Genome Assessment of Carbapenem- and Colistin-Resistant Escherichia coli from Patients in a Sentinel Hospital in China

Antimicrobial-resistant (AMR) pathogens are a significant threat to public health worldwide. However, the primary carrier of AMR genes, particularly against last-resort antibiotics, is still only partially studied in Chinese hospitals. In a sentinel hospital in China, we collected 157 E. coli strains from patients between January and July 2021. One blaNDM-1-, nine blaNDM-5-, and one mcr-1-positive E. coli recovered from inpatients were identified as resistant to meropenem and colistin. There are 37 virulence genes discovered in the 11 strains, including astA in strain EC21Z-147 (O128: H4), which belongs to the enteroaggregative E. coli (EAEC). The blaNDM gene is distributed into distinct ST types, including ST48, ST616, ST410, ST711, and ST2003, while the mcr-1 gene was identified in ST117. The conjugative plasmids IncX3, IncI1-I, and IncI2 mediated the blaNDM-5 and mcr-1 genes detected among inpatients. Notably, the youngest age at which mcr-1-positive E. coli has been reported was at one day old, in a child in which the strain is closely related to strains with animal origins. Hospitals are major environments for the spread and dissemination of critical virulence and AMR genes, which requires active monitoring systems at the genome level to surveil the spread of virulence and AMR.


Introduction
Antimicrobial resistance (AMR) has become a global public health concern with the spread of multidrug resistance (MDR) bacteria worldwide. Carbapenems are the most critical antimicrobials for treating clinical Gram-negative bacterial infections. The carbapenemresistant Enterobacteriaceae (CRE), defined as a superbug that carried the bla NDM-1 gene, was identified in 2009 [1]. bla NDM is a metallo-β-lactamase (MBL) type gene that could hydrolyze most β-lactam antimicrobials [2]. Hence, they are considered last-resort antimicrobials against serious infections caused by CRE [3]. Moreover, colistin is considered among the last resort for treating MDR Enterobacteriaceae; however, its extensive use in veterinary practice has led to the development of resistance in a wide range of pathogens recovered from animals, food, the environment, and human samples [4][5][6][7]. On the other hand, tigecycline is a last-resort antibiotic used to treat severe infections caused by extensively drug-resistant pathogenic bacteria [8]. Hence, resistance to these last-resort antimicrobial drugs threatens the healthcare systems.
A total of 157 E. coli strains from 139 patients were retrospectively collected from outpatients (n = 69) and inpatients (n = 88) between January and July 2021 at a sentinel hospital in Zhejiang province, China. The isolated strains were confirmed with MALDI-TOF Mass Spectrometry. These strains were recovered from blood (n = 65), urine (n = 48), stool (n = 14), bile (n = 23), and lung (n = 7) samples. The patients' ages were between 1 day and 100 years old.

Whole-Genome Sequencing and Annotation
The genomic DNA of the 11 colistin-or meropenem-resistant E. coli strains was extracted using a DNA Extraction Kit (Genray, Shanghai, China). Whole-genome sequencing was performed by the Oxford Nanopore GridION platform for long sequencing reads and Illumina HiSeq-PE150 for High-Throughput Sequencing reads. The genome sequences were hybrid assembled using Unicycler v0.4.4. The sequences were automatically annotated by the RAST [21]. The ARGs were identified using the ResFinder 4.1 (>90% identity and >80% coverage) (https://cge.food.dtu.dk/services/ResFinder/) (accessed on 15 June 2022). The virulence genes were identified using the VirulenceFinder 2.0 (>90% identity and >80% coverage) (https://cge.food.dtu.dk/services/VirulenceFinder/) (accessed on 15 June 2022), and the incompatibility (Inc) group of plasmids were identified using the PlasmidFinder 2.1 (>95% identity and >80% coverage) (https://cge.food.dtu.dk/services/PlasmidFinder/) (accessed on 15 June 2022), as reported in previous studies [22][23][24][25][26]. Insertion sequences were identified by ISFinder [27]. 2.4. S1-Pulsed-Field Gel Electrophoresis (PFGE) and Southern Blot As described in a previous study, the 11 E. coli strains were subjected to S1-PFGE to identify the number and size of plasmids [28,29]. The overnight cultures were washed with PBS buffer and then embedded in agarose plugs. The plugs were digested with proteinase K at 37 • C, shaking at 120 rpm for 2 h, and were restricted with S1 nuclease. Salmonella H9812 was restricted with XbaI, which was used as the size marker. A Southern blot was used to confirm the positioning of the mcr-1 or bla NDM genes on the plasmids. The DNA fragments were transferred to a positively charged nylon membrane (Millipore, USA) by wet transfer and then hybridized according to the protocol as the DIG-High Prime DNA Labeling and Detection Starter Kit I (Roche, Germany).

