This is the accepted version of the journal article: Rodríguez-Navarro, Judith; Miró, Elisenda; Brown-Jaque, Maryury; [et al.]. «Comparison of commensal and clinical isolates for diversity of plasmids in Es- cherichia coli and Klebsiella pneumoniae». Antimicrobial Agents and Chemother- apy, Vol. 64, Núm. 5 (April 2020), art. e02064-19. DOI 10.1128/AAC.02064-19 This version is available at https://ddd.uab.cat/record/285185 under the terms of the license AAC Accepted Manuscript Posted Online 2 March 2020 Antimicrob. Agents Chemother. doi:10.1128/AAC.02064-19 Copyright © 2020 American Society for Microbiology. All Rights Reserved. 1 Diversity of plasmids in Escherichia coli and Klebsiella pneumoniae: a comparison 2 of commensal and clinical isolates 3 a,b 4 Judith Rodríguez-Navarro , Elisenda Miró a, Maryury Brown-Jaque c, Juan Carlos Downloaded from http://aac.asm.org/ on March 9, 2020 at UAB/FAC VETERINARA 5 Hurtado d, Albert Moreno b,e, Maite Muniesa c , Juan José González-López b,e, Jordi Vila d, a*† *† 6 Paula Espinal and Ferran Navarro a,b 7 a 8 Department of Microbiology, Hospital de la Santa Creu i Sant Pau, Institut 9 d'Investigació Biomèdica Sant Pau (IIB Sant Pau), Barcelona, Spain. b 10 Genetics and Microbiology Department. Universitat Autònoma de Barcelona, 11 Barcelona, Spain. c 12 Department of Genetics, Microbiology and Statistics, University of Barcelona, 13 Barcelona, Spain. d 14 ISGlobal, Hospital Clínic - Universitat de Barcelona, Barcelona, Spain. e 15 Department of Clinical Microbiology, Hospital Vall d'Hebron, Vall d'Hebron Institut 16 de Recerca, Barcelona, Spain. 17 † 18 Contributed equally as senior authors 19 *Corresponding address: Dr. Ferran Navarro and Dr. Paula Espinal, Servei de 20 Microbiologia, Hospital de la Santa Creu i Sant Pau, Institut d'Investigació Biomèdica 21 Sant Pau, Barcelona, Spain., C/Sant Quintí, 89, 08041 Barcelona, Spain; Phone: 22 +34935537297; E-mails: fnavarror@santpau.cat and pespinal@santpau.cat 23 24 25 1 26 ABSTRACT 27 28 In this study, the plasmid content of clinical and commensal strains was analysed and 29 compared. The replicon profile was similar in both, except for L, M, A/C and N Downloaded from http://aac.asm.org/ on March 9, 2020 at UAB/FAC VETERINARA 30 (detected only in clinical strains) and HI1 (only in commensal strains). Although I1 and 31 F were the most frequent replicons, only IncI1 ST12 was associated with blaCMY-2 in 32 both populations. In contrast, the widespread resistant IncF plasmids were not linked to 33 a single epidemic plasmid. 34 35 36 KEYWORDS: pMLST, replicon, antimicrobial resistance, Enterobacteriaceae, plasmid 37 epidemiology. 38 RUNNING TITLE: Plasmid content in commensal and clinical Enterobacteriaceae 39 40 41 TEXT 42 The most prevalent mechanism in antimicrobial resistance gene (ARG) acquisition by 43 bacterial pathogens is horizontal gene transfer by plasmids (1, 2). PCR-based replicon 44 typing (PBRT) based on plasmid incompatibility (Inc) is currently the standard method 45 for plasmid identification (3, 4). Plasmid multilocus sequence typing (pMLST) schemes 46 allow to differentiate between plasmids within incompatibility groups and to define 47 epidemiological and evolutionary relatedness (5–10) (http://pubmlst.org/plasmid/). 48 Several plasmids carrying ARGs have been characterized, most of them recovered from 49 clinically relevant bacteria (11–14). In contrast, there is limited information on plasmids 50 in the commensal microbiota of healthy humans without a selection bias for 51 antimicrobial-resistant bacteria. In this scenario, the aim of this study was to provide a 2 52 better understanding of resistant plasmid diffusion in a clinical context by comparing 53 plasmids within Escherichia coli and Klebsiella pneumoniae strains isolated from 54 healthy human faeces and patients with bloodstream infection. 