Emergence of NDM-1- and CTX-M-3-Producing Raoultella ornithinolytica in Human Gut Microbiota

Raoultella ornithinolytica is an opportunistic pathogen of the Enterobacteriaceae family and has been implicated in nosocomial infections in recent years. The aim of this study was to characterize a carbapenemase-producing R. ornithinolytica isolate and three extended-spectrum β-lactamase (ESBL)-producing R. ornithinolytica isolates from stool samples of adults in a rural area of Shandong Province, China. The species were identified using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and 16S rDNA sequence analysis. Antimicrobial susceptibility test showed that all four isolates were multidrug-resistant (MDR). The whole genome sequence (WGS) of these isolates was determined using an Illumina HiSeq platform, which revealed MDR-related genes. The S1 nuclease-pulsed-field gel electrophoresis (S1-PFGE) was used to characterize the plasmids carried by the R. ornithinolytica isolates. The blaNDM-1 and blaCTX-M-3 genes were probed using Southern blotting, which confirmed the location of both genes on the same plasmid with molecular weight of 336.5–398.4 kb. The transferability of blaNDM-1 and blaCTX-M was also confirmed by conjugation assays. Finally, BLAST analysis of both genes showed that mobile genetic elements were associated with the spread of drug resistance genes. Taken together, we report the presence of conjugative blaNDM-1 and blaCTX-M plasmids in R. ornithinolytica isolates from healthy humans, which indicate the possibility of inter-species transfer of drug resistance genes. To the best of our knowledge, this is the first study to isolate and characterize carbapenemase-producing R. ornithinolytica and ESBL-producing R. ornithinolytica isolates from healthy human hosts.


INTRODUCTION
Raoultella is a genus of encapsulated Gram-negative aerobic bacilli of the Enterobacteriaceae family (Luo et al., 2017) that was initially part of the genus Klebsiella, but later reclassified based on the 16S rDNA sequence and the rpoB, gyrA, and gyrB genes (Drancourt et al., 2001). Raoultella ornithinolytica is one of the three species of the genus Raoultella (Singh et al., 2017) and naturally exists in the soil, water, and plants (Ayoade et al., 2018). R. ornithinolytica can cause pneumonia, biliary or urinary tract infections and bacteremia and such cases are being increasingly reported (Sekowska et al., 2015;Van Cleve et al., 2018). Both organ-specific and systemic R. ornithinolytica infections can be life-threatening, especially in cancer patients after abdominal surgery (Hajjar et al., 2018). Therefore, it is vital to understand the pathogenic potential of R. ornithinolytica isolates in humans (De Petris and Ruffini, 2018). Cephalosporins, quinolones, and carbapenems are routinely used against this pathogen, and multi-drug resistant (MDR) R. ornithinolytica isolates have been reported in recent years (Wang et al., 2019).
Widespread use of third-generation cephalosporins and other β-lactam antibiotics in the past decades have led to the emergence of third-generation cephalosporin-resistant bacteria that produce extended spectrum β-lactamases (ESBLs) and AmpC β-lactamases that are, respectively, encoded by plasmids and chromosomes (Kola et al., 2012;Dohmen et al., 2015). Consistent with this, infections caused by the ESBL-producing Gram-negative bacilli (GNB) have become increasingly prevalent worldwide, both in the healthcare and community settings, and pose significant therapeutic challenges (Wang et al., 2015). The class A serine CTX-M-type is the most common plasmid-encoded ESBLs that are produced by drug-resistant pathogens (He et al., 2016). The genetic elements encoding these CTX-M enzymes constantly evolve via random mutations and recombination between different resistance genes (Canton et al., 2012). A genetic epidemiological study on ESBLs found that bla CTX-M has replaced bla SHV and bla TEM as the most common ESBL-encoding genes (Xia et al., 2014). While CTX-M-15 and CTX-M-14 are the most prevalent ESBLs worldwide (He et al., 2016), bla CTX-M-14 has been identified as the most prevalent ESBL gene in China, and epidemiological surveillance in Asia, Latin America, and Europe has revealed a dramatic increase in cephalosporinresistant Escherichia coli and Klebsiella spp. strains due to spread of the CTX-M ESBLs (Zhang et al., 2016).
R. ornithinolytica infections are largely nosocomial and have rarely been reported in a healthy community (Seng et al., 2016). Nevertheless, the high rates of antimicrobial resistance in R. ornithinolytica isolates should be characterized in order to provide a basis for treating infections. To this end, we conducted a cross-sectoral study as part of the Sino-Swedish Integrated Multisectoral Partnership for Antibiotic Resistance Containment (IMPACT) in the Shandong Province in China using a One Health approach. The aim of this project is to study the relationship between the development of drug resistance in human (symbiotic and clinical), zoonotic, food, and environmental isolates of R. ornithinolytica. We identified NDM-1 and ESBL-producing R. ornithinolytica strains from healthy subjects and analyzed the drug resistance phenotypes and underlying mechanisms. To the best of our knowledge, this is the first report detailing the presence of an NDM-producing R. ornithinolytica strain in the human gut microbiota.

