Detection and Genomic Characterization of a Morganella morganii Isolate From China That Produces NDM-5

The increasing prevalence and transmission of the carbapenem resistance gene blaNDM–5 has led to a severe threat to public health. So far, blaNDM–5 has been widely detected in various species of Enterobacterales and different hosts across various cities. However, there is no report on the blaNDM–5– harboring Morganella morganii. In January 2016, the first NDM-5-producing Morganella morganii L241 was found in a stool sample of a patient diagnosed as recurrence of liver cancer in China. Identification of the species was performed using 16S rRNA gene sequencing. Carbapenemase genes were identified through both PCR and sequencing. To investigate the characteristics and complete genome sequence of the blaNDM–5-harboring clinical isolate, antimicrobial susceptibility testing, S1 nuclease pulsed field gel electrophoresis, Southern blotting, transconjugation experiment, complete genome sequencing, and comparative genomic analysis were performed. M. morganii L241 was found to be resistant to broad-spectrum cephalosporins and carbapenems. The complete genome of L241 is made up from both a 3,850,444 bp circular chromosome and a 46,161 bp self-transmissible IncX3 plasmid encoding blaNDM–5, which shared a conserved genetic context of blaNDM–5 (ΔIS3000-ΔISAba125-IS5-blaNDM–5-ble-trpF-dsbC-IS26). BLASTn analysis showed that IncX3 plasmids harboring blaNDM genes have been found in 15 species among Enterobacterales from 13 different countries around the world thus far. In addition, comparative genomic analysis showed that M. morganii L241 exhibits a close relationship to M. morganii subsp. morganii KT with 107 SNPs. Our research demonstrated that IncX3 is a key element in the worldwide dissemination of blaNDM-5 among various species. Further research will be necessary to control and prevent the spread of such plasmids.

NDM-5 was first identified in Escherichia coli from a patient who had been hospitalized in India in 2011 (Hornsey et al., 2011). NDM-5 and NDM-1 are similar; the only difference they demonstrated is that two amino acids have been replaced (Val88Leu and Met154Leu), resulting in NDM-5 exhibiting a high level of resistance to carbapenems and expanded-spectrum cephalosporins, and thus posing a severe threat to public health (Hornsey et al., 2011). Since then, NDM-5 has spread globally, such as China (Zhang L.P. et al., 2016), the United States of America (Rojas et al., 2017), Australia (Wailan et al., 2015), Egypt (Soliman et al., 2016), and Italy (Giufre et al., 2018).
Worryingly, in China, NDM-5 has been detected in various species of Enterobacterales across various cities (Zhang F. et al., 2016;Li et al., 2017;Mao et al., 2018;Sun et al., 2018). In the current study, we identified a clinical M. morganii isolate producing NDM-5 and performed phylogenetic analysis. Further, we investigated the drug resistance profile and plasmid characteristic analysis to depict the potential transmission mechanisms of bla NDM−5 .

