Characterization of carbapenem non-susceptible Gram-negative Bacilli isolated from the feces of 10,000 inpatients in Southern China

Background Carbapenem non-susceptible Gram-negative bacilli (CNS-GNB) were dominant pathogen causing clinical infections. The human intestine was important reservoir of GNB, but there were few studies to analysis the prevalence of fecal colonization with them. Fecal samples were collected from hosiptal screening test for GNB was conducted by using home-made MacConkey agar. Antimicrobial susceptibility was determined by the automatic microbiology analyzer and drug-resistant genes were characterized by polymerase chain reaction assays and DNA sequencing. The whole genome sequencing were used to analysis the characteristic of genetic structure of the isolates. A total of 680 CNS-GNB were collected. Acinetobacter spp. were the dominant species (33.8%) of the 22 genera. Carbapenemase genes were identied in 307 isolates (45.1%), including 206 (30.3%) bla NDM ; 51 (7.5%) bla VIM−2 , 48 (7.1%) bla IMP , and seven (1.0%) bla KPC−2 . The bla NDM genes were rst detected in three isolates, Providencia vermicola, Achromobacter spp., and Cupriavidus gilardii. Co-existence of bla VIM and bla IMP genes was detected in ve isolates; Achromobacter co-producing VIM and IMP has not been previously reported. The mcr-1 gene was identied in ve strains of Acinetobacter and one strain of K. pneumoniae. In addition, we detected seven isolates harboring the bla AFM−1 gene, a novel metallo-β-lactamase gene. This was rst genomic analysis of ST11 K. pneumoniae co-producing NDM-5 and mcr-1, which revealed that blaNDM-5 and mcr-1 are located on two different plasmids. The plasmid harboring blaNDM-5, which was composed of a typical IncX3-type backbone, and the mcr-1 gene, was located between an IS30-like element ISApl1 and a PAP2-like encoding gene in the IncHI2-type colistin. We detected ve strains of Acinetobacter and one strains of K. pneumoniae co-producing mcr-1 and NDM-5 in the present study. both bla NDM and mcr genes are commonly present in E. coli isolated from different sources, the co-existence of the bla NDM and mcr genes in K. pneumoniae rarely been reported. In this study, the plasmid harboring the mcr gene in K. pneumoniae was similar to other IncHI2-type plasmids present in E. coli, and upstream of the mcr gene was the common ISApl1 transposon. NDM-5


Introduction
Gram-negative Bacilli (GNB) are implicated in a wide range of diseases, such as pneumonia, meningitis, septicemia, and urinary tract infections [1]. According to China Antimicrobial Resistance Surveillance System (CARSS) monitoring data (http://www.carss.cn/), GNB have been shown to account for 70.6% of the total number of bacteria in hospitals, which are resistant to most available antibiotics and have developed built-in abilities to acquire new ways of resistance [2]. Carbapenem antibiotics have been reserved as drugs of last resort for salvage treatment of infections caused by multidrug-resistant Gram-negative bacteria, but the emergence of carbapenem-resistant GNB (CR-GNB) poses a global healthcare challenge because therapeutic options are limited. CR-GNB represent di cult-to-treat infections in hospitalized patients and are associated with high mortality [3]. In recent years, the resistance to carbapenem of GNB has spread rapidly, which has led to increased CR-GNB, and asymptomatically colonized patients might act as important reservoirs for transmission.
The main carbapenem-resistance mechanism of GNB is the production of carbapenemases capable of hydrolyzing carbapenems; these enzymes have been characterized into various classes, including Ambler class A β-lactamases, such as KPC and GES; Ambler class B metallo-β-lactamases, such as IMP, VIM, and NDM [4,5]; and Ambler class D oxacillinases (OXAs). Carbapenemase genes are generally located on plasmids, which can disseminate resistance horizontally by mobile genetic elements or mobile plasmids.
Colistin has been used as an effective clinical therapeutic against carbapenem-resistant bacteria. However, colistin resistance poses a substantial public health risk because it further limits treatment options in patients with infections caused by multidrug-resistant Gram-negative bacteria, including CR-GNB. The recent discovery of transferable plasmid-mediated colistin resistance genes between bacteria has further increased the risk of spread of colistin resistance [6]. The mcr gene has been linked to colistin resistance and can be transferred via plasmids [7]. Strains that are not sensitive to carbapenems and show resistance to colistin can signi cantly increase the risk of death from clinical infection, so it is necessary to detect the presence of colistin resistance genes.
Few studies have described the prevalence of fecal colonization with carbapenem non-susceptible GNB in China. In the present study, we collected 680 carbapenem non-susceptible Gram-negative bacilli (CNS-GNB) isolated from fecal survey samples of 10,000 patients and studied the drug-resistant and transmission mechanism by analyzing resistance genes and the genomic structure.

