Genetic contexts related to the diffusion of plasmid-mediated CTX-M-55 extended-spectrum beta-lactamase isolated from Enterobacteriaceae in China

CTX-M-55 extended-spectrum beta-lactamases are being rapidly disseminated and transmitted in clinical practices around the world. The genetic contexts of the transferable plasmid-mediated blaCTX-M-55 gene in Enterobacteriaceae were detected and characterized in this study. Isolates were obtained from the First Affiliated Hospital of Zhengzhou University between September 2015 and March 2016. Based on polymerase chain reaction and BLAST analysis, resistance genes and genetic context of the blaCTX-M-55 gene were investigated. Conjugation experiments and multilocus sequence typing were performed to demonstrate plasmid-mediated blaCTX-M-55 transmission. Thirteen blaCTX-M-55-positive isolates of Enterobacteriaceae were obtained. Seven isolates were Escherichia coli, 3 were Klebsiella pneumoniae, 1 was Citrobacter freundii, 1 was Morganella morganii and 1 was Serratia marcescens. The blaCTX-M-55 gene has not previously been identified from C. freundii and M. morganii. Four different blaCTX-M-55 genetic contexts were identified, and all of them harbored ISEcp1 in the region upstream of blaCTX-M-55 (in two cases, ISEcp1 was truncated by IS26, and in one case, it was truncated by IS1294), whereas ORF477 was detected downstream of the blaCTX-M-55 gene from 12 of 13 strains. The novel genetic context of ISEcp1∆-blaCTX-M-55-∆IS903 was firstly detected the IS903 element which was identified downstream of blaCTX-M-55. A conjugation assay revealed that all blaCTX-M-55 plasmids were quickly and easily transferable to recipient E. coli, which then presented resistance to multiple antibiotics. Numerous blaCTX-M-55-positive strains were isolated in a short period of 7 months. The findings indicate that blaCTX-M-55 was rapidly disseminated. The genetic context and conjugative transfer found in this study demonstrate that there is active transmission of blaCTX-M-55 among strains of Enterobacteriaceae in China, which could give rise to an urgent global public health threat.


Background
Since the first reports of CTX-M extended-spectrum beta-lactamases (ESBLs) in 1989 [1], at least 26 bacterial species across the world have been referenced in the "CTX-M pandemic" [2]. More than 190 diverse variants of CTX-M have been recorded to date. Among these variants, CTX-M-55 pertains to the CTX-M-1 cluster, which is a variant of CTX-M-15 with only one amino acid substitution (Ala-80-Val) [3]. This variant was first reported in 2006 [4] and was identified in Thailand as well as in the UK [3][4][5]. Over the past decade, the isolate rate of CTX-M-55 in Escherichia coli from animals has been increasingly raised. However, CTX-M-55 was not identified

Annals of Clinical Microbiology and Antimicrobials
in clinical practices in China until 2010, when it was detected from a person who traveled to China [6]. Since then, plenty of surveys have confirmed the emergence of bla CTX-M-55 among clinical pathogenic in China [7][8][9][10][11].
Conjugative plasmids are one of the most important mechanisms for the appearance and spread of bla CTX-M . These plasmids facilitate horizontal transfer to other isolates and even cross-species barriers [12]. Insertion sequences (ISs), which cause insertion mutations and genome rearrangements, are the smallest mobile elements (< 2.5 Kb) independent transposition in an organism and competent to promote translocation, and the transferability of a resistance gene will largely increased under the mediated of ISs [13]. Various types of genetic platforms are associated with bla CTX-M genes, and ISEcp1 is frequently recorded upstream of bla CTX-M . ISEcp1 can transpose the bla CTX-M gene and act as a strong activator for the high expression of it [12,14,15]. In addition, other insertion sequences, including IS26, IS903 and ORF477, are also frequently detected surrounding bla CTX-M [16,17].
Thus, this study intends to inquire into the prevalent trend of bla CTX-M-55 genes and their transferability and genetic contexts among clinical strains in Henan Province in central China.

