Prevalence and characteristics of ST131 clone among unselected clinical Escherichia coli in a Chinese university hospital

Escherichia coli clinical sequence type 131 (ST131) has emerged as an extensively antimicrobial resistant E. coli clonal group in recent years throughout the world. The aim of this study was to investigate the prevalence and molecular characteristics of ST131 among unselected E. coli clinical isolates in a Chinese university hospital. Seven hundred consecutive E. coli isolates were collected at a Chinese university hospital between 2014 and 2015. Isolates belonging to ST131 were identified by PCR and multilocus sequence typing (MLST), and then characterized for antibiotic resistance, CTX-M-type extended-spectrum β-lactamase genes, fluoroquinolone resistance genes, O types, phylogenetic groups, virulence factors and PFGE patterns. Overall, 83 (11.6%) isolates were identified as ST131 group. The H30 lineage accounted for 53 (63.9%) of the ST131 isolates, including 13 H30-Rx and 40 H30 non-Rx. The remaining 30 isolates belonged to H41 lineage. Two O types were identified in this study: O25b (66.3%) and O16 (33.7%). Compared with O25b-B2-ST131 isolates, O16-B2-ST131 isolates harbored less virulence factors of adhesins. ST131 H30 Rx isolates had significantly higher virulence score than those of other isolates. O16-B2-ST131 isolates were shown to have a lower resistance to quinolones than O25b-B2-ST131 isolates. 5 nonsynonymous mutations (GyrA S83 L, D87N, ParC S80I, E84V and ParE I529L) were strongly associated with ST131 H30 and O25b isolates. Results of PFGE demonstrated that these isolates were classified into 68 pulsotypes and these subtypes were grouped into 23 different PFGE clusters using 70% similarity cut-off value. This is the first study to reveal the prevalence and molecular characteristic of ST131 clonal group among consecutive clinical E. coli isolates in China. Our findings demonstrated that ST131 lineage accounts for a small proportion of clinical E. coli isolates in China, which included two major groups: O25b-B2-ST131 and O16-B2-ST131. Our results implies that O16-B2-ST131 subclone may be an important type of E. coli ST 131 in China, which suggests that future studies should not ignore such clone in this country.


Background
Escherichia coli sequence type 131 (E. coli ST131) was identified among extended-spectrum β-lactamases (ESBL)producing isolates in Asia, Europe and North America in 2008, and has rapidly emerged globally to become an important pathogen causing various infections in humans [1,2]. E. coli ST131 is becoming the predominant extraintestinal pathogenic E. coli clone which drives multidrug resistance [1].
Regarding the spread of E. coli ST131, most investigations have focused on antimicrobial-resistant isolates, especially those resistant to fluoroquinolones and cephalosporins [1,2]. E. coli ST131 has been detected among extendedspectrum β-lactamase-producing or fluoroquinoloneresistant isolates in China [7][8][9]. However, the prevalence of ST131 among unselected E. coli isolates remains unknown. The aim of this study was to investigate the prevalence and molecular characteristics of ST131 in a collection of 700 unselected clinical E. coli isolates collected at a university hospital in Southern China.

Bacterial isolates and screening for ST131
A total of 700 non-duplicate E. coli clinical isolates were consecutively collected at a Chinese university hospital (Fujian Medical University Union Hospital, Fuzhou, Fujian province, China) between August 2014 and August 2015. These isolates were obtained from urine (51.0%), blood (14.7%) and miscellaneous sources (34.3%). Isolates were identified by the Vitek-2 system GNI card (BioMèrieux, Missouri, France). All isolates were screened for ST131 by PCR-based method for ST131-associated SNPs in mdh and gyrB as previous described [10]. All the non-O25b, non-O16 and non-phylogenetic group B2 ST131 strains would be confirmed by multilocus sequence typing (MLST) to be ST131 according to the Achtman scheme using seven housekeeping genes (adk, fumC, gyrB, icd, mdh, purA and recA) (http://mlst.ucc.ie/mlst/dbs/Ecoli). All ST131 isolates detected were further tested by PCR using specific primers for the fimH30 allele for identification of the H30 subclone [11]. The H30-Rx subclone was identified using allele-specific PCR as previously described [11]. All the H30-PCR negative isolates underwent direct sequenceing of fimH as previously described [3].

