The use of human specimens and all related experimental protocols were approved by the Committee on Human Research of indicated institutions and carried out in accordance with the approved guidelines. S. boydii species was identified by slide agglutination using monovalent antisera of Denka Seiken (Tokyo, Japan) and monoclonal antibody reagents of Reagensia AB (Solna, Sweden). The agglutination was sensitively scored according to the following scale: +++, 100% agglutination of the cells; ++, >50% agglutination; +, <50% agglutination; -, no agglutination detected.

The major horizontally acquired quinolone-resistance genes, extended-spectrum β-lactamase (ESBL) genes, and macrolide-resistance genes were screened by PCR (Supplementary Table S1), followed by amplicon sequencing on ABI 3730 Sequencer (LifeTechnologies, Carlsbad, CA, USA).

Plasmid conjugal transfer were performed with Escherichia coli EC600 (LacZ^-, Nal^R, and Rif^R) being used as recipient for selection of bla_CTX-M-positive transconjugants (Chen et al., 2015). Plasmid DNA was isolated from E. coli transconjugant using Qiagen large construct kit (Qiagen, Hilden, Germany), and sequenced by whole-genome shotgun strategy in combination with Illumina HiSeq 2500 (Illumina, San Diego, CA, USA) sequencing technology. The contigs were assembled with Velvet version 1.2, and the gaps were filled through combinatorial PCR and Sanger sequencing on ABI 3730 Sequencer. The genes were predicted with GeneMarkS^TM and RAST and further annotated by BLASTP and BLASTN against UniProt and NR databases. Annotation of mobile elements was based on the databases ISfinder, INTEGRALL, Tn Number Registry, and ISCR Elements. Gene organization diagrams were drawn with Inkscape version 0.48.

Enzymatic activity of ESBL was determined by the combined disk test as recommended by Clinical and Laboratory Standards Institute (CLSI; CLSI, 2015). Bacterial antimicrobial susceptibility was tested by ETEST (BioMérieux, Hazelwood, MO, USA) for erythromycin and azithromycin and by VITEK 2 (BioMérieux Vitek, Hazelwood, MO, USA) for all the other drugs (Table 1), and interpreted as per CLSI guidelines (CLSI, 2015).

Bacteria were cultured overnight in Mueller-Hinton broth with addition of indicated antibiotics. Total RNAs were extracted from harvested bacterial cells using TRIzol Reagent (Life Technologies). The oligonucleotide primers 5′-GCA CTCTCTTTGTCACCATCTC-3′, 5′-CTACATAACGCATTTGA TAACGC-3′, and 5′-CCATGTCGGGCTGCAAGTGCGTACAG TTGGG-3′ were designed to be complementary to the RNA transcripts of bla_CTX-M-14, erm(B)L and mph(A), respectively, and end-labeled with [γ-³²P] ATP. About 10 μg of end-labeled primer were annealed with 3 μg of RNA for the primer extension assay with a Primer Extension System (Promega) as described previously (Zhang Y. et al., 2011). The end-labeled primer was used for sequencing the PCR amplicons generated by the primer pairs 5′-TTAAGTATCATTGCAGCAAAG-3′/5′-GCA CTCTCTTTGTCACCATCTC-3′, 5′-AACGCTGAACCCAGCG GTAAATCGT-3′/5′-TACTCCTGAAGTGATTACATCT-3′, and 5′-ATGGCAAACTGAAACGGAT-3′/5′-CCTCTGGTTCGACC TTCG-3′ for bla_CTX-M-14, erm(B)L, and mph(A), respectively. DNA sequencing was carried out using the AccuPower & Top DNA Sequencing Kit (Bioneer). The primer extension products and sequencing materials were analyzed on an 8 M urea-6% polyacrylamide gel electrophoresis. Radioactive species were detected by autoradiography.

The complete sequence of p2246-CTXM was submitted to GenBank under accession number KX646543.