Bacterial Isolation and Identification
A total of 1,380 fecal samples were collected from healthy people in rural communities in July 2017 according to a previously described sampling procedure (Sun et al., 2018). Briefly, the samples were collected into ESwab tubes (Copan, Brescia, Italy) and stored at −80°C until cultivation. After thawing, the fecal samples were cultured on ChromID CARBA agar and ChromID ESBL agar plates (bioMérieux, Marcy l'Etoile, France) for 18 h at 37°C to respectively screen for the carbapenemase-and ESBL-producing R. ornithinolytica strains. The suspected R. ornithinolytica colonies identified based on color and morphology were picked and sub-cultured on CHROMagar Orientation agar (CHROMagar Company, Paris, France) overnight at 37°C. The resulting isolates were identified using MALDI-TOF MS and then confirmed by 16S rDNA sequence analysis against the bacterial 16S rDNA gene sequence in GenBank. The genomic average nucleotide identity (ANI) was calculated as described previously (Jiang et al., 2018).

DNA Extraction and PCR
DNA was extracted from pure cultures of R. ornithinolytica using a Gentra Puregene Yeast/Bact. Kit (QIAGEN, Hilden, Germany), and the 16S rDNA sequences were amplified by PCR with primers designed using the Primer 5.0 software (3′-AGAGTTTGATCCTGGCTCAG-5′/3′-GGTTACCTTGTTA GGACTT-5′). The optimized cycling conditions were: initial denaturation at 94°C for 5 min followed by 30 cycles of amplification each with 94°C for 1 min, 52°C for 1 min, and 72°C for 1 min and final extension at 72°C for 5 min. The PCR product was detected by capillary electrophoresis as described previously . The bla NDM-1 , bla CTX-M-3 , and bla CTX-M-14 genes were amplified by PCR using primers published previously (Shimizu et al., 2017;Chetri et al., 2019), and the products were confirmed by capillary electrophoresis followed by Sanger sequencing.

Antimicrobial Resistance Test
To verify carbapenemase production by the isolates, resistance against imipenem, ertapenem, and/or meropenem were determined using the modified Hodge test. ESBL-production was verified using the double disk diffusion method with cefotaxime, ceftazidime, and/or clavulanic acid according to Clinical and Laboratory Standards Institute (CLSI, 2017).

Antimicrobial Susceptibility Test
A total of 17 antibiotics belonging to 13 antimicrobial classes were tested, including cephalosporins (cefotaxime and ceftazidime), cephamycins (cefoxitin), β-lactam/β-lactamase inhibitor complexes (amoxicillin-clavulanate and piperacillintazobactam), carbapenems (imipenem and meropenem), penicillins (ampicillin), aminoglycosides (gentamicin and amikacin), fluoroquinolones (ciprofloxacin), folate metabolic pathway inhibitors (trimethoprim-sulfamethoxazole), tetracycline, chloramphenicols (florfenicol), colistin, furantoin, and tigecycline. The minimal inhibitory concentrations (MICs) of colistin and tigecycline were determined by the broth microdilution method and of other antibiotics using the agar dilution method. The results were interpreted according to CLSI (2017) and European Committee on Antimicrobial Susceptibility Testing (EUCAST) (version 8.1, 2018) guidelines. E. coli ATCC25922 was used as the quality control strain. Since clinical breakpoints of florfenicol are not available for Enterobacteriaceae in EUCAST or CLSI, the resistance breakpoint of >16 mg/L was selected based on the epidemiological cut-off values for the closely related E. coli and Salmonella spp. (Chi et al., 2019).

Whole-Genome Sequencing
Whole-genome sequencing was performed on the extracted DNA by Sangon Biotech (Shanghai, China) using the Illumina HiSeq sequencing platform. The quality of the high-throughput sequence data was assessed by FastQC 1 . SPAdes 3.11.0 was used for assembling raw sequences (Hu et al., 2011). All draft genomes were deposited in the NCBI database under accession number VJYE00000000-VJYH00000000. Acquired antimicrobial resistance genes and plasmid replicons were identified using ResFinder 2.1 and PlasmidFinder 1.3 2 , respectively. The gene sequences surrounding bla NDM-1 and bla CTX-M were annotated using RAST 3 and Easyfig 2.2.3 (Chi et al., 2019). To find the core genes of the R. ornithinolytica genomes, Roary was used for SNPs analysis (Page et al., 2015). Maximum likelihoodbased phylogenetic reconstruction was performed with RAxML version 8.2.10 (Stamatakis, 2014) and visualized with FastTree (Price et al., 2009).

