A Novel Structure Harboring blaCTX-M-27 on IncF Plasmids in Escherichia coli Isolated from Swine in China

The aim of this study was to elucidate the prevalence of blaCTX-M-27-producing Escherichia coli and transmission mechanisms of blaCTX-M-27 from swine farms in China. A total of 333 E. coli isolates were collected from two farms from 2013 to 2016. Thirty-two CTX-M-27-positive E. coli were obtained, and all were multidrug-resistant. Pulsed field gel electrophoresis (PFGE) and multilocus sequence typing (MLST) profiles indicated a wide range of strain types that carried blaCTX-M-27, and the sequence type ST10 predominated. Conjugation, replicon typing, S1-PFGE and hybridization experiments confirmed that 28 out of 32 CTX-M-27 positive isolates carried blaCTX-M-27 genes on plasmids F18:A-:B10 (16) and F24:A-:B1 (12).The blaCTX-M-27 genes for 24 isolates were transmitted by plasmids with sizes ranging from 40 to 155 kb. A comparative analysis with blaCTX-M-27-plasmids indicated that the tra-trb region of F24:A-:B1 plasmids was destroyed by insertion of a complex region (eight isolates) and a novel structure containing blaCTX-M-27 in the F18:A-:B10 plasmids (12 isolates). The novel structure increased the stability of the blaCTX-M-27 gene in E. coli. This study indicated that the predominant vehicle for blaCTX-M-27 transmission has diversified over time and that control strategies to limit blaCTX-M-27 transmission in farm animals are necessary.


Introduction
Production of extended-spectrum β-lactamases (ESBLs) is the principal mechanism of resistance to cephalosporins. CTX-Ms was the largest group of ESBLs, and they have become globally disseminated [1]. A recent study indicated that food-producing animals represent an important source of bla CTX-M -producing E coli isolates [2]. CTX-M-15 and CTX-M-14 are the most prevalent. Additionally, bla CTX-M-27 has been detected as increasing rapidly in prevalence [1]. The detection of CTX-M-27 in Escherichia coli patient isolates has been increasing, and is especially alarming because of its presence in clonal groups such as ST10, ST69 and ST131 [3][4][5][6]. In particular, E. coli ST131 isolates are the primary hosts responsible for CTX-M-27 transmission in E. coli from companion animals, the environment, farm animals and animal products [3,[7][8][9][10][11][12][13]. IncF plasmids and transposons are frequently associated with transfer of bla CTX-M-27 in E. coli and are frequently associated with IncF plasmids and transposons [14,15]. The pressure of antibiotics could facilitate the evolution of the transmission mechanism of bla CTX-M . The prevalence of bla CTX-M-27 in Salmonella isolated from food animals is also increasing, and transduction of bla CTX-M- 27 in Salmonella isolated from pork has been recently demonstrated to be mediated by a P1-like bacteriophage that had integrated a Tn1721-like structure [16]. A prospective study utilizing salmonella and E. coli isolates from 2003 to 2014 indicated that bla CTX-M-27 could be mobilized via a Tn1721-like structure between E. coli plasmids and P1-like bacteriophage in salmonella [17].
Intestinal carriage of CTX-M-producing bacteria (especially pathogenic bacteria) in food-producing animals and retail meat or dairy products contaminated by CTX-M β-lactamases might contribute to increased occurrence of infections with ESBL-producing bacteria in humans [18]. The prevalent vehicle for bla CTX-M transmission has diversified over time. This would lead to the further spread of the bla CTX-M-27 gene and further highlights the importance of controlling the spread of bla CTX-M-27. In the current work we investigated the prevalence and transmission mechanisms of CTX-M-27 E. coli from swine in Guangdong province, China from 2013 to 2016. We identified a novel structure containing bla CTX-M-27 that contributed to the stability of bla CTX-M-27 in E. coli.

