Two novel Salmonella genomic island 1 variants in Proteus mirabilis

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1 AAC Accepted Manuscript Posted Online 27 April 2015 Antimicrob. Agents Chemother. doi: /aac Copyright 2015, American Society for Microbiology. All Rights Reserved. 1 2 Two novel Salmonella genomic island 1 variants in Proteus mirabilis isolates from swine farms in China Chang-Wei Lei, An-Yun Zhang, Bi-Hui Liu, Hong-Ning Wang #, Li-Qin Yang, Zhong-Bin Guan, Chang-Wen Xu, Dong-Dong Zhang, Yong-Qiang Yang Animal Disease Prevention and Food Safety Key Laboratory of Sichuan Province; Key Laboratory of Bio-resources and Eco-environment, Ministry of Education; 985 Project Science Innovative Platform for Resource and environment Protection of Southwestern China; College of Life science, Sichuan University, Chengdu, P. R. China # Corresponding author. Mailing address: College of Life science, Sichuan University, NO. 29 Wangjiang Road, Chengdu, Sichuan, China, Phone: Fax: whongning@163.com. Running title: Two novel SGI1 variants in P. mirabilis. Keywords: Salmonella, genomic island, multidrug resistance, integron

2 Abstract Four different Salmonella genomic island 1 (SGI1), including two novel variants, were characterized in one Salmonella enterica serovar Rissen ST1917 isolate and three Proteus mirabilis isolates from swine farms in China. One novel variant was derived from SGI1-B with the backbone gene S021 disrupted by a kb IS26-composite transposon containing the dfra17-aada5 cassettes and macrolide inactivation gene cluster mpha-mrx-mphr. Another one was an integron-free SGI1 and contained 183-bp truncated S025 next to IS6100 and S044. Salmonella enterica is a zoonotic pathogen and is one of the worldwide primary causes of human infections. Salmonella genomic island 1 (SGI1) is an integrative 42.4-kb chromosomal element first identified in the multidrug resistance (MDR) S. enterica serovar Typhimurium phage type DT104 clone that has been epidemic among humans and domestic animals since the 1990s (1, 2). The MDR region in SGI1 is a complex In4-type class 1 integron (named In104) clustering five antibiotic resistance genes that confer resistance to ampicillin, chloramphenicol and florfenicol, streptomycin and spectinomycin, sulphonamides and tetracycline (3). SGI1 was unexpectedly detected in a Proteus mirabilis clinical isolate in 2007 (4). Sequence analysis showed that SGI1 in S. enterica and P. mirabilis had the same chromosomal integration site corresponding to the last 18 bp of the 3 end of the trme (also named thdf) gene (5). It has been comfirmed that SGI1 in S. enterica could be transferred by conjugation with the help of IncA/C plasmid (6, 7). 2

3 Many SGI1 variants result from the homologous recombination of gene cassettes within the MDR regions (3, 8-11). A few variations in SGI1 backbone are also described due to the deletion, insertion and transposition (3, 9, 10, 12-15). Furthermore, several novel resistance genes, including ESBL gene bla VEB-6 as well as fluoroquinolone resistance genes qnra1 and qnrb2, have been reported in SGI1 (9, 11), suggesting that SGI1 could act as a mobilizable element to disseminate the critical resistance genes. In the present study, we characterized SGI1 among S. enterica and P. mirabilis isolates from swine farms in China. A total of 24 S. enterica and 61 P. mirabilis strains were isolated from samples of swine stools and diseased tissues in 35 swine farms from 16 provinces in China between May 2012 and February Antimicrobial susceptibility test was performed by the disc diffusion method according to the CLSI guideline (16). Primers used in this study are listed in Table S1. The left and right junctions of SGI1 were detected in one S. Rissen and three P. mirabilis strains. The multiple locus sequence typing for SGI1-containing S. Rissen strain Z4 showed that the types of the seven house-keeping genes were 92 (aroc), 137 (dnan), 8 (hemd), 524 (hisd), 206 (pure), 313 (suca) and 330 (thra) never reported to date. It was submitted to the website and assigned as a new ST1917. To the best of our knowledge, this is the first report of the SGI1 in S. Rissen. Three SGI1-containing P. mirabilis strains belonged to different clusters by pulsed field gel electrophoresis after SmaI digestion (Fig. S1). The origin, antibiotic resistance profiles and cassette genes of the four SGI1-containing strains are listed in Table 1. 3