In October 2013, a 51-year-old male with watery diarrhea and liver dysfunction (further diagnosed as liver cirrhosis) was admitted to a public hospital in Beijing, China. Bacterial growth could be observed after culture of fresh stool specimens of the patient on the Salmonella-Shigella agar. The bacterial isolate, designated as 2246, was identified as S. boydii using slide agglutination. Based on the antimicrobial susceptibility test results, the patient received oral administration with piperacillin/tazobactam, and his symptoms of diarrhea progressively disappeared.

As determined by PCR screening for the major plasmid-borne quinolone-resistance, ESBL, and macrolide-resistance genes, strain 2246 harbored bla_CTX-M-14, erm(B), and mph(A) rather than any of the other genes tested. These three resistance markers could be co-transferred from strain 2246 into EC600 through conjugation, generating a transconjugant 2246-CTXM-EC600. Strains 2246 and 2246-CTXM-EC600 had the ESBL enzyme activity (Supplementary Figure S1). Both 2246 and 2246-CTXM-EC600 were resistant to piperacillin, cefazolin, cefuroxime, and ceftriaxone, but remained susceptible to piperacillin/tazobactam, imipenem and meropenem, gentamicin and amikacin, and trimethoprim/sulfamethoxazole; 2246 rather than 2246-CTXM-EC600 was resistant to ciprofloxacin and levofloxacin (Table 1). Notably, both 2246 and 2246-CTXM-EC600 possessed greatly elevated minimal inhibitory concentration (MIC) values (≥256 mg/L) against erythromycin and azithromycin (Table 1).

High-throughput sequencing analysis using plasmid DNA sample isolated from 2246-CTXM-EC600 generated the entire nucleotide sequence of a single plasmid p2246-CTXM (Figure 1) with a mean coverage of 135. The p2246-CTXM genome was manifested as an 111,559 bp circular DNA molecular with a mean G+C content of 51.9% and carried a total of 143 predicted open reading frames (ORFs).

The modular structure of p2246-CTXM could be divided into two major regions, namely, a 55-kb plasmid R100 (accession number AP000342)-derived backbone region with insertion of three different insertion sequences, and a 56.4-kb mosaic region composed of several plasmid backbone sequences and accessory mobile elements of different evolutionary origins. The R100-derived backbone sequences contained DNA regions for plasmid replication (repA2, repA6, and repA1 of the IncFII incompatibility group), maintenance (repA4, tir, pemIK, etc) and conjugal transfer (tra, trb, yfiABC, etc). One copy of each of IS2, IS903D and IS1 was inserted into the R100-derived backbone sequences, disrupting them into four separate portions. The transposition of IS2, IS903D and IS1 leaved three different direct repeats (DRs: target site duplication signals of transposition), i.e., GTTTA, GCATAAATC, and CGCGACGGG, which flanked the corresponding inserted elements, respectively. The insertion of IS2 and IS903D truncated the backbone genes traJ and yfhA, respectively.

Located at different sites of the backbone of R100 were three copies of repeat D, around 438 bp in length, two of which were found in p2246-CTXM. It seemed that the repeat D-mediated homologous recombination led to the deletion of one copy of repeat D together with a 14-kb region containing plasmid maintenance genes ssb, ydeAB, ydgA, yefA, psiBA, sok, mok, hok, and yehA.

The 56.4-kb mosaic region of p2246-CTXM (Figure 2) could be divided into two parts and the first part was composed of an intact IS1 element, and a novel transposon designated Tn6285 bordered by 40 bp imperfect terminal inverted repeats (IRs) at both ends.

Tn21, as observed in plasmid R100, was a Tn3-family unit transposon which was sequentially organized as the core transposition module tnpA (transposase)-tnpR (resolvase), tnpM, the Tn402-related class 1 integron In2 that was delimited by 25 bp IRs [inverted repeat initial (IRi) plus inverted repeat terminal (IRi)] associated 5 bp DRs, urf2, and the mer gene cluster (Liebert et al., 1999).Tn21 was bordered by 38 bp IRs [inverted repeat left (IRL) plus inverted repeat right (IRR)] at both ends. It was though that the insertion of In2 into Tn21 disrupted a presumed ancestral urf2M gene into two separate ones urf2 and tnpM.

