be required to functionally characterize the newly described variant of the aadE-sat4-aphA-3 gene cluster and to determine its spread among bacterial pathogens. 4. Materials and Methods 4.1. Bacterial Strains, Plasmids, and Growth Conditions The C. jejuni strains H01 and 81-176 were isolated from human patients suffering from campylobacteriosis. The strain H01 was previously isolated by our research group [10]. The strain 81-176 carries the plasmids pVir and pTet, conferring the pTet plasmid resistance to tetracycline [9]. The C. jejuni A3S strain is nalidixic acid-resistant, streptomycin-resistant, and tetracycline-susceptible [7], and it was used as the recipient strain during the mating experiments. All C. jejuni strains were grown on Columbia blood agar base (CBA, Ox- oid) supplemented with 5% of defibrinated sheep blood (Oxoid, Hampshire, UK). When required, culture media was supplemented with tetracycline (TET), kanamycin (K), gen- tamicin (G), nalidixic acid (Nal), and streptomycin (S) at 20, 25, 10, 50, and 15 µg/mL, respectively. CBA plates were incubated for 48 h at either 37 or 42 ◦ C under microaerophilic conditions using a CampyGenTM atmosphere generation system (Oxoid, Hampshire, UK). Antibiotics 2022, 11, 466 11 of 14 4.2. Mating Experiments Mating experiments were performed essentially as described earlier [7]. Donor and recipient strains were grown for 48 h at either 37 or 42 ◦ C on CBA plates supplemented with tetracycline and streptomycin, respectively. Bacterial cells were collected in phosphate- buffered saline (PBS) supplemented with MgCl2 (100 µM), and the OD550 of the cell suspension was normalized to 1.5. Equivalent volumes (50 µL) of recipient and donor cell suspensions were mixed in the presence of DNAse I (100 U/mL) (Roche, Basel, Switzer- land) to avoid natural transformation events during the assay. Aliquots of 15 µL were spotted on CBA plates supplemented with DNAse I (100 U/mL) and incubated at either 37 or 42 ◦ C for four hours. Cells were recovered in PBS, serially diluted, and spread on CBA plates supplemented with the required antibiotics for the selection of both donor (tetracycline) and transconjugant (tetracycline plus streptomycin) cells. Control mating experiments with only donor or recipient cells were included in all experiments. Plates were incubated for 48 h at 42 ◦ C and the frequency of conjugation was calculated as the number of transconjugants per donor cell. We show the results of at least three independent experiments in a scatter dot plot graphic with the average. Plasmid isolation, restriction profile characterization, and strain genotyping by the detection of the wlaN gene were used to confirm transconjugant selection. 4.3. DNA Techniques Genomic DNA (total DNA content in the cells) and plasmid DNA were extracted using InstaGeneTM Matrix (Bio-Rad, Hercules, CA, USA) and E.Z.N.A.® Plasmid DNA kit (Omega Bio-tek, Norcross, GA, USA), respectively, following supplier indications. DNA was isolated from bacterial cultures grown at 42 ◦ C for 48 h on CBA plates. The BglII restriction pattern was used to characterize the isolated plasmids. PCR amplification was performed with PCR MasterMix (2×) (Thermo Scientific, Waltham, MA, USA). All the primers used are described in Table S1. 4.4. S1-PFGE and DNA Hybridization S1 digestion of genomic DNA was carried out as described previously in order to convert supercoiled plasmids into full-length linear molecules [34]. DNA samples were subjected to separation by PFGE using the CHEF-DR III Pulsed Field Electrophoresis Systems (Bio-Rad, Hercules, CA, USA) and following the standard operating protocol from PulseNet (https://pulsenetinternational.org/assets/PulseNet/uploads/pfge/PNL0 3_CampyPFGEprotocol.pdf (accessed on 27 February 2022)). The DNA within the PFGE gel was transferred onto a positively charged nylon membrane and UV light cross-linked by standard methods [35]. Specific digoxigenin-labeled probes for tet(O) and aph(30 )-IIIa genes were obtained using PCR (primer pairs indicated in Table S1) and the digoxigenin PCR DIG Probe Synthesis kit (Roche, Basel, Switzerland). Southern blot hybridization was carried out under high-stringency conditions according to the manufacturer’s instructions. 