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The emergence of blaCTX-M-15-carrying Escherichia coli of ST131 and new sequence types in Western China
Annals of Clinical Microbiology and Antimicrobialsvolume 12, Article number: 35 (2013)
blaCTX-M-15, the most widely distributed gene encoding extended-spectrum β-lactamases globally, was not common in China. This study was performed to characterize blaCTX-M-15-carrying Escherichia coli in western China.
Out of 144 Escherichia coli isolates from 20 hospitals in western China, 8 were found carrying blaCTX-M-15. blaCTX-M-15 was carried by isolates of ST131and 5 new STs (ST3342, ST3513, ST3516, ST3517 and ST3518). The 5 new STs shared 5 identical alleles out of 7 but only had up to 2 alleles identical to ST131. blaCTX-M-15 was located on plasmids of IncI1 (ST16) or IncFII-related group (four replicon types). The co-transfer of a few antimicrobial resistance genes including qnrA, qnrB, qnrS, qepA, aac (6′)-Ib-cr, aac (3)-II, tetA, blaTEM and blaOXA-1 with blaCTX-M-15 were examined but only blaTEM-1 was found co-transferring with blaCTX-M-15.
Five new STs of E. coli and three new types of IncFII-related plasmids carrying blaCTX-M-15 were identified. This study together with several reports suggested that blaCTX-M-15 has emerged in China and the interruption of both vertical and horizontal transmission of blaCTX-M-15 is required to hurdle its further spread.
Escherichia coli producing extended-spectrum β-lactamases (ESBLs) is a challenge for clinical treatment and infection control. blaCTX-M-15 has emerged as the most widely distributed gene encoding ESBLs globally, but it was not common in China . We performed a snapshot survey on the molecular epidemiology of E. coli carrying blaCTX-M-15 in Sichuan province, western China.
All non-duplicated E. coli clinical isolates (n = 144) that were recovered in 20 hospitals in Sichuan, western China, from June 22 to 25, 2011 were collected regardless of their antimicrobial susceptibility profiles and types of infections. The presence of blaCTX-M genes were detected using both a single PCR with universal primers CTX-U1/U2 and a multiplex PCR [2, 3] and 25.7% (37/144) isolates were found carrying blaCTX-M. A total of 17 isolates had a blaCTX-M gene belonging to the blaCTX-M-1 group and the complete sequence of the blaCTX-M-1-like gene in all 17 isolates was obtained using PCR with primers ISEcp1IR-F and orf477-R . Sequencing revealed blaCTX-M-15 in 8 isolates, corresponding to 21.6% of blaCTX-M variants, and the remaining 9 isolates had either blaCTX-M-55 (n = 8) or blaCTX-M-57 (n = 1). The 8 isolates carrying blaCTX-M-15 were from urine (n = 5), sputum (n = 2) and ascites (Table 1). Although blaCTX-M-15 has become the dominant gene encoding ESBLs in many countries and E. coli of ST131 carrying blaCTX-M-15 was widely distributed , blaCTX-M-15 had only been occasionally found in isolates recovered before 2007 in China . However, several studies on E. coli isolates obtained in 2007 and afterwards in China have found that blaCTX-M-15 accounted to 14% to 24% of blaCTX-M variants detected from human [6, 7]. The present study and previous reports therefore suggested the emergence of E. coli carrying blaCTX-M-15 in China in recent 5 years.
Six of the isolates carrying blaCTX-M-15 belonged to the phylogenetic group B2 and the remaining two were of group A1 and D, respectively, determined as described previously  (Table 1). Using multi-locus sequence typing (MLST) , the 8 isolates carrying blaCTX-M-15 were assigned to 6 sequence types (ST) including ST131 and 5 new STs (Table 1). Of note, E. coli of ST131 was found carrying blaCTX-M-3, -14 and -65 but not -15 in our local settings previously . This study demonstrated the emergence of the globally-spread ST131 carrying blaCTX-M-15 in our region. The 5 new STs (ST3342, ST3513, ST3516, ST3517 and ST3518) shared 5 identical alleles (gyrB, icd, mdh, purA and recA) out of 7 and these STs might therefore belong to a common clonal complex. In contrast, the 5 STs had only up to 2 alleles identical to ST131, suggesting the 5 STs and ST131 had different clonal origins.