Identification of resistance genes and the genetic contexts of bla CTX-M-55
To verify the emergence of plasmid-mediated ESBL genes, all ESBL-positive strains were further characterized, and plasmid DNA was extracted utilizing a Tiangen Plasmid Purification Mini Kit (Tiangen Biotech, China) referring to the protocol of manufacturer. The primer sequences presented in Table 1 were used for the bla TEM , bla SHV , and bla CTX-M-1-groups to determine the genetic context of bla CTX-M-55 . Purified PCR productions were sequenced immediately from two ends and compared with genes in GenBank (http://www.ncbi.nlm.nih.gov/ genebank/).

Multilocus sequence typing (MLST)
MLST for clinical E. coli and K. pneumoniae strains were detected basis on the assay discussed above [19,20]. The

Conjugation experiments
Conjugative assays were performed using the methods discussed above [7]. The bla CTX-M-55 -positive isolates served as donors, and E. coli C600 functioned as a recipient. Transconjugants were screened on Mueller-Hinton agar containing 750 μg/ml rifampin and 100 μg/ml ampicillin. The existence of bla CTX-M-55 in the transconjugants was identified through antimicrobial susceptibility, PCR and DNA sequencing.

MLST and conjugal transfer of the bla CTX-M-55 gene
MLST was detected for bla CTX-M-55 -positive E. coli and K. pneumoniae strains. Nine types of MLST were detected among the 7 E. coli strains (ST156, ST305, ST182, ST381, ST446 and ST2) and 3 K. pneumoniae strains (ST148, ST269 and ST37). Two E. coli isolates (EC32 and EC45) shared the same ST type (ST305) ( Table 2). Conjugative assays indicated that all bla CTX-M-55 plasmids were transmitted to E. coli C600 from 13 donors successfully through conjugation. Although all transconjugants exhibited resistance to cefotaxime and ceftazidime, they were all sensitive to fluoroquinolones. Additionally, the bla TEM and bla SHV resistance genes were transformed to E. coli C600 with the bla CTX-M-55 for some isolates (Table 3).