Strain Screening
Since January 2016, we collected various clinical samples from patients based at the First Affiliated Hospital of Zhejiang University in Hangzhou (FAHZU). The samples were spread on the surface of MacConkey agar (OXOID, Hampshire, United Kingdom) plates that contained 2 mg/L meropenem (Meilunbio, Dalian, China) for 18-24 h at 37 • C for the preliminary screening of carbapenem-resistant Enterobacterales (CRE) isolates (CLSI, 2018). The CarbaNP test and modified carbapenem inactivation method (mCIM) with EDTA-modified carbapenem inactivation method (eCIM) were used to detect carbapenemase activity (CLSI, 2018). Identification of species was performed using both matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/MS) (Bruker Daltonik GmbH, Bremen, Germany) and 16S rRNA gene sequencing. Carbapenemase genes (bla KPC , bla NDM , bla OXA−48 , bla VIM , and bla IMP ) were identified using PCR and DNA sequencing as described previously (Zheng et al., 2015). Finally, M. morganii L241 isolate was detected and its details were described in the results.
Location of bla NDM−5 Gene and Transferability of Plasmid Carrying bla NDM−5 The number and size of plasmid of M. morganii L241 were determined with the S1 nuclease pulsed field gel electrophoresis (S1-PFGE) method, as described previously (Zheng et al., 2015). Southern blotting and hybridization using DIG-labeled bla NDMspecific probe were performed to estimate the location of bla NDM gene, while the transferability of NDM-carrying plasmid from the isolate was determined through the use of conjugation experiments, with rifampicin-resistant E. coli C600 as the recipient strain. Further to this, transconjugants were selected on Mueller-Hinton agar (OXOID, Hampshire, United Kingdom) plates that contained both 200 mg/L rifampicin (Meilunbio, Dalian, China) and 2 mg/L meropenem. Finally, a combination of MALDI-TOF/MS identification, bla NDM gene detection and antimicrobial susceptibility testing of the transconjugants were performed in order to confirm whether the plasmid was successfully transferred to the recipient.

Whole Genome Sequencing and in silico Analyses
Genomic DNA was extracted using the OMEGA Bacterial DNA kit (Omega Bio-tek, Norcross, United) and was then sequenced on both the llumina HiSeq 4000-PE150 (Illumina, San Diego, CA, United States) and the PacBio RS II platforms (Pacific Biosciences, California, United States). We created a complete genome sequence for M. morganii L241 using Unicycler (Wick et al., 2017) by combining our llumina sequencing reads with PacBio sequencing reads. By using Unicycler (Wick et al., 2017), raw llumina reads were assembled using SPAdes, semiglobal alignment was then performed by aligning PacBio reads to the assembly data, the llumina sequenceing reads were finally used to polish the genome assembly with Bowtie2 and Pilon. Additionally, online tools 1 were used to identify acquired antimicrobial resistance genes and replicon type of plasmid. This genome was annotated by the RAST server (Aziz et al., 2008), while the IS Finder database 2 was used to identify transposon and IS elements. The circular image of multiple plasmids comparisons was generated by the BLAST Ring Image Generator (BRIG) (Alikhan et al., 2011). Finally, the comparison figures of the genetic context of bla NDM−5 on multiple plasmids were performed with a Python application Easyfig (Zheng et al., 2017).

Comparative Genomic Analysis
Genome sequences for 41 strains of M. morganii were downloaded from Pathogen Detection 3 . These genomes, plus the M. morganii L241 genomic sequence, were then analyzed using Snippy 4 , a process in which raw reads were mapped against the reference M. morganii genome (no.ALJX00000000) (Chen et al., 2012). A phylogenetic tree based on concatenated, qualified single nucleotide polymorphisms (SNPs) was then performed using Harvest (Treangen et al., 2014). Characteristics of all the M. morganii strains included in this study are summarized in Supplementary Table S1.

Accession Numbers
The genome sequences of both M. morganii L241 chromosome and plasmid pNDM5-L241 were deposited in the GenBank with the accession numbers CP033056 and CP033057.

Isolation and Identification of NDM-5-Producing M. morganii L241 Strain
A male patient of 53 years old was admitted to surgical ward of FAHZU in January 2016 and initially diagnosed as recurrence of liver cancer. The patient received hepatectomy 2 days after hospitalization and developed acute diarrhea on the third day after surgery. Since then, diarrhea has always existed. On the sixth day after surgery, a rod shaped Gram-negative bacterium, designated as L241, was recovered from the selective medium, which was found to be positive for the CarbaNP test, and mCIM with eCIM assay. Then it was confirmed as M. morganii and found to harbor bla NDM−5 after PCR and sequencing .