Phenotypes of bla CTX-M-27 -Carrying Isolates
We surveyed two swine farms in Guangdong province and confirmed the presence of bla CTX-M-27 in 32 E. coli isolates including 62.5% (20) from farm 1 and 37.5% (12) from farm 2 ( Figure 1). CTX-M-27 has been only sporadically detected in E. coli isolates from food-producing animals, and its prevalence has remained constant at <8% over the past decade [17,19]. The prevalence of bla CTX-M-27 in our study (9.61%) was higher than in previous studies. Importantly, all 32 CTX-M-27 isolates were multidrug-resistant (MDR) strains (Table S1). The prevalence of ciprofloxacin-resistance in these isolates was 93.75% compared with 77.78% from isolates in 2003 to 2009 in China on swine farms [17], and the prevalence of ciprofloxacin resistance in bla CTX-M-14 or bla CTX-M-15 -producing E. coli almost above 80% [20,21]. Additionally, we detected QRDR (Quinolone Resistance Determining Regions) mutations in gyrA (S83L and D87N) and parC (S80I) in high-level quinolone resistant isolates (28), and 14 of these coharbored oqxAB ( Figure 1). This was consistent with a previous study where blood isolates of bla CTX-M-27 -producing ST131 E. coli possessed QRDR mutations that enabled high level resistance to quinolones [22]. Similarly, bla CTX-M-14 , bla CTX-M-55 , bla CTX-M-15 and bla CTX-M-9 -producing E. coli possessed QRDR mutations and carried PMQR genes that enabled high level resistance to quinolones [23]. In contrast, our isolates possessed a lower MIC CIP range of 0.25 to 1 µg/mL but did not contain QRDR mutations although either qnrS or oqxAB were present. This was consistent with a previous study reporting that bla CTX-M-55 -producing salmonella with an MIC CIP in the 0.5 to 4 µg/mL range did not contain QRDR mutations but all contained qnrS [24]. In addition, 9/32 (28.1%) of our isolates coharbored mcr-1, and these exhibited resistance to colistin at MIC = 8 µg/mL ( Figure 1).
The coexistence or cotransfer of other resistance genes in CTX-M-producing E. coli strains increases their probability of survival in the presence of other antibiotics, including the cephalosporins [24][25][26]. Infections caused by MDR bacteria are becoming common and represents a serious public health concern [27].

Molecular Typing Analysis
In our group of 32 CTX-M-27-producing E. coli isolates, we identified seven sequence types (ST). The ST131 was previously found to be associated with bla CTX-M-27 in Japan, North America and Europe [28][29][30]. However, ST131 was not present in any of our isolates. We found that ST10 was most prevalent (50%, 16/32) followed by ST224 (28.1%, 9/32), ST101 (9.4%, 3/32). ST10 and ST224 were detected on both farms. The isolates containing ST46, ST162, ST93 and ST58 were represented by one isolate each ( Figure 1 Previous studies showed that although the epidemiology and population dynamics of Global Extraintestinal Pathogenic E. coli (ExPEC) are complex, ST10 was the major ST the ExPEC (Extraintestinal Pathogenic Escherichia coli) group, and has remained at a constant level. ST10 has now been linked to multidrug resistance gene carriage or ESBL production [38]. Increased numbers of antibiotic resistance genes have been found and are carried by pathogenic bacteria and this is a human health concern. The 32 CTX-M-27 isolates we found could also be divided into 14 different PFGE clusters. These data indicated that the transmission of bla CTX-M-27 among E. coli isolates occurred via clonal expansion and horizontal transmission. The results of both clonal spread of resistant strains and horizontal transmission of the resistance plasmids contributed to the dissemination of bla CTX-M-27 -positive salmonella or E. coli isolates were also obtained in previous studies [16,17,39].

Molecular Typing Analysis
In our group of 32 CTX-M-27-producing E. coli isolates, we identified seven sequence types (ST). The ST131 was previously found to be associated with blaCTX-M-27 in Japan, North America and Europe [28][29][30]. However, ST131 was not present in any of our isolates. We found that ST10 was most prevalent (50%, 16/32) followed by ST224 (28.1%, 9/32), ST101 (9.4%, 3/32). ST10 and ST224 were detected on both farms. The isolates containing ST46, ST162, ST93 and ST58 were represented by one isolate each ( was the major ST the ExPEC (Extraintestinal Pathogenic Escherichia coli) group, and has remained at a constant level. ST10 has now been linked to multidrug resistance gene carriage or ESBL production [38]. Increased numbers of antibiotic resistance genes have been genes were all located on plasmids. Isolate names that are underlined indicate that bla CTX-M-27 plasmids could be transferred to strain C600 by conjugation. *: Only the ∆ISECP1-bla CTX-M-27 -IS903B region was sequenced. Pulsed field gel electrophoresis (PFGE) patterns with a cutoff at 85% similarity (the dashed line) are considered to be the same PFGE cluster and are indicated as groups I-XIV respectively.