4 Four different SGI1 variants were identified through PCR-mapping and sequencing (Table 2). Two known SGI1 variants, SGI1-I and SGI1-PmABB, have previously been reported (10, 17). Two novel SGI1 variants, SGI1-B2 (45.93-kb) in PmSC17 and SGI1-Z (24.30-kb) in PmSC42, were characterized in this study for the first time (Fig. 1). Two gene cassettes, bla PSE-1 (1.20-kb) and dfra17-aada5 (1.66-kb), were detected in strain PmSC17. However, the MDR region in SGI1-B2 contained only the bla PSE-1 cassette identical to SGI1-B (3). PCR amplicon was negative by using primers S020-R and S024-outF in the PCR-mapping of SGI1-B2 backbone. Through primer walking and PCR linkage, a kb IS26-composite transposon was found inserted in the backbone gene S021 never reported to date in SGI1 (Fig. 1). The transposition event occurred in the region between 128-bp and 175-bp of the S021 gene, resulting in a 48-bp target site duplication surrounding the IS26-composite transposon. It contained the dfra17-aada5 cassettes and the macrolide inactivation gene cluster mpha-mrx-mphr, which differed only by 4 single-base changes from the corresponding regions in the Escherichia coli plasmid pek499 (18). Therefore, SGI1-B2 was derived from SGI1-B with S021 disrupted by an IS26-composite transposon. SGI1-Z (24.30-kb) contained backbone genes S001-S024 and 183-bp truncated S025 next to IS6100 and S044. Although the strain PmSC42 harbored the dfra17-aada5 cassettes, they were not located in SGI1. The 183-bp truncated S025 was exactly close to the left 14-bp inverted repeat region of IS6100. So SGI1-Z is an 4

5 integron-free SGI1 and does not contain any resistance genes. We hypothesize that an IS6100-mediated transposition event might occur in the backbone gene S025 and the homologous recombination happened subsequently between the two copies of IS6100 resulting in the loss of the integron. The mobility and stability of the SGI1 have previously been confirmed in S. enterica (6, 19, 20). Many SGI1s can be excised from the chromosome and the resulting free circular form may be transferable with the helper plasmid (6, 20). The circular extrachromosomal form was detected in all four SGI1s after two rounds of PCR amplification by using the same circ1/2 primers (Fig. S2) (6), implying the mobility of the SGI1. Four SGI1-containing strains were propagated lasting for 20 days (40 passages) in the absence of antimicrobial pressure. None SGI1-negative clone was detected from the picked 827 clones (each strain picked about 200 clones) in the 41 st passage, suggesting that SGI1 was stable in both S. enterica and P. mirabilis. Nevertheless, the homologous recombination within SGI1 integrons could occur in few clones. Four SGI1-I changed to SGI1-C containing only the aada2 cassette in MDR region already described (19, 20). The exchange of gene cassettes between the two integrons in SGI1-B2 was detected in nine clones, generating a new SGI1 MDR region containing the dfra17 and aada5 cassettes (Fig. S3). In conclusion, four SGI1, including two novel variants, were characterized in S. enterica and P. mirabilis isolates from swine farms in China. The dfra17 and aada5 cassettes, as well as mpha-mrx-mphr cluster, were reported in SGI1 for the first time. The persistence of SGI1 in S. enterica and P. mirabilis might threaten public health 5

