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Molecular Characteristics of Escherichia coli Causing Bloodstream Infections During 2010–2015 in a Tertiary Hospital, Shanghai, China

Background The bloodstream infections (BSI) caused by Escherichia coli pose a serious threat to human health. To explore molecular characteristics of E. coli causing BSI, we collected E. coli isolates causing BSI in Huashan Hospital, Shanghai, China during 2010–2015. Methods In all E. coli isolates causing BSI collected from this study, polymerase chain reaction (PCR) was used to detect ESBLs and carbapenemase genes, and minimum inhibitory concentrations (MICs) were determined with agar dilution method. Outer membrane proteins were examined by SDS-PAGE in carbapenem-resistant strains. The genetic background of blaKPC gene was investigated by combining next-generation sequencing with a PCR mapping approach. Conjugation and transformation experiments were performed to verify the mobilization of blaKPC. The transcription levels of the blaKPC gene were measured by RT-PCR. Results During 2010–2015, a total of 207 E. coli BSI strains were isolated. The positive rates of β-lactamase resistant genes were 0.48% (blaKPC), 57% (blaTEM), 23.67% (blaCTX-M-1), 18.84% (blaCTX-M-9), and 1.93% (blaSHV). High rates of blaTEM, blaCTX-M-1, and blaCTX-M-9 were consistent with the poor activity of third-generation cephalosporins and aztreonam in vitro, except for carbapenem and β-lactamase inhibitor combinations. Low susceptibility rates were observed for piperacillin (25.1%) in contrast to the increased susceptibility when combined with β-lactamase inhibitors, namely piperacillin-tazobactam (90.8%). Only one KPC-producing E. coli strain was detected. Despite the combination of OmpC loss, the low expression level of KPC may be responsible for its lower resistance to carbapenems compared to E. coli DH5α (pKP12-100). Conclusion E. coli strains isolated from BSI were still highly susceptible to carbapenems and β-lactamase inhibitor combinations, and blaCTX-M was the dominant genotype of ESBLs. The low expression of blaKPC may be the reason for the low resistance to carbapenems.


Introduction
Escherichia coli, a gram-negative, motile, facultative anaerobic, rod-shaped bacterium, is one of the most common hospital-acquired pathogens which could cause urinary tract infections, abdominal infections, bloodstream infections (BSI), etc. 1 Bacteremia represents a major cause of death with large increases in incidence and mortality. 2 E. coli is a leading cause of bloodstream infection, it ranks first as a cause of community-acquired episodes and second as a cause of hospital-acquired BSI in different world regions. 3 In addition, the incidence of E. coli BSI is increasing with associated high morbidity and mortality. 4 In a study from England, all-cause mortality rate in individuals with E. coli bacteremia was 18.2%. 5 And in China, one study showed that in 45 episodes of E. coli bacteremia, the 30-day allcause mortality was 22.2%. 6 β-lactams are commonly used in the treatment of BSI caused by E. coli. β-lactamase production remains the most important contributing factor to β-lactam resistance. 7 Extended-spectrum β lactamases (ESBLs), one group of β lactamases, have the ability to hydrolyze and cause resistance to various types of the β-lactam antibiotics, including the third-generation cephalosporins and monobactams except the cephamycins and carbapenems. 8,9 The most common ESBLs belong to three groups: TEM, SHV, and CTX-M types. 8 The CTX-M β lactamases, now exceeding 50 different types, can be divided into five groups based on their amino acid identities: CTX-M-1, CTX-M-2, CTX-M-8, CTX-M-9, and CTX-M-25. 10 Since their first description, class A extended-spectrum β-lactamases (ESBLs) producing E. coli continue to thwart our best clinical efforts. ESBLsproducing E. coli remains an important reason for therapy failure with cephalosporins and have serious consequences for infection control. 7 Based on data from a multicenter randomized controlled trial, carbapenem is recommended as first-line treatment for infections outside of the urinary tract caused by ESBLs-producing E. coli. 11 In E. coli, carbapenem resistance is typically caused by two main mechanisms: production of carbapenemases and βlactamase activity combined with structural mutations. 12 When combined with the mutation of outer membrane proteins or drug efflux pumps, ESBLs and AmpC are capable of conferring carbapenem resistance. 12 According to the Ambler classification method, carbapenemases are members of the molecular class A, B, and D beta-lactamases. Class A and D enzymes have a serine-based hydrolytic mechanism, while class B enzymes are metallo-beta-lactamases that contain zinc in the active site. 13,14 Of these, the KPC carbapenemases are the most prevalent, found mostly on plasmids in Klebsiella pneumoniae. 15 Given the increasing importance and the fact that BSI caused by ESBLs-producing E. coli are an increasing therapeutic challenge, we investigated the molecular characteristics and antimicrobial susceptibility profiles of BSI caused by E. coli during 2010-2015 in Huashan Hospital, Shanghai, China.