Molecular characterization of ST131
Phylogenetic groups were determined using PCR method described by Clermont et al. previously [13]. Molecular O types were performed on all ST131 isolates by PCR with primers as described previously [14]. The ST131-associated O25b rfb variant was detected by a separated PCR assay [15]. The presence of 26 virulence factors (VFs) genes was evaluated by a multiplex PCR method [16].
The virulence score (VF score) was calculated for each isolate as the sum of all virulence-associated genes detected in this study [17]. The sum of all the VF scores of the isolates was then calculated, and finally this sum was divided by the number of isolates to give the mean VF score. Pap and sfa-foc were counted only once regardless of the number of elements or subunits identified [17].
Pulsed-field gel electrophoresis analysis (PFGE) and dendrogram construction ST131 isolates were subjected to PFGE analysis using XbaI digestion [18]. A PFGE dendrogram was constructed with BioNumerics software (Applied Maths, Sint-Martens-Latem, Belgium) according to the unweighted pair group method based on Dice coefficients. Isolates with a Dice similarity index ≥70% were considered to belong to the same PFGE cluster.

Statistical analysis
Data were analyzed using the SPSS 19.0. Comparisons of proportions were performed using χ 2 or Fisher's exact test (two tailed) or Mann-Whitney test. For each comparison, p < 0.05 was considered to be statistically significant.

Bacterial clonal relatedness
PFGE analysis of the 83 E. coli ST131 isolates demonstrated that these isolates were classified into 68 pulsotypes (named 1-68, Fig. 1) and these subtypes were grouped into 23 different PFGE clusters (named A-W, in Fig. 1) using 70% similarity cut-off value. Two PFGE clusters (C and D) were predominant, grouping 13 and 14 isolates, respectively, whereas the other 21 PFGE clusters contained up to 9 ST131 isolates each (Fig. 1). Cluster C contained 92.3% (12/ 13) of H30-Rx isolates. The majority of O16 ST131 isolates belonged to two clusters (H and I), grouping 5 and 9 isolates. As shown in Fig. 1, at 63% similarity level, O25b-ST131 isolates were clustered into six major clonal groups, while 92.9% of the O16 isolates were clustered into two clonal groups (group one contained clusters H, I, J and K, and group two contained clusters O, P, Q, R and S).

Discussion
To our knowledge, this is the first molecular survey reporting the prevalence and characteristics of E. coli ST131 and its subclones among unselected clinical isolates in China. We found E. coli ST131 accounted for 11.6% of all E. coli isolates, which is similar to rates reported previously [1,22,23]. In addition, there was a similar distribution of ST131 among urine and blood samples. Meanwhile, the PFGE study indicated that ST131 isolates in this study were highly diverse at the pulsotype level and there was no predominated clone (Fig. 1).
O25b was previously considered the predominant ST131 clone type, while O16 isolates accounted for a small percentage [1,2,24]. Similar to previous studies [23,24],   [25], these findings suggest that O16-B2-ST131 may emerge as an important type of E. coli ST131 in China. The rate of O25b-B2-ST131 was not striking in this study. However, the O16-B2-ST131 may need special attention for the high percentages found in our results and previous study [25]. As discussed before, the clonal group should be investigated in the future study in other geographical regions in China [25,26]. The H30 lineage was a very important ST131 subclone, which firstly appeared in the year of 2000 and expanded rapidly to become the most dominant and extensively distributed multidrug-resistant lineage of E. coli worldwide [1,2]. Within the H30 subclone, the H30-Rx subset is a major drug-resistant pathogen among fluoroquinoloneresistant E. coli isolates and is associated with bla CTX-M-15 [1,2]. In this study, we also explored the prevalence of H30 lineage and H30-Rx sublineage among E. coli ST131 isolates. H30 lineage accounted for the majority of ST131 isolates in our study, which is similar to those in previous studies [22,23]. However, the prevalence of H30-Rx ST131 was obviously lower than those reported in other researches [23,27]. Meanwhile, the great majority of H30-Rx isolates harbored bla CTX-M-15 in this study. Resistances to 11 most commonly used antimicrobials were evaluated in this study. Our findings support the strong association of H30-Rx sublineage with multidrug resistance and the presence of bla CTX-M-15 (Table 1). We also identified the H30-Rx with the presence of aac(6′)-Ib-cr (Table 1). Resistance of fluoroquinolones is mainly due to chromosomal mutations in the QRDR regions, especially in gyrA and parC [28,29]. In this study, A set of 3 conserved mutations in QRDR regions (GyrA S83 L, D87N, and ParC S80I) could be found in all the CIP-resistant ST131 isolates. These chromosomal mutations have been previously linked to fluoroquinolone resistance [23,30]. Meanwhile, 5 mutations (GyrA S83 L, D87N, ParC S80I, E84V and ParE I529L) were strongly associated with H30 ST131 isolates, which is similar to the previous studies [23,31].
In conclusion, to our knowledge, this is the first report on the prevalence and molecular characteristic of ST131 clonal group among consecutive clinical E. coli isolates in China. Our findings demonstrated that ST131 lineage accounts for a small proportion of clinical E. coli isolates in China, which included two major groups: O25b-B2-ST131 and O16-B2-ST131. Our results implies that O16-B2-ST131 subclone may be an important type of E. coli ST 131 in China, which suggests that future studies should not ignore such clone in this country.