4.5. Genome Sequencing, Plasmid Assembly, and Alignment For the whole-genome sequencing of the C. jejuni isolates H01, the DNA was ex- tracted using the Wizard DNA-Purification kit (Promega, Madison, WI, USA). Genomic libraries and sequencing were performed in ADM Life Sequencing (https://www.biopolis- microbiome.com/ (accessed on 27 February 2022)) using the Illumina NextSeq 500 platform and Nextera XT 150PE/TrueSeq DNA kit for library preparation. The pCjH01 plasmid was assembled from the trimmed reads with SPAdes v3.13.0 [35], with the plasmid option in addition to default parameters. The pCjH01 plasmid was assembled from the trimmed 150 bp paired-end reads (cu- tadapt with parameters: -f fastq -e 0.1 -q 10 -O 1 -a AGATCGGAAGAGC) with Unicycler v0.4.6 [36], which in turn ran SPAdes v3.13.0 [37], with default parameters. An assem- bly with two connected components was obtained. The smallest connected component consisted of two contigs with lengths of 47,651 and 107 bp, which, after inspection of the Antibiotics 2022, 11, 466 12 of 14 assembly graph, were further joined into a single contig of 47,758 bp. The origin was then rotated to begin with the tet(O) gene. We annotated the assembled plasmid with Prokka v1.12 [38]. Resistance Gene Iden- tifier (RGI), with CARD database version 3.0.2 [39] was used to predict resistomes from the genome assembly. The Prokka and RGI annotations were combined to produce a final protein-coding gene set. To compare the assembled plasmids with the reference plasmid pTet (accession: AY394561.1) as well as pGB19 (accession: CP071593), pairwise BLAST [40] alignments were performed (blastn e-value cutoff of 10−5 ) and additional pairwise com- parisons were made with Mummer v3.22 [41] to determine percent identity. To assign gene names to their corresponding orthologue in pTet, we performed an all versus all Blastp with all of the protein sequences. Additional homologous plasmids were identified using the NCBI BLAST service against nt with default blastn parameters. To visualize the homology of pCjH01, pTet, and pGB19, we aligned the plasmids with Minimap2 [42] and parsed the alignments in order to construct a pangenome sequence of pTet and pCjH01 for use as reference with the CGView Comparison Tool [43]. The protein-level homology of the antibiotic resistance cassette of pCjH01 (region: 1–7156) to pGB19 and an additional plasmid, pN29710-1 (accession: CP004067), were compared with Clinker v0.0.23 [44]. Supplementary Materials: The following supporting information can be downloaded at: https:// www.mdpi.com/article/10.3390/antibiotics11040466/s1, Figure S1: Overexposed caption from the gel shown in Figure 2A; Table S1: Primers used in this study; Table S2: Result of a blastn homology search using the region I sequence (3292 to 7302) from pCjH01 as a query sequence; Table S3: Result of a blatn homology search using the pCjH01-sequence carrying from g1776 to g1779 (3309–5938) as a query sequence; Table S4: Result of a blatn homology search using the pGB19 (CP071593.1) aadE-sat4-aphA-3 cluster as a query sequence; Table S5: Result of a blastp homology search using the predicted amino acid sequence of g1778 from pCjH01 (4313–4942) as a query sequence; Table S6: Result of a blatn homology search using the pCjH01 sequence from 5939 to 7302 as a query sequence; Table S7: Result of a blatn homology search using the pCjH01 sequence from 5939 to 7810 as a query sequence. References [45–47] are cited in the supplementary materials Author Contributions: Conceptualization, P.G., E.M., Y.I.-T., F.N., S.C., T.S.A., J.G.-G., C.M. and C.B.; formal analysis, P.G., E.M., F.N., T.S.A., J.G.-G., C.M. and C.B.; investigation, P.G., E.M., Y.I.-T., T.S.A., J.G.-G., C.M. and C.B.; software, T.S.A., J.G.-G., C.M. and C.B.; writing—original draft preparation, C.M. and C.B.; writing—review and editing, P.G., E.M., Y.I.-T., F.N., S.C., T.S.A., J.G.-G., C.M. and C.B.; supervision, E.M., C.M. and C.B.; project administration, C.M. and C.B.; funding acquisition, C.M. and C.B. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by the Spanish Ministry of Economy and Competitiveness [grant AGL2013-45339R], the Spanish Ministry of Science, Innovation and Universities (MCIU), State Bureau of Investigation (AIE), the European Regional Development Fund (FEDER) [grant PGC2018-096958-B- I00], and the Catalonian government [grant 2017SGR499]. PG was the recipient of an ADR fellowship of the University of Barcelona. Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: The data presented in this study are openly available in GenBanK data base (https://www.ncbi.nlm.nih.gov/genbank/, accessed on 1 March 2022) accession number ON007234. Acknowledgments: We thank Marta Cerdà-Cuellar from IRTA-CReSA for fruitful discussion. Conflicts of Interest: The authors declare no conflict of interest. References 1. European Food Safety Authority; European Centre for Disease Prevention and Control. 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