Sources of Strains
A total of 207 non-duplicate E. coli isolates were collected from blood cultures of the inpatients of Huashan Hospital, Fudan University from 2010 to 2015. E. coli was identified using the Vitek 2 system. E. coli A49, selected from 207 strains mentioned previously, was used as the positive reference for outer membrane proteins with complete OmpC, OmpF, and OmpA. Plasmid pKP12-100 (Supplementary Data 1) was extracted from KPC-producing K. pneumoniae KP100-12 isolated from Huashan Hospital, not belonging to the 207 strains mentioned previously. This plasmid was used for transformation.

MIC Determination
In 207 E. coli strains in this study (Supplementary Data 2), minimum inhibitory concentrations (MICs) of cefotaxime, cefepime, ceftazidime, cefoxitin, ampicillin, aztreonam, piperacillin, piperacillin-tazobactam, meropenem, imipenem, ertapenem, fosfomycin, ciprofloxacin, amikacin, and gentamicin were determined with agar dilution method according to Clinical and Laboratory Standards Institute (CLSI) guidelines. Escherichia coli ATCC25922 and Pseudomonas aeruginosa ATCC27853 were used as routine controls for agents mentioned previously. The criterion of the susceptibility of fosfomycin was based on EUCAST (European Committee on Antimicrobial Susceptibility Testing) (Resistance standard: MIC ≥ 32μg/mL). We investigated the effect of efflux pump inhibitors cyanide 3-chlorophenylhydrazone (CCCP) on the carbapenems' susceptibility in the carbapenem-resistant E. coli. The concentration of CCCP was 25μg/mL.

Detection of Resistance Genes
Polymerase chain reaction (PCR) was used to detect βlactmase such as bla TEM , bla CTX-M-1 , bla CTX-M-9 , bla CTX-M-2 , bla CTX-M-8 , bla CTX-M-25 , bla SHV and the carbapenemases genes such as bla KPC , bla NDM , bla IMP , bla SPM, bla AIM , bla VIM , bla OXA , bla GIM , bla BIC , bla SIM , bla DIM . 16 And in our laboratory, we possessed isolates which were used as positive controls for the following genes: bla KPC , bla NDM , bla IMP , bla OXA , bla CTX-M-1 , bla CTX-M-9 , bla SHV and bla TEM. Amplification was carried out as follows: initial denaturation at 94°C for 5 min; 30 cycles of 94°C for 30s, 60°C for 30s and 72°C for 1 min; and a final elongation step at 72°C for 7 min. Primers were listed in Supplementary Data 3.

Analysis of Outer Membrane Proteins (OMPs) of KPC-Producing Strain
Briefly, the suspension was sonicated on ice for about 10 minutes (60 cycles for 5 seconds with 5-second intervals). The cell extracts were centrifuged at 15,600 g 4°C for 60 minutes, then we removed the supernatant and added 200 μL 1×PBS, 25 μL10% Sarcosyl to resolve the protein. We repeated the procedure and suspended the OMP with 80 μL 1×PBS. Porins were loaded onto 15% SDSpolyacrylamide gel. After a 150-min electrophoresis of 80 V, the membrane was stained with 0.1% Coomassie brilliant blue (Beyotime, China). 17

Conjugation and Transformation Experiments
Conjugation and transformation experiments were performed to verify the transferability of bla KPC . Plasmid pKP12-100 was extracted from a bla KPC -positive isolate through phenol-chloroform method and then transformed into the recipient strain E. coli DH5α. E. coli J53, an azide-resistant strain was used for conjugation experiments. 18 Agar plate containing ampicillin (50μg/mL) was used to screen for transformants. Conjugation strain was selected on LB agar plates supplemented with 50μg/mL ampicillin and 150μg/mL sodium azide. PCR with primers Kpc-RT (listed in Table 1) and sequencing were used to verify transformants and conjugation strain.

Genetic Environment of bla KPC Positive Strain
DNA was extracted from EC-A59 (TIANamp Bacteria DNA Kit) and next-generation sequencing was performed (Supplementary Data 4). Flanking sequences of bla KPC were extracted from the contig harboring bla KPC and analyzed by Blastn. The genetic background of bla KPC gene in E. coli A59 obtained from this study was investigated by combining nextgeneration sequencing with a PCR mapping approach with the primers listed in Table 1

OMP Profile of bla KPC -Positive Strain
SDS-PAGE analysis revealed different OMP profiles among the two isolates ( Figure 3). OmpC loss was observed in isolate E. coli A59 compared to E. coli A49.