Conjugation Test
Colistin-or meropenem-resistant E. coli containing the mcr-1, bla NDM-1 , or bla NDM-5 genes was used as the donor strain, and E. coli J53 (sodium azide-resistant) was used as the recipient. Fresh cultures of the donor and recipient strains were adjusted in concentration and co-incubated on the LB agar plate at 37 • C overnight. The mixed cultures were collected and diluted with PBS. Then, the mixture was inoculated onto MHA plates containing 2 µg/mL of colistin with NaN 3 (100 µg/mL) or 2 µg/mL meropenem with NaN 3 (100 µg/mL) at 37 • C overnight. The number of transconjugants and recipients was recorded, which was used to calculate the transfer frequency as described previously [18].

Discussion
Hospital and community settings are important reservoirs for the spread of pathogens and ARGs, such as ESBL, bla NDM , bla KPC , mcr, etc. Here, we investigated the prevalence of last-resort antimicrobial-resistant E. coli strains from inpatients in a hospital. Ten (6.37%) carbapenem-resistant-, one (0.64%) colistin-resistant-, and zero (0%) tigecycline resistant-strains were identified. All of them were found in inpatients instead of outpatients, indicating that the ARGs were spread in the hospital environment. Additionally, we observed that inpatients carry significantly more E. coli against last-resort antibiotics than outpatients.
The prevalence of bla NDM (6.37%, 10/157) was higher than the global prevalence in 55 countries (0.28%, 290/103,960) [15]. It is also higher than the prevalence of CREC (2.38%, 92/3895) in healthy people in healthcare centers located in 19 provinces across China [16]. The bla NDM gene was also detected from the specimens of various environments, vegetables (0.28%), companion animals (2.3%), and livestock and poultry (0.88%), the amounts of which were significantly lower than that in patients [33][34][35]. However, it has a similar prevalence in India (6.22%) [15]. Unlike bla NDM , the prevalence of the mcr and tet(X) genes in patients was significantly lower than that in animals, especially for the tet(X) gene. As expected, except the tet(X) gene identified in this study, these genes were more widespread in freshwater fishes (24.7%), chickens (23.6%), cattle (19.3%), and pigs (8.95%) than in patients (0.3%) [36,37]. Only one mcr-1-positive E. coli strain (0.64%) was identified in patients, similar to the positive rates in patients (0.62%, 36/5828) from several provinces of China [38]. This rate is significantly lower than those in livestock and poultry (14.81%, 270/1823), and environmental strains (5.43%, 5/92) [35,38]. It is important to note that this mcr-1-positive E. coli was isolated from the lungs in a one-day-old child. This child is the youngest reported case of mcr-1-positive E. coli infection.
The virulence gene co-located with ARGs in Enterobacteriaceae has received extensive attention in recent years. The virulence determinants were identified in various bacteria harboring mcr, bla NDM , or other genes from foods, animals, humans, and environments and caused a serious threat to public security [39][40][41]. The EAEC harboring heat-stable enterotoxin gene astA has been discovered in E. coli serotype O7: H4, O untypeable: H10, O4: H34, etc. The novel serotype O128: H4 E. coli harboring astA was identified in the mcr-1-positive E. coli EC21Z-14 belonging to ST117. The E.coli ST117 was an emerging foodborne zoonotic pathogen that has been isolated from the poultry, meat, human, and sea ecosystem [42][43][44][45]. A recent study showed that the ST117 strains isolated from different hosts had higher genetic similarity, suggesting that ST117 could transfer among different hosts [46]. In this study, the EC21Z-14 isolated from the one-day-old child was associated with the pathogenic E. coli from avian animals, reminding us that MDR pathogens from animals could spread in human and cause disease.