A Tn21 remnant, which comprised two separate fractions IRL_Tn21-mer-urf2-DR_In2- IRi_In2-ΔtniA and IRt_In2-DR_In2-tnpM, was found in Tn6285 (Figure 2). Separation of these two fraction was likely resulted from the insertion of two consecutive resistance elements Tn6295 (name given in this study) and IS26-mph(A)-mrx-mphR(A)-IS6100, which were found at two different locations in pETN48 (accession number FQ482074), a CTX-M-14-encoding plasmid of the FII-FIB replicon type and originating from an E. coli O102-ST405 strain (Billard-Pomares et al., 2011).

Tn6295 (Figure 2) was a composite transposon, which had two flanking copies of IS26 oriented in the same direction and was further delimited by DRs of GGA at both ends; moreover, an ISCR14 element and a macrolide-resistance operon erm(B)L (leading peptide)-erm(B) (methylase) were found in Tn6295.The IS26-mph(A)-mrx- mphR(A)-IS6100 unit was known as a mobile element, which harbored another macrolide-resistance operon mph(A)-mrx-mphR(A) encoding a phosphotransferase, a positive regulator and a negative transcription factor, respectively (Partridge, 2011). Notably, the upstream IS26 copy of Tn6295 and the counterpart of the IS26-mph-IS6100 unit overlapped each other in Tn6285, indicating that the connection of Tn6295 with IS26-mph(A)-mrx-mphR(A)-IS6100 was mediated by homologous recombination in IS26.

Upstream of tnpM in Tn6285 was a 9.5 kb region, which was composed in order of ΔIn153, a Tn1722 remnant, a truncated version of the ISEcp1-bla_CTX-M-14-based transposition unit (IB14TU), and IS26 (Figure 2). This region was highly similar to that from nucleotide position 80171–70422 of pETN48. The tnpM gene was flanked by IRt_In2 and IRi_In153, and it appeared that the homologous recombination mediated by these two 25 bp IRs characteristic of class 1 integrons led to the inversion of tnpM in Tn6285 relative to Tn21. The ΔIn153 in Tn6285 contained two resistance gene cassettes aacA4:attC_aacA4 and cmlA1:attC_cmlA1 and this class 1 integron had undergone the truncation of sulI and the loss of IRt in the 3′-conserved segment. Tn1722 was a Tn3-family cryptic transposon initially identified in E. coli, with a modular structure IRL_Tn1722-tnpA-tnpR-mcp (methyl-accepting chemotaxis protein)-IRR_Tn1722 (Allmeier et al., 1992). The Tn1722 remnant in Tn6285 manifested as Δmcp-IRR_Tn1722.

pETN48 carried a prototype IB14TU, bound by 14 bp terminal IRs, which contained bla_CTX-M-14 together with an upstream ISEcp1 and a downstream IS903D followed by ΔiroN. Similar structures of IRL_ISEcp1-tnpA_ISEcp1-IRR_ISEcp1-bla_CTX-M-IS903D-ΔiroN- IRR_ISEcp1 were found in various plasmids of global origin (Zong et al., 2011). Compared with pETN48, Tn6285 harbored a truncated IB14TU element, which had undergone the loss of tnpA_574to973 (position 574 to 973 of the total 1263 bp coding region of tnpA in ISEcp1) and ΔiroN-IRR_ISEcp1, and the inversion of tnpA_974to1263-IRR_ISEcp1-bla_CTX-M-14-IS903D (Figure 2).

