The emergence of blaCTX-M-15-carrying Escherichia coli of ST131 and new sequence types in Western China

Background 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. Findings 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. Conclusions 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.

Keywords: Escherichia coli, Plasmids, MLST, Beta-lactamases, Antimicrobial resistance Findings Escherichia coli producing extended-spectrum β-lactamases (ESBLs) is a challenge for clinical treatment and infection control. bla CTX-M-15 has emerged as the most widely distributed gene encoding ESBLs globally, but it was not common in China [1]. We performed a snapshot survey on the molecular epidemiology of E. coli carrying bla CTX-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 bla CTX-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 bla CTX-M . A total of 17 isolates had a bla CTX-M gene belonging to the bla CTX-M-1 group and the complete sequence of the bla CTX-M-1 -like gene in all 17 isolates was obtained using PCR with primers ISEcp1IR-F and orf477-R [4]. Sequencing revealed bla CTX-M-15 in 8 isolates, corresponding to 21.6% of bla CTX-M variants, and the remaining 9 isolates had either bla CTX-M-55 (n = 8) or bla CTX-M-57 (n = 1). The 8 isolates carrying bla CTX-M-15 were from urine (n = 5), sputum (n = 2) and ascites (Table 1). Although bla CTX-M-15 has become the dominant gene encoding ESBLs in many countries and E. coli of ST131 carrying bla CTX-M-15 was widely distributed [5], bla CTX-M-15 had only been occasionally found in isolates recovered before 2007 in China [1]. However, several studies on E. coli isolates obtained in 2007 and afterwards in China have found that bla CTX-M-15 accounted to 14% to 24% of bla CTX-M variants detected from human [6,7]. The present study and previous reports therefore suggested the emergence of E. coli carrying bla CTX-M-15 in China in recent 5 years.
Six of the isolates carrying bla CTX-M-15 belonged to the phylogenetic group B2 and the remaining two were of group A1 and D, respectively, determined as described previously [8] (Table 1). Using multi-locus sequence typing (MLST) [9], the 8 isolates carrying bla CTX-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 bla CTX-M-3 , −14 and −65 but not −15 in our local settings previously [10]. This study demonstrated the emergence of the globally-spread ST131 carrying bla CTX-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 bla CTX-M-15 were obtained from 7 out of the 8 isolates using mating as described previously [4]. The remaining isolate V12 did not yield transconjugants carrying bla CTX-M-15 despite repeated attempts but transformants carrying bla CTX-M-15 were obtained from V12 by electroporation with plasmids prepared using alkaline lysis [11]. This suggests that bla CTX-M-15 was carried by a non-conjugative plasmid in isolate V12. Plasmids carrying bla CTX-M-15 were prepared using alkaline lysis and were subjected to PCR-based replicon typing [12]. 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) [13] and IncI1 plasmid MLST (pMLST) [14] were employed to investigate the relatedness of these plasmids carrying bla CTX-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 bla CTX-M-15 in isolates from cattle in the UK but were not found in human isolates carrying bla CTX-M-15 before. The identification of ST16 IncI1 plasmids in both cattle and human isolates suggested the transfer of bla CTX-M-15 between animal and human, which implicates that the control of the transmission of bla CTX-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 bla CTX-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 bla CTX-M-15 , suggesting that the spread of bla CTX-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 bla CTX-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.
In summary, although bla CTX-M-14 was the most common bla CTX-M variant in China, the present study together with recent reports [6,7] suggested that bla CTX-M-15 has emerged in China. The prevalence of bla CTX-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 bla CTX-M-15 in our local settings is mediated by two clonal complexes and by self-transmissible The allele number of the IncI1 pMLST profile (repI, ardA, trbA, sogS and pill) is indicated. 2 ND, not determined. 3 Resistance genes that were co-transferred with bla CTX-M-15 are in bold.
plasmids of IncI1 or IncF. The spread of isolates carrying bla CTX-M-15 in China warrants more studies. The interruption of both vertical and horizontal transmission of bla CTX-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.

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