S1 Nuclease-Pulsed-Field Gel Electrophoresis and Southern Blotting
The location of bla NDM-1 , bla CTX-M-3 , and bla CTX-M-14 on the plasmids was validated by S1-PFGE and southern blotting. Briefly, the isolates were embedded in 10 g/L Seakem Gold gel, digested with endonuclease S1 nuclease (TakaRa, Dalian, China), and subjected to pulsed-field gel electrophoresis (Parameters: 14°C, voltage 6 V/cm, electric field angle 120°, conversion time 2.16-63.8 s, and electrophoresis 16 h). The DNA fragments were transferred horizontally to a nylon membrane (Millipore, USA), and hybridized with three digoxinlabeled probes obtained by PCR amplification (Yu et al., 2002) and the Dig High Prime DNA Labeling and Detection Starter Kit (Roche Diagnostics). The genomic DNA of Salmonella enterica serovar Braenderup H9812 strain cut with XbaI was used as the DNA marker.

Conjugation Assay
The horizontal transferability of bla NDM-1 , bla CTX-M-3 , and bla CTX-M-14 were examined using conjugation assay using E. coli J53 (azide-resistant) or E. coli EC600 (rifampicin-resistant) as the recipient strains. The recipient bacteria and the target single colony were inoculated into 2 ml LB liquid medium and cultured at 37°C with constant shaking for about 6.5 h till the logarithmic growth phase. The recipient and donor bacteria culture broths were mixed at 1:2 v/v (200 μl and 400 μl), inoculated in 2 ml LB liquid medium, and incubated at 37°C for 12 h. The following day, 100 μl of the culture broth was uniformly spread on sodium azide-resistant agar containing cefotaxime (imipenem) and rifampicin and cultured at 37°C for 18 h. The single colonies were picked and purified overnight. Conjugants were identified by the presence of antibiotic resistance genes that were detected using PCR as described.

Multidrug-Resistant Raoultella ornithinolytica Strains Were Isolated From the Human Fecal Samples
One carbapenemase-producing (ROF058) and three ESBLproducing (ROE007, ROE058, and ROI014) R. ornithinolytica strains were isolated from 1,380 fecal samples ( Table 1) and confirmed by MALDI-TOF MS, 16S rDNA sequencing, and ANI analysis ( Figure 1A). Phylogenetic analysis revealed that isolates ROE007 and ROE058 are clonally related although they were recovered from different villagers in the same natural village (Figure 1B). The bla NDM-1 , bla CTX-M-3 , and bla CTX-M-14 genes were subsequently identified in all the strains, and multidrug resistance was confirmed by antimicrobial susceptibility tests. ROF058 was resistant to 12 antibiotics other than carbapenem, but sensitive to amikacin, tigecycline, furantoin, colistin, and trimethoprim-sulfamethoxazole. The resistance profiles of the three ESBL-producing isolates were similar, and all were resistant to gentamicin, tetracycline, cefotaxime, trimethoprimsulfamethoxazole, and ampicillin. Furthermore, all transconjugants exhibited MDR phenotypes similar to the donor strain ( Table 1).

The Raoultella ornithinolytica Strains Harbor Multidrug-Resistant Genes
MDR was defined as acquired non-susceptibility to at least one agent in three or more antimicrobial categories, and XDR was defined as non-susceptibility to at least one agent in all but two or fewer antimicrobial categories (Magiorakos et al., 2012). Whole genome sequencing showed that ROF058 is an extensively drug-resistant (XDR) strain, while ROE007, ROE058, and ROI014 are MDR strains (Table 1). In addition to the carbapenemase-encoding bla NDM-1 , ROF058 also carries genes encoding for other ESBLs (bla CTX-M-3 , bla OXA-1 , bla TEM-1B , and bla DHA-1 ), as well as resistance factors against, aminoglycosides [aac (3)