6 given that the SGI1-containing strains could spread from animal farms to humans through meat consumption (5, 8, 21). Nucleotide sequence accession numbers. The complete nucleotide sequences of four SGI1s detected in this study were submitted to GenBank and assigned accession numbers KM (SGI1-I in S. Rissen Z4), KP (SGI1-B2 in P. mirabilis PmSC17), KP (SGI1-Z in P. mirabilis SC42) and KP (SGI1-PmABB in P. mirabilis PmXJF). ACKNOWLEDGMENTS This work was supported by 973 National Basic Research Program of China (project number 2013CB127200) and Science & Technology Pillar Program in Sichuan Province (grant number 2013NZ0025, 13ZC2578 and 2012GZ0001-1). REFERENCES 1. Boyd D, Peters GA, Cloeckaert A, Boumedine KS, Chaslus-Dancla E, Imberechts H, Mulvey MR Complete nucleotide sequence of a 43-kilobase genomic island associated with the multidrug resistance region of Salmonella enterica serovar Typhimurium DT104 and its identification in phage type DT120 and serovar Agona. J Bacteriol 183: Threlfall EJ Epidemic Salmonella typhimurium DT 104--a truly international multiresistant clone. J Antimicrob Chemother 46: Hall RM Salmonella genomic islands and antibiotic resistance in 6

7 Salmonella enterica. Future Microbiol 5: Ahmed AM, Hussein AI, Shimamoto T Proteus mirabilis clinical isolate harbouring a new variant of Salmonella genomic island 1 containing the multiple antibiotic resistance region. J Antimicrob Chemother 59: Boyd DA, Shi X, Hu QH, Ng LK, Doublet B, Cloeckaert A, Mulvey MR Salmonella genomic island 1 (SGI1), variant SGI1-I, and new variant SGI1-O in Proteus mirabilis clinical and food isolates from China. Antimicrob Agents Chemother 52: Doublet B, Boyd D, Mulvey MR, Cloeckaert A The Salmonella genomic island 1 is an integrative mobilizable element. Mol Microbiol 55: Douard G, Praud K, Cloeckaert A, Doublet B The Salmonella genomic island 1 is specifically mobilized in trans by the IncA/C multidrug resistance plasmid family. PLoS One 5:e Bi S, Yan H, Chen M, Zhang Z, Shi L, Wang H New variant Salmonella genomic island 1-U in Proteus mirabilis clinical and food isolates from South China. J Antimicrob Chemother 66: Siebor E, Neuwirth C The new variant of Salmonella genomic island 1 (SGI1-V) from a Proteus mirabilis French clinical isolate harbours bla VEB-6 and qnra1 in the multiple antibiotic resistance region. J Antimicrob Chemother 66: Siebor E, Neuwirth C Emergence of Salmonella genomic island 1 (SGI1) among Proteus mirabilis clinical isolates in Dijon, France. J Antimicrob 7

8 Chemother 68: Lei CW, Zhang AY, Liu BH, Wang HN, Guan ZB, Xu CW, Xia QQ, Cheng H, Zhang DD Molecular characteristics of Salmonella genomic island 1 in Proteus mirabilis isolates from poultry farms in China. Antimicrob Agents Chemother 58: Doublet B, Praud K, Bertrand S, Collard JM, Weill FX, Cloeckaert A Novel insertion sequence- and transposon-mediated genetic rearrangements in genomic island SGI1 of Salmonella enterica serovar Kentucky. Antimicrob Agents Chemother 52: Doublet B, Praud K, Weill FX, Cloeckaert A Association of IS26-composite transposons and complex In4-type integrons generates novel multidrug resistance loci in Salmonella genomic island 1. J Antimicrob Chemother 63: Hamidian M, Holt KE, Hall RM The complete sequence of Salmonella genomic island SGI1-K. J Antimicrob Chemother 70: Hamidian M, Holt KE, Hall RM The complete sequence of Salmonella genomic island SGI2. J Antimicrob Chemother. Doi: /jac/dku CLSI Performance Standards for Antimicrobial Susceptibility Testing, 22th Informational Supplement. CLSI document M100-S22. Clinical and Laboratory Standards Institute, Wayne, PA, USA. 17. Levings RS, Lightfoot D, Partridge SR, Hall RM, Djordjevic SP The genomic island SGI1, containing the multiple antibiotic resistance region of 8

