Escherichia Coli Isolate From Hospital Sewage Is Carrying blaNDM-1 and blaoxa-10

Zimeng Hu Nanjing Agricultural University College of Veterinary Medicine Weiye Chen Nanjing Agricultural University College of Veterinary Medicine Genglin Guo Nanjing Agricultural University College of Veterinary Medicine Chen Dong Jiangsu Province CDC: Jiangsu Province Center for Disease Control and Prevention Yun Shen Jiangsu Province CDC: Jiangsu Province Center for Disease Control and Prevention Si Qin Jiangsu Province CDC: Jiangsu Province Center for Disease Control and Prevention Long Chen The a liated Zhangjiagang Hospital of Soochow University Wei Zhang (  vszw@niau.edu.cn ) Nanjing Agricultural University College of Veterinary Medicine https://orcid.org/0000-0002-3575-6225


Introduction
Among β-lactam antibiotics, carbapenem antibiotics are considered to be the "last line of defense" against gram-negative bacterial infections and are used to treat bacteria produced by ESBLs (Extendspectrum β-lactamases) bacterial infection [1]. Recently, carbapenem-resistant bacteria can infect humans and animals and spread widely around the world, posing a serious public health threat to the world [2]. In the Ambler nomenclature, carbapenemase is divided into A, B, C and D enzymes, B is a metalloenzyme, and NDM is a B enzyme [3]. Since the rst report of NDM in an Indian patient in Sweden in 2008, the bla NDM−1 gene has been shown to be easily spread between different types of gram-negative bacteria and can spread to many countries in a short time, becoming one of the most worrying drug resistance genes in the world [4].
NDM-1 and NDM-5 are the two most common NDM variants in Enterobacteriaceae in China [4]. Bla NDM−1 gene and its variants have been widely popular among of the Enterobacteriaceae, the most common members are Escherichia coli [5], Klebsiella pneumoniae[6], Enterobacter cloacae etc. [5][6][7]. Bla OXA−10 is a CHDLs (Carbapenem-hydrolyzing class D carbapenemases) previously detected in human pathogens, usually produced by Pseudomonas aeruginosa. It is a narrow-spectrum enzyme and has the greatest hydrolytic capacity to penicillin [8,9]. Initially, these enzymes were highly resistant to penicillin, but now they are more resistant to carbapenem antibiotics, which should pay more attention on it [9].
Horizontal gene transfer plays an important role in introducing new genes into micropopulation [10]. Due to the continuous discharge of feces and urine, hospital sewage is an ideal place to spread and develop antibiotic resistance genes [11]. Despite the obvious importance of hospital sewage in the development and spread of drug resistance, the presence of ESBLs and carbapenemase-resistant bacteria in hospital sewage has not been well studied. The plasmid mediated spread of antibiotic-resistant bacteria in hospital sewage is a problem with great impact on public health, ecology and economy.
Therefore, the purpose of this research is to isolate carbapenem-resistant bacteria from hospital sewage and conduct a modi ed Hoge test to verify whether they produce carbapenemase, conduct a conjugate transfer test to study whether the drug-resistant plasmids are mobile, and perform genome-wide sequencing analysis (including analysis of their serotypes, ST types, and prediction of drug-resistant genes, etc.) to assess their risk of transmission to public health through the aquatic environment.

Bacteria and culture methods
Escherichia coli ATCC25922 is a quality control strain for drug susceptibility test; EC600 as recipient bacteria for plasmid conjugation transfer test and was donated by the Second A liated Hospital of Soochow University. Bacteria were routinely cultured in Luria-Bertani (LB) broth at 37℃.

Sample collection and bacterial identi cation
In September 2019, the sewage was collected from the sewage outlet of a hospital in eastern China, and then the drug-resistant bacteria were isolated on a meropenem resistant plate (200 µg/ml). Resistant colonies were collected and set for further researches.