Patients and specimens
All samples were obtained randomly and were not selected for on the basis of suspected enteric infection or diarrhea. A total of 10,000 fecal survey samples from in-patients who underwent routine stool examinations on the rst day of hospitalization were prospectively and consecutively collected from July 2013 to June 2015 at Nanfang Hospital, a large, tertiary-level teaching hospital with 2,200 beds in Guangzhou, China.

Bacterial Isolates
To screen for carbapenem non-susceptible GNB, approximately 0.5 mL or 0.5 g of stool sample was suspended in 0.5 mL of 0.9% sterile saline, and 10 µL of the resulting suspension was inoculated onto MacConkey agar medium (Beijing Land Bridge Technology, Beijing, China) containing 2 mg/L meropenem (Dainippon Sumitomo Pharma, Osaka, Japan) at 37 °C for 18-24 h. Colonies surviving on MacConkey agar medium were stored at − 80 °C in nutrient broth containing 30% (v/v) glycerol. The study was approved by the Medical Ethics Committee of Nanfang Hospital Southern Medical University and conducted in compliance with the Declaration of Helsinki.

Species identi cation and anti-microbial susceptibility testing
Species identi cation and anti-microbial susceptibility testing was conducted with the BD Phoenix 100 Automated Microbiology System (Becton Dickinson and Co., Franklin Lakes, NJ, USA), and the results were interpreted according to Clinical and Laboratory Standards Institute (CLSI) guidelines categories and minimum inhibitory concentration (MIC) breakpoints. Ampli cation and sequencing of a ~ 996 base pair fragment from the partial 16S ribosomal RNA (rRNA) gene sequence were performed to con rm the genus or species level of the low con dence (< 90 value) value isolates. A standard nucleotide BLAST search of the NCBI sequence database (https://blast.ncbi.nlm.nih.gov/Blast.cgi) was performed to identify related 16S rRNA sequences. E. coli ATCC 25922 and P. aeruginosa ATCC 27853 were used as control strains.

Conjugation Assay
Conjugation experiments were performed in broth by using a sodium azide-resistant E.coli strain J53 as the recipient. The transconjugants were selected on Mueller-Hinton (MH) agar plates containing 4 µg/ml meropenem and 150 µg/ml sodium azide and MH agar plates supplemented with 4 µg/ml colistin and 150 µg/ml sodium azide. The MICs of imipenem, meropenem, ceftazidime, aztreonam and colistin against the donor, recipient and transconjugants were determined by using E-test strips (BioMérieux SA, La Balme-les-Grottes, France) obtained from Tian Kangxin (Beijing) Technology Co. Ltd. (Beijing, China) on MH agar plates.