55 One hundred and fifty faecal samples were collected during 2014-2015 from healthy Downloaded from http://aac.asm.org/ on March 9, 2020 at UAB/FAC VETERINARA 56 humans who did not consume antibiotics and were not hospitalized. A total of 145 E. 57 coli and 12 K. pneumoniae strains were isolated. In addition, 202 strains from blood 58 cultures, 99 of E. coli and 103 of K. pneumoniae, from three hospitals in Barcelona 59 were analysed (one per patient). All strains underwent antimicrobial susceptibility 60 testing using disk diffusion (Table S1) and the results were interpreted according to 61 CLSI guidelines (15). The characterization of ESBLs, AmpCs and carbapenemases (16– 62 20) detected in both populations is shown in Table 1. The prevalence of ESBL- 63 producing E. coli in healthy carriers (4.7%) was higher than in a previous study in 64 Barcelona in 2005 (3.3%) (21), but still within the 3-6% average of Europe (22). 65 Plasmid identification was performed using the PBRT-kit (Diatheva, 2014) and simplex 66 PCR for ColE, X3, X4, L and M replicons (23–25). Twenty-nine replicons were 67 analysed, and only FIIS, W, T, U and HI2 were not detected in any strain. A total of 68 978 replicons were identified in the 359 studied strains: 84.1% (302/359) harboured 69 from one to four replicons and 10.9% (39/359) from five to seven. In 5% (18/359) of 70 the strains no replicon was detected. Overall, the results suggest that the replicon 71 content of E. coli strains followed a similar trend in patients and healthy individuals, 72 and the most prevalent in both sample groups were ColE, FII and FIB (Fig. 1A). 73 Nevertheless, replicons M, A/C and N were only detected in clinical strains, in 74 accordance with the literature (26, 27), while FIIK and HI1 were observed only in faecal 75 strains (Fig. 1A). Hence, it might be hypothesized that the hospital environment, where 76 there is a high antimicrobial use and an intense interhuman transmission, selects for 3 77 plasmids more adapted to these settings. The plasmid content in K. pneumoniae isolates 78 seems to follow similar trends (Fig. 1B) to that of E. coli, but this could not be 79 confirmed due to the low number of strains obtained from faecal samples. Notably, both 80 the diversity and frequency of replicons were higher in E. coli than in K. pneumoniae, Downloaded from http://aac.asm.org/ on March 9, 2020 at UAB/FAC VETERINARA 81 except for R and FIIK (Fig. 1C). 82 IncF and IncI1 plasmids have been reported in Enterobacteriaceae as promoters of beta- 83 lactamase gene dissemination in multiple environments, specifically blaCTXM-15 and 84 blaCMY-2 (28–33). In this study, 56 beta-lactamase genes were detected (Table 1). After 85 S1-PFGE and Southern hybridization (19, 34), 75% of ESBL, AmpC and 86 carbapenemase genes identified in E. coli (27/36) and K. pneumoniae (15/20) were 87 located on a plasmid, the most prevalent being IncF and IncI1 (37% both) in E. coli, and 88 IncF (47%) and IncR (20%) in K. pneumoniae. The predominant genes were blaCTX-M- 89 15/14/27 and blaCMY-2 in IncF and InI1 plasmids of E. coli (Fig. S1A) and blaCTX-M-15 in 90 IncF plasmids of K. pneumoniae (Fig. S1B). Figure 2 summarises the 49 ESBL-, 91 AmpC- and/or carbapenemase-producing strains detected in the study, the plasmids they 92 harboured, and the location of the beta-lactamase genes. 