9 Salmonella enterica serovar Typhimurium DT104 or variants of it, is widely distributed in other S. enterica serovars. J Bacteriol 187: Woodford N, Carattoli A, Karisik E, Underwood A, Ellington MJ, Livermore DM Complete nucleotide sequences of plasmids pek204, pek499, and pek516, encoding CTX-M enzymes in three major Escherichia coli lineages from the United Kingdom, all belonging to the international O25:H4-ST131 clone. Antimicrob Agents Chemother 53: Djordjevic SP, Cain AK, Evershed NJ, Falconer L, Levings RS, Lightfoot D, Hall RM Emergence and evolution of multiply antibiotic-resistant Salmonella enterica serovar Paratyphi B D-tartrate-utilizing strains containing SGI1. Antimicrob Agents Chemother 53: Kiss J, Nagy B, Olasz F Stability, entrapment and variant formation of Salmonella genomic island 1. PLoS One 7:e Seiffert SN, Tinguely R, Lupo A, Neuwirth C, Perreten V, Endimiani A High prevalence of extended-spectrum-cephalosporin-resistant enterobacteriaceae in poultry meat in Switzerland: emergence of CMY-2- and VEB-6-possessing Proteus mirabilis. Antimicrob Agents Chemother 57:

10 Tables and figure legends Table 1. SGI1-containing strains characterized in this study. Table 2. Four different SGI1 variants characterized in this study Fig. 1. Schematic view of the two novel SGI1 variants characterized in this study. Genes and ORFs are shown as arrows and their orientations of transcription are indicated by the arrowheads. DR-L and DR-R represent the 18-bp direct repeats at the ends of SGI1. CS: Conserved segment; IRi and IRt: Inverted repeats defining the left and right hands of integron; orf: Open reading frame. Downloaded from on June 28, 2018 by guest 10

11 Table 1. SGI1-containing strains characterized in this study. Province Strain Date of isolation Origin Integron cassette(s) Antibiotic resistance profile of isolation S. Rissen Z4 Anhui 28 October 2012 liver aada2, dfra1-orfc CHL, FFC, STR, SPT, DOX, TMP, SUL, SXT P. mirabilis SC17 Sichuan 12 April 2013 liver dfra17-aada5, bla PSE-1 AMP, CHL, FFC, NAL, STR, SPT, DOX, TMP, SUL, SXT P. mirabilis SC42 Sichuan 15 November 2013 stool dfra17-aada5 CHL, FFC, NAL, CIP, STR, STP, GEN, DOX, TMP, SUL, SXT P. mirabilis XJF Henan 09 January 2014 stool aaca5-aada7 AMP, STR, STP, GEN, DOX, TMP, SUL, SXT AMP, ampicillin; CHL, chloramphenicol; FFC, florfenicol; NAL, nalidixic acid; CIP, ciprofloxacin; STR, streptomycin; SPT, spectinomycin; GEN, gentamicin; DOX, doxycycline; TMP, trimethoprim; SUL, sulfizoxazole; SXT, trimethoprim-sulfamethoxazole. 11

12 Table 2. Four different SGI1 variants characterized in this study. Amplification of PCR product (bp) a SGI1 variants Strain Resistance genes in MDR region S005-S010 S020-S024 S024-S025 res-inti1 S024-S044 Type Size (kb) S. Rissen Z ND aada2, florc, tet (G), dfra1, sul1 SGI1-I P. mirabilis SC NE ND dfra17, aada5, bla PSE-1, sul1, mpha, mrx, mphr SGI1-B P. mirabilis SC NE NE 3399 None SGI1-Z P. mirabilis XJF ND aacca5, aada7, sul1 SGI1-PmABB a Primers are listed in Table S1. NE, negative. ND, not detected. 12

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