The Multidrug-Resistant Genes Are Located on Mobile Genetic Elements
Whole genome sequencing of strain ROF058 generated 5,747,150 clean reads, which were then assembled to 209 contigs with a GC content of 51.38%; WGS of strain R0E007 generated 5,214,213 clean reads, which were then assembled to 203 contigs with a GC content of 49.36%; WGS of strain ROE058 generated 5,235,762 clean reads, which were then assembled to 199 contigs Frontiers in Microbiology | www.frontiersin.org with a GC content of 50.28%; WGS of strain ROI014 generated 5,510,178 clean reads, which were then assembled to 215 contigs with a GC content of 54.66%. The plasmids in the different isolates were identified using the Center for Genomic Epidemiology program (Luo et al., 2017), which showed that ROF058 harbored the IncFIB(K)-type plasmid, ROE007 and ROE058 contained IncFIB(K) and IncFII-type plasmids, and ROI014 carried IncFIB(K) and IncFII plasmids. In addition, S1-PFGE and southern blot hybridization revealed three different plasmids in the R. ornithinolytica isolates ranging from 104.5 to 398.4 kb (Figure 3). ROFO58 contained the larger plasmid (336.5-398.4 kb) harboring the bla NDM-1 and bla CTX-M-3 genes. R0E007 and ROE058 had identical plasmid profiles, and the bla CTX-M-14 gene was located on a 138.9 kb plasmid in both isolates. The bla CTX-M-14 gene in ROI014 was located on a 216.9-244.4 kb plasmid. Furthermore, the bla NDM-1 and bla CTX-M-3 genes could be transferred from each of the isolates into recipient E. coli strains via conjugation (Table 1), which was confirmed by PCR (data not shown).