Whole genome sequencing and analysis
It has been reported that the bla NDM gene and mcr gene were mainly existed in E. coli isolated from different sources, but the co-existence of bla NDM gene and mcr gene in K. pneumoniae was rarely reported. In our study, one K.pneumoniae strain carrying blaNDM-5 gene and mcr-1 gene was detected in human feces, and which was resistant to carbapenem and colistin (Supplementary Table 2). Thus, the strain was selected for the whole genome sequencing. The whole genome was sequenced using the Single Molecule Real-Time (SMRT) sequencing platform with the PacBio sequencer and Illumina HiSeq at the Health Time Gene Institute (Shenzhen, China). The reads were denovo assembled using the HGAP (version 3.0). The prediction and annotation of the genome was achieved using GeneMarkS (version 4.6b, http://topaz.gatech.edu/GeneMark/) and BLAST (https://blast.ncbi.nlm.nih.gov/). The plasmids were typed using the PlasmidFinder 2.1 (https://cge.cbs.dtu.dk/services/PlasmidFinder/). The comparative and synteny analysis were generated using by BLAST and MUMmer (version 3.23, http://mummer.sourceforge.net/).

Clinical data collection
The electronic medical records system were reviewed systematically for the clinical characteristics of the patients carrying fecal CNS-GNB. The following information was included: demographics (age and sex); geographical distribution of patients; colonization/infection status, type of infection, therapy received and clinical outcome; Data on transplantation and immunosuppression, prior receipt of glucocorticoid, prior exposure to carbapenems, prior hospitalization, and invasive procedures such as surgery, use of mechanical ventilation, use of peripherally inserted central catheter were also recorded.  Table 1). Table 1 The carbapenemase genes and colistin-resistant genes of 680 carbapenem-non-susceptible GNB Antimicrobial susceptibility testing

Identi cation of isolates
Most of the isolates were resistant to cephalosporin, but they were sensitive to colistin. Amikacin resistance was identi ed in 147 isolates (22.5%). Most of the carbapenem-resistant non-fermenting Bacillus had a sensitivity rate of more than 80%, and sensitivity to amikacin of A. baumannii was 54.8%. Most of CRE strains had a resistance rate of less than 10% to amikacin; E.coli had a resistance rate of 25.8%. A total of 490 (75.2%) isolates were cipro oxacin resistant, 391 (57.5%) isolates were levo oxacin resistant, and the susceptibility of different CRE species to cipro oxacin and levo oxacin varied. CRE species were more sensitive to trimethoprim/sulfamethoxazole than non-fermenting Bacillus, and 527 (77.5%) isolates showed resistance to trimethoprim/sulfamethoxazole. A total of 354 (54.3%) isolates were resistant to tetracycline, and non-fermenting Bacillus species were more sensitive to tetracycline than CRE (Table 2).    13.5%) also surpassed other general underlying diseases. Bacterial infections were prominent, and most of the cases had a history of antibiotic use.  Genomic analysis of CRKP co-producing bla NDM−5 and mcr-1 The chromosome was 5,235,159 bp with 57.55% GC content; it harbored 5186 genes, including ve drug resistance genes encoding a class A broad-spectrum beta-lactamase, multidrug e ux transporter transcriptional repressor, tetracycline repressor protein, multidrug e ux MFS transporter, and fosfomycin resistance protein (Table 5). It harbored the mcr-1 gene, which was located between the IS30-like element ISApl1 and a PAP2-like encoding gene (Fig. 1). The plasmid co-carried the resistance gene blaCTX-M-14. BLASTn analysis of the complete nucleotide sequence against the NCBI database indicated the highest similarity (95% query cover and 99% nucleotide identity) to plasmid pMCR_WCHEC050613 (CP019214.2), which was from E. coli isolated from sewage in Chengdou. The genetic structure of mcr-1 in plasmid pMCR_WCHEC050613 was also the same as pAN65-MCR. BLAST homology analyses revealed that pAN65-MCR has 93% identity and 99% query coverage with pHNSHP45 [8], which was isolated from pigs. Comparative analysis of the genetic structures of mcr-1 in the reference plasmids pHN6DS2 and pMCR1-PA revealed there were identical to pAN65-1, and pHNSHP45 had lost the PAP2 gene downstream of mcr (Fig. 2).