Antimicrobial Susceptibility Testing
Broth dilution method was used for antimicrobial susceptibility testing of isolated bacteria, and results were interpreted according to the criteria recommended by Clinical and Laboratory Standards Institute (CLSI, 2019). A total of 10 kinds of antibiotics were selected for antibiotics sensitivity test, including chloramphenicol, fosfomycin, tetracycline, ampicillin, gentamicin, o oxacin, cefotaxime, rifampicin polymyxin B and meropenem (HANGWEI, Hangzhou, China). Each experiment was repeated three times.

Whole Genome Sequencing
Genomic DNA was extracted with the SDS method [12]. A total amount of 1µg DNA per sample was used as input material for the DNA sample preparations. The whole genome of isolated bacteria was sequenced using Illumina NovaSeq PE150 at the Beijing Novogene Bioinformatics Technology Co., Ltd.
The raw data obtained by sequencing (Raw Data) had a certain proportion of low-quality data. In order to ensure the accuracy and reliability of the subsequent information analysis results, the original data must be ltered to obtain valid data (Clean Data). The genome was assembled from Clean Data of each sample after quality control.

Phylogenetic analysis
MEGAX was used to construct adjacent phylogenetic trees of the whole genome sequence of isolates and other similar E.coli.

Carbapenemases detection
The modi ed Hodge test (MHT) was performed on a MHA (Mueller-Hinton Agar) plate with meropenem as substrate for the detection of carbapenemases (CLSI, 2019). Speci c steps are as follows: prepare ATCC25922 suspension with 0.5 MCF in MH and dilute it with MH at 1:10. Inoculate into the MHA plate according to the drug sensitivity test, let the plate dry for 3 ~ 10 minutes, and then stick the meropenem disk in the middle of the plate. 3 ~ 5 isolates that had been cultured overnight on blood AGAR were selected by inoculation ring, and a straight line was drawn from the edge of the plate to the center during inoculation, the line must be at least 20 mm ~ 25 mm long.

Transferability of Plasmids with Carbapenems Resistance
In order to evaluate the transferability of plasmids with carbapenem resistance, EC600 (Rifampicin resistance) was chosen as the recipient bacteria. Two isolated donor strains and EC600 were cultivated to 0.4 OD, mix 50 µl of donor bacteria with 150 µl of recipient bacteria (EC600) and cultivated in an incubator at 37℃ about 18 h respectively. The cultured mixed bacteria were cultivated on the doubleresistant MH plates of meropenem and rifampicin respectively. The determination method was the mixed bacteria grew colonies on the double-resistant plate.

Bacterial isolation and identi cation
The draft sequencing results of the tested bacteria were uploaded to NCBI for bacterial homology comparison. The similarity rate with E. coli reached 97.166% and the isolate was named as AH001.

Antimicrobial Susceptibility Testing
In addition to meropenem, AH001 exhibited resistance to 7 kinds of antibiotic include chloramphenicol, fosfomycin, tetracycline, ampicillin, gentamicin, o oxacin, and cefotaxime, expect, rifampicin and polymyxin B ( Table 1). The results showed that the multi-drug resistance of E. coli was serious. After uploading the 16s rDNA sequence of AH001 to NCBI for comparison, bacteria with higher similarity were selected for evolution analysis (Fig. 1). The results show that the similarities between AH001 and different strains including Acinetobacter baumannii are very high.

Transferability of Plasmids
The growth of the isolate of AH001 was that there was no colony growth on the dual-resistant meropenem and rifampicin plates, which proved that the resistant plasmid was not conjugated to the recipient strain EC600.