93 As IncF and IncI1 were two of the most frequently detected plasmids in both faecal and 94 clinical samples (27, 35, 36), they were further characterised using the pMLST method 95 (5, 10). In E. coli strains, 29 different IncI1 sequence types (STs) were detected, 59% of 96 which were assigned as new STs. This result reflects the great diversity within this 97 plasmid family, with only ST12 and ST36 being present in both clinical and faecal 98 populations (Fig. S2). Moreover, some of the most frequently reported STs worldwide 99 (ST2, ST12, ST26 and ST36) (37) were only found in E. coli from healthy humans. The 100 detection of many newly assigned STs in the clinical isolates and a scarce number of the 4 101 most reported STs suggests the latter may have been over-reported due to their 102 involvement in ARG dissemination, resulting in an epidemiological bias. 103 In addition, IncI1 plasmids have been associated with the carriage of blaCMY-2, 104 particularly IncI1 ST2, ST12, and ST23 (5, 38–40). In the current study, all identified Downloaded from http://aac.asm.org/ on March 9, 2020 at UAB/FAC VETERINARA 105 ST12 plasmids harboured blaCMY-2 and were detected in E. coli from both populations 106 (Fig. 2). These results support the suggestion that some IncI1 plasmids have been able 107 to evolve and persist in clinical settings, thanks to particular features that provide 108 resistance, persistence and adaptive success, which would explain why they are more 109 frequently reported and described as epidemic plasmids (38–40). 110 After defining the final number of IncF plasmids (n=279; 211 in E. coli and 68 in K. 111 pneumoniae) by Southern hybridization of F-replicons within each strain, 112 subtypification using replicon sequence typing (RST) was performed. In E. coli strains, 113 86 different FAB formulas from 205 typable plasmids (45 from faecal samples, 24 from 114 blood and 17 from both) were defined, where F29:A-:B10, F2:A-:B1, F2:A-:B- and 115 F24:A-:B1 were the most frequent (Fig. S3). Some of these formulas have been 116 previously identified in different environments, such as avian-pathogenic E. coli strains 117 and uropathogenic and extraintestinal pathogenic E. coli (27, 41), indicating a broad 118 distribution. In K. pneumoniae, 16 different FAB formulas from 68 typable plasmids 119 (12 from blood and 4 from both) were detected, K1:A-:B- being the most frequent (Fig. 120 S3). 121 IncF plasmids, including F2:A-:B-, F2:A1:B-, F31:A4:B1 and F1:A2:B20, have been 122 associated with the worldwide emergence of CTX-M-15 (10, 12, 42–44). Although 123 these four plasmids were detected in our study, only F2:A-:B- harboured the blaCTX-M-15 124 gene in a clinical strain and blaCTX-M-27 in a faecal strain. All the other CTX-M-encoding 125 genes were located in different IncF plasmids (Fig. 2). Thus, according to our results 5 126 and in agreement with those of previous reports (33, 35), there is no evidence for the 127 persistence of a unique IncF. These highly versatile plasmids are able to adapt to 128 intracellular environments by the rapid evolution of replicon regulatory sequences (10) 129 and they were widely distributed in the Enterobacteriaceae before antimicrobial use, Downloaded from http://aac.asm.org/ on March 9, 2020 at UAB/FAC VETERINARA 130 facilitating the persistence and spread of beta-lactamases (10, 33). Their co-existence 131 with other resistance determinants also contributes to the dissemination of IncF-CTX-M 132 plasmids (45). 