Self-transmissible plasmids carrying blaCTX-M-15 were obtained from 7 out of the 8 isolates using mating as described previously . The remaining isolate V12 did not yield transconjugants carrying blaCTX-M-15 despite repeated attempts but transformants carrying blaCTX-M-15 were obtained from V12 by electroporation with plasmids prepared using alkaline lysis . This suggests that blaCTX-M-15 was carried by a non-conjugative plasmid in isolate V12. Plasmids carrying blaCTX-M-15 were prepared using alkaline lysis and were subjected to PCR-based replicon typing . Four plasmids were of IncI1 and the other four belonged to the IncFII-related group, two of which contained replicons of IncFIA and IncFIB in addition to the IncFII-related replicon. IncF plasmid replicons sequence typing (RST)  and IncI1 plasmid MLST (pMLST)  were employed to investigate the relatedness of these plasmids carrying blaCTX-M-15 (Table 1). All of the IncI1 plasmids except the non-conjugative one from isolate V12 were of ST16 (1-5-10-8-6). According to the IncI1 pMLST database (http://pubmlst.org/plasmid/), ST16 IncI1 plasmids have been found carrying blaCTX-M-15 in isolates from cattle in the UK but were not found in human isolates carrying blaCTX-M-15 before. The identification of ST16 IncI1 plasmids in both cattle and human isolates suggested the transfer of blaCTX-M-15 between animal and human, which implicates that the control of the transmission of blaCTX-M-15 should address sources beyond human. Two alleles, ardA and sogS, both of which are associated with conjugation, were unable to be amplified from the non-conjugative plasmid (pV12) carrying blaCTX-M-15 from isolate V12. Therefore, the ST could not be assigned for pV12 but the remaining three alleles of pV12 were identical to those of ST16, suggesting that pV12 might be derived from a ST16 IncI1 plasmid. Four different RST profiles were present for the four IncF plasmids carrying blaCTX-M-15, suggesting that the spread of blaCTX-M-15 in our local settings might have been mediated by multiple IncF plasmids. Among the four RST types of IncF plasmids identified here, F2:A-:B- plasmids carrying blaCTX-M-15 appeared to be widely distributed and have been found in Canada, France, Italy and the UK (http://pubmlst.org/plasmid/), while the remaining three types, F1:A2:B1, F1:A2:B20 and F31:A-:B- have not been deposited in the RST database, representing new IncF RST types.
The co-transfer of a few antimicrobial resistance genes including qnrA, qnrB, qnrS, qepA, aac(6′)-Ib-cr, aac(3)-II, tetA, blaTEM and blaOXA-1 with blaCTX-M-15 were examined for transconjugants and transformants by PCR. Unlike previous studies, the co-transfer of resistance genes with blaCTX-M-15 except for blaTEM-1 was not common in this study (Table 1).
In summary, although blaCTX-M-14 was the most common blaCTX-M variant in China, the present study together with recent reports [6, 7] suggested that blaCTX-M-15 has emerged in China. The prevalence of blaCTX-M-15-carrying isolates would compromise the efficacy of the widely-used broad-spectrum cephalosporins and therefore represents a serious challenge for clinical treatment and public health. The spread of blaCTX-M-15 in our local settings is mediated by two clonal complexes and by self-transmissible plasmids of IncI1 or IncF. The spread of isolates carrying blaCTX-M-15 in China warrants more studies. The interruption of both vertical and horizontal transmission of blaCTX-M-15 using infection control measures such as standard precautions and contact precautions appear to be the key to hurdle the further spread of this antimicrobial resistance determinant.
Availability of supporting data
The data set supporting the results of this article is included within the article.
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The authors are grateful for Yanyu Gao for the technical assistance. This work was supported by a grant from the National Natural Science Foundation of China (project no. 30900052) and was also partially supported by a grant from the US-China Collaborative on Emerging and Re-emerging Infectious Diseases.
The authors declare that they have no competing interests.
ZL carried out the study, participated in the sequence alignment and helped to draft the manuscript. LX participated in the design of the study and helped to draft the manuscript. ZZ conceived of the study, participated in the sequence alignment and coordination and drafted the manuscript. All authors read and approved the final manuscript.