Discussion
Since the CTX-M-55 firstly reported in 2006, it has been identified in E. coli, K. pneumoniae, S. flexneri and Salmonella enteritidis [3,7,10]. For all we know, bla CTX-M-55 in C. freundii and M. morganii is firstly detected in this study. In addition, 13/227 isolates were identified as bla CTX-M-55 -positive in just 7 months. This rate far surpasses other ESBLs [21][22][23], which demonstrates the rapid dissemination of bla  . Notably, all bla CTX-M-55 -positive isolates were identified as multiple drugresistant (MDR) bacteria that are strongly resistant to ceftazidime and cefotaxime (MIC > 256 μg/ml). More significantly, molecular characterization also revealed that most of the bla CTX-M-55 -positive isolates harbored bla TEM . In addition, some isolates contained bla SHV . These results imply that the spreading of bla CTX-M-55 over many different genera of Enterobacteriaceae is activated in hospitals     in Henan Province, which represents a public health issue due to the inability to treat these bacteria. Two E. coli isolates (EC32 and EC45) isolated from two different departments (Gastroenterology and ICU) shared the same ST type (ST305), which suggests that they are clonally related. However, the data indicate that the CTX-M-55-positive E. coli and K. pneumoniae strains identified in our study were not clonally related by MLST, which indicates that there is no specific ST in Henan Province. This finding contrasts with observations in the region of European and North American, where a high prevalence of ST131 has been observed [24]. Furthermore, this study demonstrates the association of eight STs [ST305, ST182, ST381, ST446 and ST2 (E. coli) and ST148, ST269 and ST37 (K. pneumoniae)] with the products of CTX-M-55 first time, which means bla CTX-M-55 has been actively spreading among Enterobacteriaceae in China. Given our focus on conjugative assays, the 13 transconjugants all exhibited resistance to cefotaxime and ceftazidime but sensitivity to fluoroquinolones, which was consistent with the original isolates. These results suggest that the plasmid-mediated bla CTX-M-55gene is to answer for an ESBL phenotype with poor susceptibility to cefotaxime and ceftazidime and exhibits a strong transferability of resistance. This finding also indicates that fluoroquinolones should be used for the therapy of bla CTX-M-55 -positive pathogen infections in clinical settings. Interestingly, our data indicate that some original isolates were resistant to cefepime, but the transconjugants were susceptible, which suggests that the original isolates may include other resistance genes that promote resistance to cefepime. We did not detect these genes in our study. These resistance genes cannot be transmitted through conjugative assays and are not located on the chromosome. Thus, this mechanism requires further study.
The sporadic existence of CTX-M-55-positive strains in mainland China has been occasionally detected. In some surveys, CTX-M-55 incidence has surpassed that of CTX-M-15 [25]. Heterogeneous genetic contexts may indicate the dissemination and mobilization of bla CTX-M-55 . As shown in Fig. 1, all isolates were detected ISEcp1, locating upstream of bla CTX-M-55 ; this region contain the promoter sequence (− 35 and − 10) and act as a significant role in the expression and mobilization of the β-lactamase genes [12,15,26]. Moreover, the presence of ISEcp1 in this cross-species study indicates that the complete or partial insertion sequence was probably excised along with CTX-M-55 during horizontal transfer. Previous reports demonstrated that the disruption of the ISEcp1 element by IS26 was linked to the promotion of bla CTX gene dissemination [27,28]. Interestingly, as previously reported, ISEcp1 disruption by IS1294 in bla CTX-M-55 was detected from a chicken in China, which may contribute to the mobilization of bla CTX-M-55 [29]. Remarkably, the two E. coli strains [EC30 (this study) and E. coli C21 [29] ] shared the same MLST type (ST156), which suggests that these isolates are clonally related. This coincidence implies that bla CTX-M-55 is likely to be transferred from animals to the clinical setting. Fey et al. found that a 12-year-old boy acquired ceftriaxone-resistant Salmonella enterica serotype Typhimurium from cattle [30]. Jing Zhang et al. reported that CTX-M-55 had already been transmitted to humankind from animals and is distributed among both hospitals and community in China. The findings of our investigation and previous studies indicate that bla CTX-M-55 can be transmitted to humankind from food and can enhance clinical resistance. Notably, the novel arrangement ISEcp1∆-bla CTX-M-55 -∆IS903 is characterized by the element of IS903 which is detected downstream of bla CTX-M-55 first time and often identified by the context of other bla CTX-M genes [31]. The mechanism responsible for its presence remains unclear. According to Poirel et al., ISEcp1, bla CTX and IS903 form a putative transposon, and this block of genes could be disseminated by transposition [26,32]. This finding implies that IS903 contributes to the dissemination of bla CTX-M-55 , which requires further study. Therefore, our findings strongly suggest that genetic elements (ISEcp1, ORF477, IS26, IS1294, and IS903) are involved in the inter-species and intra-species mobilization and dissemination of bla CTX-M-55 . Additionally, CTX-M-55-harboring isolates in animals may act as a potential storage of bacterial that is spread in clinical.

Conclusions
This investigation reminds a high occurrence rate of CTX-M-55-producing ESBLs in patients from different departments at the First Affiliated Hospital of Zhengzhou University in Henan Province. These plasmidmediated bla CTX-M-55 -positive isolates are contributed to the transmission of bla CTX-M-55 to new species and new hosts by conjugation. Data obtained in this study suggest that the genetic context of bla CTX-M-55 , especially ISEcp1, act as a vital part in the mobilization, dissemination and expression of drug resistance determinants. We also demonstrated a novel arrangement of bla CTX-M-55 (ISEcp1∆-bla CTX-M-55 -∆IS903). Thus, the presence of MDR Enterobacteriaceae contains conjugative plasmids that co-harbor other IS elements, such as ISEcp1, should be surveilled worldwide because the active transfer and high prevalence of these pathogenic will significantly decrease our further selection of clinical therapies. Further studies on this issue should be performed to help us obtain a deeper understanding of the transmission and