133 Additionally, E. coli strains were assigned to phylogenetic groups following the 134 procedure of Clermont et al (46) (Table 2), and the presence of 15 virulence factors 135 (VFs) was determined (47, 48) (Fig. 3). In commensal E. coli, the prevalence of 136 phylogenetic groups varies among studies (36, 49). It has been reported that the highly 137 diverse hosts and environmental factors, the determinants of virulence and the 138 antimicrobial pressure can modify prevalence for a better adaptation to commensal 139 habitats (49). In our study, even though commensal E. coli presented a higher diversity 140 of phylogroups compared to the clinical samples (Table 2), a predominance of the 141 phylogroup B2 carrying high rates of VFs was found in both populations. Although no 142 evident association has been reported between plasmids and phylogroups (35), our 143 results indicate a possible association of HI1 plasmids with phylogroup A (p≤0.007, 144 Bonferroni's correction was applied). 145 All VFs studied were detected in both populations. As expected, the clinical strains had 146 a higher diversity (9 to15 VFs) and frequency of VFs compared to faecal samples (1 to 147 8 VFs) (Fig. 3). Finally, as supported by other authors (50), an association between 148 some VFs (fyuA, iutaA, hlyF, iss and traT) and strains carrying F plasmids was 149 determined (p≤0.003, Bonferroni's correction was applied). 6 150 In conclusion, new information is provided about the plasmid background in strains 151 isolated in a non-hospital setting. Although a similar trend was observed in the Inc 152 groups from both populations, IncL/M, IncA/C and IncN plasmids were only detected 153 in clinical strains, whereas HI1 was only present in faecal strains. Also, two different Downloaded from http://aac.asm.org/ on March 9, 2020 at UAB/FAC VETERINARA 154 evolutionary pathways followed by plasmids were observed: specific IncI1 plasmids, 155 such as IncI1 ST12, seem to have evolved by acquiring persistence, adaptive and 156 antibiotic resistance features relevant in clinical settings, whereas the more widespread 157 multireplicon IncF plasmids have randomly acquired resistance genes. Additionally, the 158 findings from this study confirm that strains from healthy individuals have less 159 antimicrobial resistance and fewer VFs and display a higher diversity of phylogenetic 160 lineages (in E. coli) than strains causing infection. 161 162 ACKNOWLEDGMENTS 163 This study was supported by the Plan Nacional de I+D+i and Instituto de Salud Carlos 164 III (grant PI13/00329); Ministerio de Economía y Competitividad, Spanish Network for 165 Research in Infectious Diseases (REIPI RD12/0015/0017); and co-financed by 166 European Development Regional Fund ‘‘A way to achieve Europe’’. P. Espinal is 167 funded by the Instituto de Salud Carlos III [‘‘Sara Borrell’’ contract number 168 CD15/00017]. 169 170 Ethical considerations 171 The study was approved by the Hospital de la Santa Creu i Sant Pau ethics committee 172 (13/051/1439). 173 174 7 175 REFERENCES 176 1. Wright GD. 2011. Molecular mechanisms of antibiotic resistance. Chem 177 Commun 47:4055–4061. 178 2. 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Microbiol Mol Biol Rev 73:750–774. 355 15 356 Figure legends 357 358 Figure 1. Replicon prevalence: (a) comparison between replicons detected in E. coli 359 from clinical and faecal samples; (b) comparison between replicons detected in K. Downloaded from http://aac.asm.org/ on March 9, 2020 at UAB/FAC VETERINARA 360 pneumoniae from clinical and faecal samples; (c) comparison between replicons 361 detected in E. coli and K. pneumoniae from the total strains. Tables show the number of 362 each replicon detected. 363 ND, not detected 364 *Statistical differences (p<0.05) between each population, faecal and blood samples or 365 E. coli and K. pneumoniae. 