DISCUSSION
Intermittent but lethal nosocomial infections by R. ornithinolytica have been increasingly reported in the past decade (De Petris and Ruffini, 2018), and the advent of techniques like MALDI-TOF MS has enabled identification of new isolates (De Jong et al., 2013;Seng et al., 2016). Notable reports of R. ornithinolytica infections include sepsis in a patient with mitochondrial disease in Spain (Sanchez-Codez et al., 2019), urinary tract infection in a pediatric patient in Turkey (Buyukcam et al., 2019), and ventilator associated pneumonia in two immunocompetent trauma patients in USA (Van Cleve et al., 2018). R. ornithinolytica has also been implicated in joint infections and appendicitis in the clinical setting (Beye et al., 2018;Hajjar et al., 2018). In addition, MDR strains of this pathogen have emerged in recent years, primarily due to the spread of the resistance genes. The first NDM-1-producing R. ornithinolytica strain was isolated from the pus of hospitalized patients in India in 2013 (Khajuria et al., 2013) and was followed by reports of resistant R. ornithinolytica in USA, China, India, Spain, Korea, Bangladesh, and Brazil. In addition, some resistant strains have also been isolated from animals, vegetables, wastewater, and river water. The major drug resistance genes identified in these isolates include bla NDM-1 , bla KPC-2 , mcr-1, bla CTX-M-15 , bla IMP-4 , and bla OXA-1 (Zhou et al., 2014;Hasan et al., 2015;Zheng et al., 2015;Hernandez-Garcia et al., 2018;Miao et al., 2018;Yoon et al., 2018;Carramaschi et al., 2019;Wang et al., 2019). However, little is known regarding the carbapenemase-and ESBL-producing R. ornithinolytica strains. To the best of our knowledge, this is the first report on resistant R. ornithinolytica isolated from human gut microbiota.
Studies show that bacterial strains producing NDM-1 are resistant to most of the antibiotics used clinically and are only sensitive to a few such as colistin and tigecycline (Khan et al., 2017). However, Wang et al. (2018)  Frontiers in Microbiology | www.frontiersin.org 6 November 2019 | Volume 10 | Article 2678 that was positive for the NDM-1 gene and showed resistance to polymyxin and colistin . Consistent with this, our drug susceptibility test showed that ROFO58 was resistant to cefotaxime, ceftazidime, gentamicin, tetracycline, and ciprofloxacin. R. ornithinolytica is naturally resistant to aminopenicillin due to the presence of the chromosomal class A β-lactamase gene (Vasaikar et al., 2017), and all isolates did exhibit resistance to ampicillin. The presence of drug resistance genes strongly correlated with resistant phenotypes. Therefore, the WGS approach can rapidly detect antibiotic resistance genes that have been annotated and predict drugresistance in a particular isolate. Although all trans-conjugants showed an MDR phenotype similar to that of the donor strain in our study, the NDM-1-producing isolate had lower resistance to gentamicin and were sensitive to imipenem, ciprofloxacin, and amoxicillin-clavulanic acid. This could be due to factors other than the resistance genes. For example, studies have shown that the same resistance gene confers different susceptibility patterns when under the control of different promoters .
A previous report showed that the bla NDM-1 gene in Enterobacteriaceae is located on a rapidly transferable 50-200 kb plasmid belonging to several incompatibility groups such as IncL/M, IncHI1, IncFIIs, IncF, or untypable (Ahmad et al., 2018). In this study, the plasmid carrying bla NDM-1 was larger than 300 kb and of type IncFIB. Furthermore, this plasmid could be transferred from R0F058 to E. coli EC600 but not to E. coli J53. We surmised therefore that the plasmid was rare and selectively transferred. Epidemiological and genetic studies have shown that plasmid ligation and transposition of mobile genetic elements play an important role in the horizontal transmission of bla NDM-1 (Khajuria et al., 2013). BLAST analysis showed the presence  Frontiers in Microbiology | www.frontiersin.org of the aminoglycoside acetyltransferase gene [aac(3)] and bleomycin resistance gene upstream and downstream of bla NDM-1 , respectively. There were two insertion sequences (ΔIS91) flanking both resistance genes, indicating its important role in the spread of bla NDM-1 and other resistant genes. In addition, a remnant of ΔISAba125 was located upstream of bla NDM-1 (Figure 2). A previous study has shown that partial ISAba125 is a promoter of the bla NDM-1 gene (Poirel et al., 2011). The bla NDM-1 and bla CTX-M-3 genes were detected in the ROF058 trans-conjugant, indicating that bla NDM-1 can be co-transferred with bla CTX-M-3 and across species, and thereby disseminate drug resistance. Although clinically important bacteria such as Enterobacter cloacae and Klebsiella pneumoniae produce both NDM-1 and CTX-M-3 (Rubin et al., 2014;Hu et al., 2016), we have shown the co-existence of bla NDM-1 and bla CTX-M-3 in one plasmid for the first time.
The isolates in our study carried the bla CTX-M-3 and bla CTX-M-14 genes, which is similar to the CTX-M types of ESBLproducing K. pneumoniae isolated from environmental samples (Chi et al., 2019). This likely indicates spread of resistant bacteria between human hosts and the environment. The bla CTX-M genes are usually present on the IncF, IncI, IncN, IncHI2, IncL/M, and IncK plasmid types (Zhang et al., 2016). WGS analysis showed that three ESBL-producing R. ornithinolytica isolates in our study carry the IncFIC plasmid, along with a ΔISEcp1 sequence upstream of bla CTX-M-3 in ROF058, and bla CTX-M-14 in ROE007 and ROE058. A study showed that 97.6% of the bla CTX-M genes have an upstream ΔISEcp1 sequence (Adamski et al., 2015), which plays an important role in the transposition of bla CTX-M and other ESBL genes (Decano et al., 2019). R0E007 and ROE058 isolated from the same village showed an identical sequence flanking bla CTX-M-14 , i.e., ΔISEcp1-bla CTX-M-14 -ΔIS903, which could be the result of either the clonal expansion of the original host bacteria or lateral transfer of the genetic elements. A linear fecE-fecD-fecC-fecB-fecA-fecR-fecI ferric citrate genes were detected upstream of bla CTX-M-14 in ROI014, which is very similar to that reported in an environmental K. pneumoniae isolate (accession no. KF914891) from the same area (Chi et al., 2019). These findings strongly indicate the spread of drugresistance genes between K. pneumoniae and R. ornithinolytica.
To summarize, we characterized R. ornithinolytica isolates from healthy human feces in terms of their antibiotic susceptibility, drug resistance genes, and the transfer mechanism of the mobile genetic elements. The strains co-producing NDM-1 and CTX-M can confer higher levels of resistance to multiple antibiotics and can transfer the genes to other strains via plasmids by conjugation. Therefore, the patterns of antibiotic resistance and transmission should be closely monitored for R. ornithinolytica, especially in healthy individuals.

DATA AVAILABILITY STATEMENT
The datasets generated for this study can be found in the National Center for Biotechnology Information, VJYE00000000-VJYH00000000, https://submit.ncbi.nlm.nih.gov/.

ETHICS STATEMENT
The studies involving human participants were reviewed and approved by Ethics Committee for Shandong Center for Disease Control and Prevention; Shandong Center for Disease Control and Prevention. The patients/participants provided their written informed consent to participate in this study.

AUTHOR CONTRIBUTIONS
SW and LX contributed to perform the experiments, data analysis, and manuscript writing. XC and YL contributed to perform the experiments. XC, ZK, PH, HX, ZB, and BZ contributed to the sample collection and data analysis. BZ, ZB, and HX contributed to conceive and design the experiments and reviewed the article.