Antimicrobial Susceptibility Testing
The minimum inhibitory concentration (MIC) values of antimicrobials for M. morganii L241 are shown in Table 1  also demonstrated resistance to fosfomycin (MIC = 256 mg/L) and chloramphenicol (MIC = 32 mg/L) but was shown to be susceptible to amikacin, tetracycline, gentamicin, ciprofloxacin, levofloxacin, and trimethoprim/sulfamethoxazole.

Genomics Features of M. morganii L241
The genomic features of the M. morganii L241 are shown in Table 2. It was found that the M. morganii L241 genome consists of a 3,850,444 bp circular chromosome with an average G+C content of 51.1% and one plasmid. Further, the chromosome contained 3,831 protein coding genes, 82 tRNAs, and 22 rRNAs. A screening for acquired resistance determinants found that chromosome possess the resistance gene catA2 encoding phenicol resistance and bla DHA−17 encoding β-lactam resistance; while the plasmid encoding acquired resistance gene confers

Comparative Genomic Analysis
While previous studies have reported the genomes of M. Morganii (Khatri et al., 2013;Nash and Young, 2015), there are few studies which have linked the genetic information of multiple strains of M. morganii to explore their evolutionary relationships and the internal structure of the genome. Therefore, we performed a comparative genomic analysis. As shown in Figure 1 and Supplementary Table S1, all of M. morganii isolates were found in various specimen types, including stool, rectal swab, wound, urine, blood, sputum, abscess, pericardial fluid, lettuce leaves, cheese, phytotelma, freshwater lake and roots from different hosts (homo sapiens, animal, plant, food, and environment). At the same time, they were detected across various countries, including India, Japan, Austria, Brazil, Russia, United States, South Africa, Viet Nam, United Kingdom, Malaysia, South Korea, Portugal, Switzerland, Canada, and China from 1800 to 2017, suggesting that M. morganii isolates are widely distributed. Resistance genes present on all M. morganii isolates showed that there was a common β-lactamases resistance gene bla DHA. In addition, no plasmid replication type was detected in most M. morganii isolates.
In   (Chen et al., 2012). These nine clustered strains were isolated from different specimen types and were detected at different times and in different countries. Further analysis of genomic information showed that all of these strains contained bla DHA−17 and catA2 genes, but there was no common plasmid replicon type. It is noteworthy that M. morganii L241 and M. morganii subsp. morganii KT, which is the first genome sequence of M. morganii, are the most closely related isolates, differing by just 107 SNPs.

Characterization of Plasmid Bearing bla NDM−5
The S1-PFGE result showed that only a ∼46-Kb plasmid was found in M. morganii L241 (Figure 2A). Subsequently, Southern blotting revealed that the bla NDM−5 gene was located on this plasmid ( Figure 2B) (Table 1), with considerable increases in the MICs of carbapenems when compared with the recipient strain E. coli C600. These results indicate that the bla NDM−5 -encoding plasmid of M. morganii L241, designated as pNDM5-L241, was successfully transferred into recipient E. coli C600 strain. In addition, results suggest that this was a self-transmissible plasmid. Owing to M. morganii L241 only possessing plasmid pNDM5-L241, the antimicrobial resistance phenotypes of the transconjugant were acquired from pNDM5-L241.
In silico analysis identified that plasmid pNDM5-L241 is an IncX3 type plasmid, with 46,161 bp in length, 64 predicted coding sequences and a GC content of 46.6%. This plasmid was home to several types of genes, such as antimicrobial resistance genes, mobile elements genes, putative genes, genes encoding replication proteins and genes encoding proteins for plasmid stability and plasmid transfer, respectively. A search of the nr/nt database found plasmid pNDM5-L241 exhibiting 99% nucleotide identity with the IncX3 bla NDM−5 encoding plasmid pTB203 (no. CP029245) and pNDM_MGR194 (no. KF220657). In all of these cases, bla NDM−5 was the only antimicrobial resistance gene. Importantly, a conserved structure sequence ( IS3000-ISAba125-IS5-bla NDM−5 -ble-trpF-dsbC-IS26) was found in the upstream and downstream of the bla NDM−5 (Figure 3B).