Location and Genetic Context of the bla CTX-M-27 Gene
The genomic locations of the bla CTX-M-27 for our 32 isolates included F plasmids of 40-155 kb for 28 isolates and four were present on nontypeable incompatibility group plasmids. Most of these endogenous plasmids (24. 75%) could be successfully transferred to a laboratory strain E. coli C600 by conjugation. This was consistent with previous studies reporting that IncF plasmids are frequently associated with bla CTX-M genes [17]. Plasmid multilocus sequence typing (pMLST) of the IncF plasmids indicated only two plasmid types: F18:A-:B10 (16) and F24:A-:B1 (12) (Figure 1).
The plasmid p1-8 (F18:A-:B10) was completely sequenced (155,669 bp), possessed a G + C content of 51.56% and possessed a full complement of DNA transfer genes in the F18:A-:B10 backbone ( Figure 2B). These transfer regions were highly similar to other F plasmids and differed by the number of CAACAGCCG tandem repeats in the traD gene. Our isolates all possessed only one of these repeats and were highly similar to that of a bla CTX-M-55 -harboring F18:A-:B1 plasmid pCREC-544_1 (NZ_CP024827.1) from a human clinical isolate from Korea, and a bla NDM-5 -harboring F18:A-:B58 plasmid pMTY18780-2 (NZ_AP023199.1) human isolate from Japan. IncF plasmids have been implicated as main players for transmission of bla CTX-M-27 [40]. So, we estimate that F18:A-:Bx plasmids are the primary vectors for animal to human transmission of most antibiotic resistance genes, and this has significant health implications for both humans and animals.
The second plasmid pM8-1 (F24:A-:B1) we identified possessed DNA transfer genes that had been altered by insertion of a 17,034 bp region that included TnAs3, IS5075, IS26, intI1, mph and IS6100, as well as two IS26 copies in the same orientation. This insertion destroyed traI and traN, and the entire trbH/F/G/B/A/E, traD/T/S/G/H/Q/F region was lost (Figure 2). A similar disruption was previously documented for pHNAH9 that lacked most parts of the tra-trb region due to multiple recombination events, and this most likely accounted for its conjugation failure. Our F24:A-:B1 plasmids were highly similar to pHNAH9 [41]. These results indicate that E. coli ST224 possessing F24:A-:B1 was disseminated prior to its acquisition of bla CTX-M-27 , similar to the bla CTX-M -carrying F1:A2:B20 plasmid in E. coli ST131-H30R1 [42].
The region (Figure 3a) containing bla CTX-M-27 (17354 bp) was present in both our plasmid types, F18:A-:B10 and F24:A-:B1. This region was identical to the variable region from pMRY15_117 (accession no: NZ_AP017618.1, Figure 3a). The insertion of ISEcp1bla CTX-M-27 -IS903B in the tnpA transposition gene of Tn2 might be expected to prevent transposition. However, IR TEM had been lost, although the IR tnp and the resolvase (res) sites were intact. Therefore, it is possible that the TnpA function was provided by another Tn3like transposon and that this mediated movement of this interrupted Tn2 region [43]. Thus, Tn2-mediated events contributed to the spread of bla CTX-M-27 in the F24:A-:B1 plasmids.
We also identified another genetic context contained within a 22,178 bp insertion (Figure 3b). This was a novel structure associated with bla CTX-M- 27 (Figure 3c), the insertion of IS15 resulted in ∆tnpR/res/tnpA∆-iroN∆ inversion. The iroN and Tn2 sequences were lost, and R1, R2, R3 and aadA5-qacE∆1-sul1 were all inverted in p1-8, while intI1 and dfrA17 from In54 were lost (Figure 3b). Taken together, these results indicate that Tn2 was incomplete and incapable of transfer of bla CTX-M-27 in these plasmids. Therefore, F18:A-:B10 was one of the primary contributors to the spread of bla CTX-M-27 in E. coli for our swine isolates and resulted in greater stability of bla CTX-M-27 in clinical isolates. This indicates that the bla CTX-M-27 gene was stable