Conjugation and Transformation Experiments of the bla KPC Positive Strain
The bla KPC-2 -carrying plasmid named pKP12-100 was extracted from K. pneumoniae 12-100 and transformed into E. coli DH5α. The MIC of the transformant E. coli DH5α (pKP12-100) can be seen in Table 3. Conjugation and transformation of E. coli A59 were failed with at least three repeats.
And an 8-fold decrease in the MIC of ertapenem was observed in DH5α-P12-100 (Table 3).

Genetic Environment of bla KPC -Positive Strain
Combining next-generation sequencing with a PCR mapping approach, genetic environment of bla KPC in E. coli A59 was ISKpn6-bla KPC-2 -ISKpn27-IS26 sharing the same core structure as that from the chromosome of ECO3385 (CP029420.1). Since bla KPC-2 was not able to be transferred to the recipient we supposed that the bla KPC in E. coli A59 may be located on the chromosome (Figure 4).

RT-qPCR of bla KPC Gene
Compared with the imipenem resistant control strain, E. coli DH5α (pKP12-100), the transcription levels of

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bla KPC gene were five-fold lower for isolate E. coli A59 ( Figure 5). At least 280-bp sequence upstream of the bla KPCs in the two strain was identical, see Supplementary Data 5, indicating that they shared the same promoter region of bla KPC.  19 ESBLs are often encoded by plasmids that are transferable from strain to strain and between bacterial species. 20,21 In our study, bla CTX-M was the dominant genotype among the ESBLs-producing E. coli which is consistent with the situation in China. 22 The occurrence of ESBLs is increasing. 8 Data from rural Thailand showed very high rates, reaching 69.3% in 2010. The great majority of CTX-M alleles identified in Thailand belonged to group 9. 23 Carbapenem-resistant E. coli is posing great challenges to human health. 12,24 The plasmid-mediated horizontal transmission of carbapenemase genes is the main cause of the surge in the prevalence of CRE. NDM, one of the metallo-β-lactamases, is the predominant carbapenemase in E. coli while KPC carbapenemases are the most prevalent ones among class A carbapenemase group and found mostly on plasmids in K. pneumoniae. 15,25 K. pneumoniae are the predominant carriers of bla KPC , mainly associated with the clonal group 258 (CG258) including ST258, ST11, ST340, ST512, and others. 26,27 One study indicated that type I-E CRISPR-Cas system targeting the backbone regions of bla KPC -bearing IncF plasmids influences the acquisition of bla KPC plasmid in K. pneumoniae. The absence of type I-E CRISPR-Cas in CG258 contributes to the dissemination of IncF epidemic resistance plasmids in this clonal complex. 28 Until now, reports about KPC-producing E. coli have been rare and  From the results of the national surveillance of CRE strains in China, it was shown that the core structure of ISKpn6-bla KPC-2 -ISKpn27 was conservative in KPCproducing K. pneumoniae and E. coli strains. 29 In this study only the chromosomes of ECO3385 (CP029420.1) and E. coli A59 shared the same core structure. In addition to the conservative sequences, they still hold another transposable element IS26, and this kind of structure was a little bit different from the previously reported pK048 (IncFIIK5) harboring non-Tn4401 elements in China. 30 Whether certain divergences between K. pneumoniae and E. coli resulted in the intergeneric diversity of transposable genetic elements should be taken into consideration.

Discussion
Previous research showed that KPC enzymes contribute to the carbapenem resistance in K. pneumoniae. 26 Compared with the KP12-100, the MIC of carbapenem for its transformation strain E. coli DH5α (pK12-100) had decreased at least 16-fold, which indicated the existence of other resistance mechanisms. E. coli A59 was a bla KPC positive strain with OmpC loss which was not resistant to meropenem and imipenem. Considering the low expression level and the failed conjugation and transformation experiments, we propose that the decreased MIC of meropenem and imipenem may be due to the low expression level of bla KPC located on the chromosome of E. coli A59. Only one KPC-producing E. coli was detected in this study. More strains will be included to clarify the overall detection rate of bla KPC in E. coli in the future.

Ethical Statement
The strains we used in this study were obtained from the biological sample and strains bank of the Institute of Antibiotics, Huashan Hospital, Shanghai, China. They came from normal clinical testing and were stored in the strains bank. The ethics committee of Huashan Hospital authorized our study and written informed consent was not required. This study would not do harm to rights, benefits, and health of the subjects, and the privacy and personal identity information of the subjects will not be included in this study.