DISCUSSION
Morganella morganii has been recognized as an increasingly important pathogen because of the increased frequency and a high mortality rate of its infections. In addition, according to a recent report, acquired resistance is increasingly observed in M. morganii (Liu et al., 2016). For example, M. morganii has shown resistance to β-lactams, aminoglycosides, phenicols, macrolides, tetracycline, trimethoprim, and fluoroquinolones (Liu et al., 2016). As a result of the intrinsic and acquired resistance of M. morganii, it poses a serious clinical threat which has limited treatment options. Nevertheless, there has not been too much attention on M. morganii so far.
The rapid development of gene sequencing technology has enabled us to have a deeper understanding of bacteria. As we know, the comparative genomic analysis based on SNPs combined with the plasmid replication type, antibiotic resistance gene content, time of isolation, geographical region, isolation source, and host is a valuable tool to conduct genomic epidemiological analyses. Therefore, in this study, the complete genome sequence and comparative genomic analysis were performed. Our analysis showed that M. morganii L241 is clustered with M. morganii MM4, M. morganii MM190, M. morganii FDAARGOS_438, M. morganii FDAARGOS_172, M. morganii NCTC232, M. morganii NCTC12028, M. morganii E042, and M. morganii subsp. morganii KT (Chen et al., 2012). The clustering phenomena and relatively small number of SNPs seen in these M. morganii isolates from different geographic locations over such a long time frame suggest that these isolates might be highly clonal. Further analysis showed that the clustering of M. morganii L241 with other M. morganii isolates was not determined by IncX3 type plasmid. Moreover, results showed that M. morganii L241 and M. morganii subsp. morganii KT are the most closely related isolates, prompting that M. morganii L241 may have evolved from M. morganii subsp. morganii KT. The pathogenicity-related factors of M. morganii subsp. morganii KT were identified, such as fimbrial adhesins, T3SS, TCS, iron acquisition system, IgA protease, and insecticidal and apoptotic toxins (Chen et al., 2012), implying M. morganii L241 has the similar toxicity characteristics.
To date, NDM-5 has been found in Proteus mirabilis (Zhang F. et al., 2016), K. pneumonia (Cho et al., 2015), E. coli (Soliman et al., 2016), Enterobacter aerogenes (Ahmad et al., 2018), and Salmonella enterica serovar Typhimuriumstrain (Li et al., 2017). As far as we are aware, the current study is the first report that has identified NDM-5 in M. morganii. This is a worrying development as it demonstrates the further spread of bla NDM−5 among different species of Enterobacterales.
In this work, we observed that a conserved structure sequence ( IS3000-ISAba125-IS5-bla NDM−5 -ble-trpF-dsbC-IS26) was found in the upstream and downstream of the bla NDM−5 in IncX3 type plasmid. Interestingly, the conserved structure sequence is consistent with the upstream and downstream of the bla NDM−5 in IncFII type plasmid (Li et al., 2017). Recently, research has proposed that IS26 element may contribute to the vertically transfer of bla NDM−5 gene among plasmids and chromosomes (Li et al., 2017).

CONCLUSION
In summary, we first identified a bla NDM−5 -positive M. morganii and reported its complete genome sequence. The bla NDM−5 gene was located on a self-transmissible IncX3 plasmid which spread among species of Enterobacterales worldwide. This study highlights the wide spread of bla NDM -encoding IncX3 plasmids, including their transmissionin to uncommon Enterobacterales strains including M. morganii. Therefore, the IncX3 plasmids must be closely monitored, and attention must be paid to uncommon Enterobacterales strains. Further research is necessary to prevent and control the spread of bla NDMencoding IncX3 plasmids.

DATA AVAILABILITY
The datasets generated for this study can be found in NCBI, CP033056 and CP033057.

ETHICS STATEMENT
Written informed consent was obtained from the participants of this study.