Discussion
Intestinal ora forms a major reservoir of bacteria, including multidrug-resistant bacteria. In this study, the overall prevalence of fecal carriage of CNS-GNB in 10,000 stool samples was 7.45%, and the rate of carrying CRE was 1.6%, of which the dominant species were K. pneumoniae and E. coli, consistent with a previous report [10]. This showed that K. pneumoniae and E. coli were the dominant species in 60 CRE isolated from 704 stool samples. However, the rate of feces carrying CRE was 8.5%; this may be due to the fact that the stool samples collected in this study were primarily from hospitalized patients in Intensive Care Unit. The fecal samples collected in the present study contained 5.0% carbapenem-resistant non-fermenting bacillus, and Acinetobacter spp. was the dominant species accounting for 2.3%; this result was similar to our previous report of the positive rate of Acinetobacter spp. [11]. In general, the abundance of Enterobacteriaceae in the normal intestine is greater than Acinetobacter, but the positive rate of carbapenem-resistant Acinetobacter in the intestine was higher than Enterobacteriaceae in our study. Acinetobacter is the main pathogenic bacteria that causes nosocomial infections and is widely distributed in the environment, which is signi cant for the spread of drug-resistant bacteria. We isolated 22 genera of CR-GNB from stool samples, some of which did not cause clinical infections, suggesting the possibility that drug resistance had spread widely between different strains.
CR-GNB isolates in the present study exhibited high resistance rates to β-lactam antimicrobial agents, including cephalosporins, carbapenems, and the monobactam aztreonam; however, most isolates were sensitive to colistin. The susceptibility of different CRE species to amikacin varied; most of the isolates exhibited low resistance to amikacin (< 20.0%). Liu et al. [10] reported that resistance to amikacin against CRE species isolated from fecal samples was less than 25%, while Wang et al. [12] reported that 1801 clinical CRE isolates showed susceptibility to amikacin (54.5%). Carbapenem-resistant strains isolated from fecal survey samples were more sensitive to amikacin than clinical strains. Pritsch et al. [13] reported that two of three carbapenamase-producing A. baumannii isolates were untreatable with locally available antimicrobial agents and were susceptible to colistin and amikacin only. In some cases, Our datas provided an effective choice for clinical treatment of infections.
A total of 307 (45.1%) isolates carried carbapenemase genes. bla NDM (67.1%, 206/307) was the predominant carbapenemase gene in the present study; the four main types included bla NDM−1 , bla NDM−4 , bla NDM−5 , and bla NDM−7 . The positive rate of the bla NDM−1 gene was higher than for any other variant, consistent with previous reports [14]. In the present study, bla NDM was identi ed in different species, and the sequencing of four different species indicated that bla NDM can be transmitted between different species through mobile genetic elements or removable plasmids, including Enterobacteriaceae and non-fermenting Bacillus. Notably, NDM was mainly present in Acinetobacter, and we detected only one type of carbapenemase NDM in Acinetobacter, indicating that NDM production is an important factor in the resistance of Acinetobacter to carbapenem. Other metal enzymes such as VIM and IMP were mainly present in Pseudomonas spp., and KPC was mainly present in K. pneumoniae. Apart from the NDM, KPC, VIM, and IMP carbapenemases, no other carbapenemase genes were found. OXA-48, a carbapenemase gene found in many European countries, is rare in China. Whether the results of the present study, and those of previous studies in which OXA-48 was not detected, are typical, will require further investigation [15,16]. In addition, 373 carbapenemase gene-negative GNB isolates were identi ed. Other mechanisms of carbapenem resistance have been recognized, which include overexpression of Extended Spectrum Beta-Lactamases (ESBLs) or AmpC β-lactamase, decreased membrane permeability due to porin loss, and expression of e ux pumps [17,18]. The complexity of carbapenem resistance mechanisms in GNB and characteristics of the human gastrointestinal tract make the dissemination and transmutation of resistance genes more complex and frequent.
The bla AFM gene was discovered in our research. This gene is a novel metallo-β-lactamase gene; its expression product can hydrolyze carbapenemase.
Therefore, strains carrying the bla AFM gene can also develop resistance to carbapenem antibiotics. In the present study, the bla AFM gene was also present in different strains of the human gastrointestinal tract. Although it was not detected in common isolates of Enterobacteriaceae, the bla AFM gene is often located on a plasmid and can spread through mobile genetic elements.