The bla NDM-1 and bla NDM-5 genes were first identified in Klebsiella pneumoniae in India in 2009 and in E. coli in the United Kingdom in 2011, respectively [1,47]. A higher prevalence of the bla NDM-1 gene (69%) than the bla NDM-5 gene (19%) was primarily reported in clinical settings [16]. However, the opposite was observed for the bla NDM-5 gene, which was dominant in CREC in China after 2016 [16]. It was observed in this study that 90% of CRECs bore the bla NDM-5 gene instead of the bla NDM-1 gene. The different hydrolysis activities may cause this to carbapenems [48]. The bla NDM-5 gene gradually replaced bla NDM-1 in China, and this phenomenon may have spread to other countries.
bla NDM -positive E. coli strains belonging to various STs have been reported. The most common CRECs worldwide were ST167, ST410, and ST617, and ST131 was the major CREC in China [15,48,49]. No obvious epidemic clones of NDM-positive E. coli were found, and only three ST410 strains were identified from one patient in this study. The strains isolated from different patients belonged to different STs. Furthermore, the mobile plasmid IncX3 is the primary replicon type for carrying various bla NDM (NDM-1, NDM-4, NDM-5, NDM-6, NDM-7, etc.) and was detected in several strains in this study [15]. IncX3 is dominant in bla NDM -positive plasmids and promote horizontal bla NDM gene transfer. It is pointed out that the IncX3 carrying bla NDM-1 unsuccessfully transferred to the recipient by conjugation, and no obvious mechanic was found for this abnormality. This may be a reason for the lower prevalence of the bla NDM-1 gene. IncHI2A and IncI1-1 were discovered as relatively uncommon replicon types for carrying bla NDM-5 [50]. Overall, the bla NDM-5 gene is the primarily genetic determinant for CREC, mainly spread by the IncX3 plasmids.
Multiple samples collected from two lymphoma patients at several points in time were further analyzed. The EC21Z-101 strains lost a plasmid (IncFIB-FIC) carrying several ARGs and virulence genes compared with the EC21Z-078, EC21Z-083, and EC21Z-097 strains from one patient that became sensitive to the antimicrobials. The mechanism behind the missing highly stable multidrug-resistant IncFIB plasmid is worth further studying [51]. On the other hand, the conjugation ability showed significant differences between two IncI1-1 plasmids (pEC21Z144-121K-NDM5 and pEC21Z151-128K-NDM5) carrying bla NDM-5 from one patient. Different shufflon regions were discovered between shufflon-specific DNA recombinase and PilV. It is widely known that shufflon is a multiple-inversion system that is closely related to the efficiency of the conjugation [52]. Shufflon comprises four segments (A, B, C, and D) which may be rearranged and inverted to influence the conjugation during liquid mating [53]. The rearrangement of shufflon usually reduces the transfer frequency instead of the loss [54]. The novel rearrangement in pEC21Z151-128K-NDM5 may provide a strategy to decrease plasmid dissemination, which is worth further investigating.

Conclusions
In summary, our data support that the hospital setting is a vital vehicle for spreading and disseminating last-resort AMR pathogens. Moreover, the acquisition of bla NDM and mcr-1 genes encoding resistance to carbapenem and colistin was mediated by horizontal transfer via plasmids. IncX3 carrying bla NDM-5 is the leading carrier of resistance in CREC. The ST117 E. coli is an emerging zoonotic pathogen carrying the mcr-1 gene transferred between humans and animals. Therefore, the horizontal transfer of ARGs among inpatients indicated the importance of preventing plasmids in clinical pathogens. The continuous surveillance of carbapenem and colistin resistance at the genome level in sentinel hospitals is highly prioritized to limit the transmission of last-resort pathogens.