366 367 Figure 2. Heat-map summary of the sources, phylogenetic groups, β-lactamase 368 resistance genes, and the corresponding Inc plasmid types and their sizes for 49 β- 369 lactam-resistant E. coli (n=32) and K. pneumoniae (n=17) strains from faecal and blood 370 samples. Black and white squares denote the presence and absence of a particular 371 feature, respectively. 372 ND, not determined; CP, carbapenemases a 373 Plasmids where the replicon hybridisation occurred in the same plasmid size (hybrid 374 plasmids). b 375 bla-genes detected in the same plasmid. 376 377 Figure 3. Frequency of virulence factors (VFs) analysed in E. coli from faecal and 378 blood sample strains and percentages of E. coli strains according to the number of VFs 379 they carry. Adhesins fimH (mannose-specific adhesin of type 1 fimbriae) and hra (heat- 380 resistant agglutinin); siderophores fyuA (yersiniabactin), iutA (aerobactin), and iroN 381 (salmochelin receptor); toxins hlyD (α-hemolysin), hlyF (hemolysin F), cnf1 (cytotoxic 16 382 necrotizing factor 1), clbB and clbN (colibactin), and the miscellaneous VF genes iss 383 (surface exclusion serum survival protein), traT (serum resistance), ompT (outer 384 membrane protease), ibeA (invasion of brain endothelium) and usp (uropathogenic- 385 specific protein). Downloaded from http://aac.asm.org/ on March 9, 2020 at UAB/FAC VETERINARA 386 *Statistical differences (p<0.05) between each population, faecal and blood samples 17 387 Table 1. ESBL, AmpC and carbapenemase genes detected in E. coli and K. 388 pneumoniae from faecal and blood samples E. coli (n=244) K. pneumoniae (n=115) Faecal samples Blood cultures Blood cultures Downloaded from http://aac.asm.org/ on March 9, 2020 at UAB/FAC VETERINARA Faecal samples a b c (n=13/145) (n=19/99) (n=0/12) (n=17/103) n=8, 5.5% n=17, 17.2% n=17, 16.5% blaCTX-M-15 (n=10) blaCTX-M-15 (n=4) blaCTX-M-15 (n=9) ESBLs blaCTX-M-14 (n=2) blaCTX-M-14 (n=3) blaCTX-M-14 (n=2) (n=43) blaCTX-M-27 (n=2) - blaCTX-M-27 (n=1) blaSHV-28 (n=5) blaCTX-M-32 (n=1) blaSHV-12 (n=1) blaSHV-2 (n=1) blaSHV-12 (n=2) n=5, 3.5% n=5, 5.0% n=1, 1.0% AmpCs blaCMY-2 (n=4) (n=11) blaCMY-2 (n=5) - blaDHA-1 (n=1) blaDHA-1 (n=1) Carbapenemases n=2, 1.9% - - - (n=2) blaKPC-3 (n=2) Total genes 14 22 0 20 (n=56) 389 a 390 one E. coli strain had blaCTX-M-15 and blaCTX-M-14 b 391 three E. coli strains had blaCTX-M-15/blaCTX-M-32, blaCTX-M-27/blaDHA-1 and blaSHV- 392 12/blaCMY-2. c 393 one K. pneumoniae strain had blaCTX-M-15 and blaCTX-M-14, and two strains blaCTX-M-15 394 and blaSHV-28. 395 18 Downloaded from http://aac.asm.org/ on March 9, 2020 at UAB/FAC VETERINARA 396 Table 2. Phylogenetic groups detected in E. coli strains isolated from faecal and blood samples Phylogenetic groups A B1 B2 C D E F Clade I Unknown Total E. coli n=244 (%) 26 (10.6) 22 (9) 124 (50.8) 3 (1.2) 32 (13.1) 15 (6.1) 17 (6.9) 4 (1.6) 1 (0.41) n=145 23 (15.9)a 11 (7.6) 59 (40.7) b 3 (2.1) 23 (15.9) 8 (5.5) 13 (8.9) 4 (2.7) 1 (0.7) Faecal samples S n= 71 16 (22.5) 1 (1.4) 24 (33.8) 3 (4.2) 10 (14.1) 7 (9.9) 9 (12.6) 1 (1.4) 0 R n= 74 7 (9.5) 10 (13.5) 35 (47.3) c 0 13 (17.6) 1 (1.3) 4 (5.4) 3 (4.1) 1 (1.3) n= 99 3 (3) a 11 (11.1) 65 (65.7) b 0 9 (9.1) 7 (7.1) 4 (4) 0 0 Blood samples S n= 9 0 1 (11.1) 6 (66.7) 0 2 (22.2) 0 0 0 0 R n= 90 3 (3.4) 10 (11.1) 59 (65.5) c 0 7 (7.8) 7 (7.8) 4 (4.4) 0 0 397 S, susceptible to all antimicrobials tested; R, resistant to at least one of the antimicrobials tested. a,b,c 398 Statistical differences (p<0.05) between each population and the different phylogroups. 399 19 Downloaded from http://aac.asm.org/ on March 9, 2020 at UAB/FAC VETERINARA Downloaded from http://aac.asm.org/ on March 9, 2020 at UAB/FAC VETERINARA Downloaded from http://aac.asm.org/ on March 9, 2020 at UAB/FAC VETERINARA