Discussion
In this study, we isolated an unknown strain of E. coli that is resistant to meropenem from medical sewage, and then studied its resistant phenotype and genotype. It was found that it contained both bla NDM and bla OXA−10 genes.
The emergence and rapid spread of antibiotic resistance is a growing threat to human health [13]. CRE (Carbapenem-resistant Enterobacteriaceae) has the ability to promote the widely spread of drug resistance genes, mainly through mobile genetic elements including natural transformation, and the process of plasmid conjugation. We found that E. coli AH001 is carrying NDM−1 and OXA−10, both of which are belong to carbapenem resistant enzymes. The aquatic environment, such as the sewage in hospital which are enriched both antibiotics and bacteria, is an important reservoir for drug resistance genes and can be used as a medium to spread ARGs (Antibiotic Resistance Genes) from one ecosystem to another, thereby increasing the risk of MDR (Multi-Drug Resistance) bacteria infection outside the hospital [14]. The widespread detection of CPO (Carbapenemase-producing Organism) in the environment is an emerging environmental problem that may seriously affect public health. Our results are consistent with previous studies, indicating that bla NDM has appeared in many different species and spread rapidly in different environments [15,16].
In China, a recent study of 10,273 clinical stool samples collected from 52 hospitals found that the total infection rate of intestinal bacteria carrying bla NDM−1 among clinical patients was 14.8% [17]. And these feces can be discharged into hospital sewage, prompting bla NDM−1 to spread in many species and environments [18]. In environments such as sewage, the discovery of two different carbapenemase isolates in the same strain of bacteria has become increasingly frequent [15,19,20].
We found the bla OXA−10 gene in AH001, OXA-10 type β-lactamase was previously considered a narrowband enzyme in E. coli and Pseudomonas aeruginosa. However, Antunes NT proved that when expressed in Acinetobacter baumannii, this enzyme showed high resistance to carbapenems, and the production of OXA-10 increased the MIC of the bacteria to ceftazidime by 32 times [21]. These data clearly demonstrate the importance of OXA-10 as CHDLs, and how to verify that OXA-10 expresses carbapenem resistance on E. coli is worth exploring.
In addition to meropenem, AH001 exhibited resistance to 7 kinds of antibiotic include chloramphenicol, fosfomycin, tetracycline, ampicillin, gentamicin, o oxacin, and cefotaxime, expect, rifampicin and polymyxin B.  [22]. It has been reported that cefotaxime resistance has been observed in patients with cephalosporin treatment of Aeromonas bacteremia [23].
The serotype of AH001 predicted on the CGE website showed three serotypes, namely O9, O89 and O162, and the coincidence rates were 99.31%, 94.1%, and 93.63%, respectively. It is reported that E. coli of the O8 serotype are commonly associated with septicemia or diarrhea in calves and pose a signi cant threat to the cattle industry worldwide [24]. It has also been reported that the O89: H9 serotype is related to E. coli that produces ESBLs and carries bla NDM−5 and mcr-1 genes [25][26][27].
Nowadays, the detection of bacteria in the sewage is often studied by metagenomics, including 16s rDNA sequencing and the whole gene assembly of all bacteria. This study is based on direct phenotypic screening, which still has a certain signi cance. To the best of our knowledge, this is the rst report of a carbapenem-resistant E. coli carrying both bla NDM−1 and bla OXA−10 genes. The sewage of hospital treatment system will continuously discharge a large number of drug resistance genes into the water environment and release it into surface water [28]. This gene can be transferred horizontally in the sewage through the MGE (Mobile Genetic Elements) carrying the gene. Therefore, more attention should be paid to the discharge of hospital sewage.

Conclusions
To our knowledge, this is the rst report of carbapenem-resistant E. coli carrying both bla NDM−1 and bla OXA−10 genes. The research in this article will help us understand the E. colii carrying both bla NDM−1 and bla OXA−10 genes in medical sewage. Figure 1 Evolutionary relationship between 16S rDNA gene of AH001 and other adjacent strains (This gure was created by MEGAX) Figure 2 The result of Modi ed Hoge (EC354 is a isolate of E. coli isolated from sewage in the same batch as a positive control. AH001 and EC354 were tested by Modi ed Hoge test, and both isolates produced carbapenems. The principle is as shown in gure 1: carbapenems from AH001 and EC354 were inactivated and diffused into meropenem medium. There was not enough meropenem in this area to