in vehicles and its natural host. The plasmid can mediate its transmission in a wider range hosts, which poses a serious threat to public health. The ISEcp1-like family has been shown to act as a promoter region for high-level expression of different CTX-M enzymes in a previous study [44]. The conserved region responsible for transmission of bla CTX-M-27 was ISECP1-bla CTX-M-27 -IS903B. Since more complex structures that mediate transmission of bla CTX-M-27 have been recently described, and a diversity of media promotes the transmission of bla CTX-M-27, it may cause a serious threat to public health.
The F18:A-:B10 plasmids may make it easier for E. coli ST10 to acquire bla CTX-M-27, and similar results have been reported for the MDR IncF group F1:A2:B20 plasmid in E. coli ST131-H30R1 [42]. Our recent work revealed that bla CTX-M-27 -plasmids isolated from 2003 to 2009 contained a genetic context with the Tn1721-like structure ∆ISEcp1B-bla CTX-M-27 -IS903D-iroN-∆map-Tn1721 [17]. In contrast, the present study indicated that the horizontal transmission of bla CTX-M-27 was related to Tn2, so that the prevalent vehicle for bla CTX-M-27 transmission has diversified over time.
MLST was performed for bla CTX-M-27 -producing isolates as previously reported [46]. The PCR and DNA sequence analysis of seven housekeeping genes (adk, fumC, gyrB, icd, mdh, purA and recA) was performed, the allelic profiles were determined, and the sequence type (ST) was assigned in accordance with http://mlst.warwick.ac.uk/mlst/ dbs/, (accessed on 15 February 2021). Pulsed field gel electrophoresis (PFGE) patterns with the XbaI enzyme were analyzed in experiments following the method of Jiang et al. for genetic relatedness of all bla CTX-M-27 -harboring isolates [18].
E. coli C600 strain (streptomycin-resistant) was used as donor strain and clinical bla CTX-M-27 -producing strains were used as recipient strains to test the transferability of the bla CTX-M-27 gene. Transconjugants were selected on MacConkey agar supplemented with 1 mg/L cefotaxime and 2000 mg/L streptomycin [18]. PCR-based replicon typing (PBRT) was performed on all transconjugants with bla CTX-M-27 -carrying plasmids using primers as previously described [47]. To determine the location of bla CTX-M-27 , all the transconjugants were subjected to pulsed-field gel electrophoresis (PFGE) with S1 nuclease (Takara) and Southern transfer and probing with a digoxigenin-labelled probes specific for the bla CTX-M-9G gene as described previously [16].
Genomic DNA samples from M8-1 and transconjugant C-1-8 were subjected to 250-bp paired-end whole-genome sequencing using the Illumina HiSeq system (Illumina, San Diego, CA, USA), and the paired-end Illumina reads were assembled by SOAPdenovo version 2.04 [48]. To obtain the complete sequence of p1-8, PCR and Sanger sequencing was applied to close all the suspected gaps. Sequence comparisons of M8-1 and pMRY15_117 (NZ_AP017618.1) were performed using BLAST (http://blast.ncbi.nlm.nih. gov (accessed on 15 February 2021)) and Mauve [49], and similar contigs were extracted from the assemblies.

Conclusions
In this study we identified ST10 E. coli as one of the dominant vehicles for bla CTX-M-27 transmission in China. We report a novel structure containing bla CTX-M-27 that contributed to the persistence of this gene in E. coli. This study identified multidrug-resistant IncF group F18:A-:B10 plasmids that contributed to the spread of bla CTX-M-27 in E. coli. E. coli ST224 possessing F24:A-:B1 were likely transmitted before acquiring bla CTX-M-27 , and Tn2related events may have contributed to the spread of bla CTX-M-27 in F24:A-:B1 plasmids. Additionally, the prevalent vehicle for bla CTX-M-27 transmission has diversified over time so that that transmission of the bla CTX-M-27 gene in farm animals should be closely monitored.