The second part of the 56.4-kb mosaic region contained four plasmid backbone-derived sequences, designated DBS-1to DBS-4 in the order that they occur (Figure 2). DBS-1 was a 926 bp region with two annotated genes encoding hypothetical proteins, which showed 93% sequence similarity to the IncFII-type plasmid pSE11-1 from E. coli (Oshima et al., 2008). Located between DBS-1 and DBS-2 was an IS1 element. The 18.7-kb DBS-2 contained the colicin Ia activity-immunity system cia–cii, and it was highly similar to a 15-gene region of pSE11-1. Two separate insertions, i.e., IS26 connected with a Tn5396 remnant, and a novel insertion sequence designated ISSbo2, occurred within DBS-2 relative to pSE11-1. Tn5396 was a Tn3-family cryptic transposon initially identified in the small plasmid pEC22 from E. coli, with a modular structure IRL_Tn5396-tnpA-tnpR-orf6 -orf7-IRR_Tn5396 (Elhai et al., 1994), and the 3′-end region ΔtnpR-orf6-orf7-IRR_Tn5396 constituted the Tn5396 remnant observed in p2246-CTXM. The 3.6-kb DBS-3 was highly similar to a 7-gene region of the E. coli genome assembly FHI74 (accession number LM996602), which contained the toxin-antitoxin system ccdBA, the colicin M immunity-activity system cmi–cma and the colicin B system cbi–cba. An IS1 element was inserted into DBS-3, leaving cmi and cba truncated. The 6.4-kb DBS-4 was found in the IncI1-type plasmid pC271 (accession number LN735561) from E. coli and contained several plasmid maintenance genes such as parA, stbAB, and impCAB.

Spacer regions between ISEcp1 and bla_CTX-M-55 from different ISEcp1-bla_CTX-M-55 isoforms displayed three different lengths, namely 45 bp (e.g., bla_CTX-M-55^p1081-CTXM) (Qu et al., 2014), 48 bp (e.g., bla_CTX-M-55^p2246-CTXM in this study), and 127 bp (e.g., bla_CTX-M-55^JQ343851 and bla_CTX-M-3^AF550415; Figure 3). A total of two promoters P1 and P2 were experimentally identified for bla_CTX-M-3^AF550415 with a 127 bp spacer and, moreover, the ISEcp1-provided promoter P1 was stronger and more important than the intrinsic P2 promoter in the 127 bp spacer (Ma et al., 2011).

Compared with the 127 bp spacer, the 45 or 48 bp spacer from bla_CTX-M-55^p1081-CTXM or bla_CTX-M-55^p2246-CTXM was a truncated form due to the loss of an 82 or 79 bp region within the spacer, respectively (Figure 3). This deletion impaired the -35 element of P2, most likely making the P2 activity undetectable for bla_CTX-M-55^p1081-CTXM and bla_CTX-M-55^p2246-CTXM. In the present study, the primer extension assay detected a single transcription start, i.e., nucleotide G located at 116 bp upstream of bla_CTX-M-55 (Figure 4), which corresponded to the P1 promoter shared by bla_CTX-M-55^p1081-CTXM (Qu et al., 2014) and bla_CTX-M-55^p2246-CTXM (Figure 3). In addition, the primer extension assay showed that the addition of increasing amounts of ampicillin during cultivation of strains 1081 and 1081-CTXM EC600 had no effect on the activity of bla_CTX-M-55^p2246-CTXM promoter (Figure 4), denoting the constitutive expression of bla_CTX-M-55^p2246-CTXM.

The primer extension assay detected a transcription start for each of erm(B)L and mph(A) from p2246-CTXM, i.e., nucleotide T located at 52 bp upstream of erm(B)L, and G at 31 bp upstream of mph(A); in addition, this assay also showed the inducible expression of erm(B)L and mph(A) upon addition of increasing amounts of amikacin and azithromycin, respectively (Figure 4). The above results were consistent with the expression patterns previously characterized for the two macrolide-resistant operons erm(B)L-erm(B) (Min et al., 2008) and mph(A)-mrx-mphR(A) (Noguchi et al., 2000).