Colistin is a last-resort antimicrobial for infections caused by multidrug resistant Gram-negative bacteria. In 680 CNS-GNB, six isolates were resistant to colistin, which indicated low prevalence of the mcr-1 gene among carbapenemase-producing fecal survey isolates. The mcr-1 gene is a transferable resistance determinant against colistin. We detected ve strains of Acinetobacter and one strains of K. pneumoniae co-producing mcr-1 and NDM-5 in the present study. It has been reported that both bla NDM and mcr genes are commonly present in E. coli isolated from different sources, but the co-existence of the bla NDM and mcr genes in K. pneumoniae has rarely been reported. In this study, the plasmid harboring the mcr gene in K. pneumoniae was similar to other IncHI2-type plasmids present in E. coli, and upstream of the mcr gene was the common ISApl1 transposon. NDM-5 has been reported in many other countries, including India [19], China [20], Denmark [21], Italy [22], America [23], and Spain [24]; it has also been isolated from pigs [25,26], dairy cows [27], and vegetables [28]. In the present study, the IncX3 plasmids harboring bla NDM−5 were highly similar in various sources, suggesting their ability to be an e cient vehicle for bla NDM−5 dissemination among humans, animals, food, and the environment via the human intestine. This strain can capture the plasmid harboring mcr or bla NDM genes, which leads to the generation of pan-drug resistance, and the phenomenon of co-existence of mcr or bla NDM genes has spread to other isolates, which poses a signi cant challenge in the treatment of clinical infections.
In the present study, individuals with CRE were primarily from the pneumology department, hematology department, intensive care unit, and rehabilitation department. There were some common feature of individuals with CRE, most of them had infectious diseases, multiple hospital admissions and higher infectious risk, the history of using antibiotics. Therefore, the frequent use of antibiotics was closely related to multidrug resistant bacteria. However, some individuals with CRE did not have underlying diseases, and there was no record of previous antibiotic use. In addition, these individuals were from different regions of China, indicating that CRE has been widely present in the intestines of normal individuals. If individuals carrying resistance genes have infections, failure of antibiotic treatment, as well as an increase in the risk of transmission to other patients, is likely.
In conclusion, the emergence of CNS-GNB in fecal matter poses a major concern. Our data showed that the overall prevalence of fecal carriage of CNS-GNB in 10,000 stool samples was 7.45%, Acinetobacter was the dominant isolate of CNS-GNB, and most of the strains were sensitive to colistin. The presence of carbapenemase genes was the main mechanism of CNS-GNB resistance to carbapenem antibiotics, mainly encoding NDM. Notably, bla NDM genes were widely distributed in various isolates. The bla NDM genes were rst detected in Providencia vermicola, Achromobacter spp., and Cupriavidus gilardii, and Achromobacter co-producing bla VIM and bla IMP genes has not been reported. Genomic sequencing and analysis of K. pneumoniae harboring bla NDM and mcr revealed that these genes were located on two different plasmids, which were similar to previously reported plasmids, suggesting their ability to be e cient vehicles for bla NDM−5 dissemination among humans, animals, food, and the environment via the human intestine. Our results highlight the fact that enhanced surveillance and health policies for the detection and control of these pathogens are urgently needed to limit the emergence and spread of such an organism.
As the nal antibiotic for the treatment of CR-GNB infection, the emergence of MCR-carrying strains reminds us to strengthen the management of antibiotic use and encourages the implementation of human feces surveillance, as well as actions to prevent and control the spread of multidrug resistant bacteria, especially bacteria resistant to carbapenems and colistin.

Declarations
Ethics approval and consent to participate: Verbal informed consent was obtained from all participants. The samples used were the specimens remaining after the patient's clinical examination. The study was approved by the Medical Ethics Committee of Nanfang Hospital Southern Medical University and conducted in compliance with the Declaration of Helsinki (No. NFEC-2014-002).
Consent to publish: Not applicable.

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