Clinical Molecular and Genomic Epidemiology of Morganella morganii in China

Objectives: Ongoing acquisition of antimicrobial resistance genes has made Morganella morganii a new clinical treatment challenge. Understanding the molecular epidemiology of M. morganii will contribute to clinical treatment and prevention. Methods: We undertook a 6-year clinical molecular epidemiological investigation of M. morganii from three tertiary hospitals in China since 2014. Antimicrobial susceptibility testing was performed using a VITEK-2 system. All isolates were screened for β-lactam and plasmid-mediated quinolone resistance genes by PCR. Isolates carrying carbapenem-resistant genes were subjected to whole-genome sequencing (WGS). The variation and evolution of these mobile genetic elements (MGEs) were then systematically analyzed. Results: Among all M. morganii isolates (n = 335), forty (11.9%) were recognized as multidrug resistant strains. qnrD1, aac(6′)-Ib-cr, blaTEM–104, and blaCTX–M–162 were the top four most prevalent resistance genes. Notably, phylogenomic and population structure analysis suggested clade 1 (rhierBAPS SC3 and SC5) associated with multiple resistance genes seemed to be widely spread. WGS showed a blaOXA–181-carrying IncX3 plasmid and a Proteus genomic island 2 variant carrying blaCTX–M–3, aac(6′)-Ib-cr coexisted in the same multidrug resistant strain zy_m28. Additionally, a blaIMP–1-carrying IncP-1β type plasmid was found in the strain nx_m63. Conclusion: This study indicates a clade of M. morganii is prone to acquire resistance genes, and multidrug resistant M. morganii are increasing by harboring a variety of MGEs including two newly discovered ones in the species. We should be vigilant that M. morganii may bring more extensive and challenging antimicrobial resistance issue.


INTRODUCTION
Morganella morganii is emerging as a significant opportunistic pathogen in the hospital settings (Liu et al., 2016). A 6-year study of Gram-negative bacterial infections in Taiwan showed that this pathogen is the ninth most prevalent cause of clinical infections (Chen et al., 2012). It is reported that M. morganii has been involved in a variety of clinical infections, such as peritonitis, septic arthritis, sepsis, infective endocarditis (van Bentum et al., 2019) and bilateral keratitis (Zhang et al., 2017). Invasive M. morganii infections are usually associated with high mortality rates due to lack of appropriate empirical antibiotic treatment (Erlanger et al., 2019).
Salmonella genomic island 1 (SGI1), is an integrative MGI that has many variants (Schultz et al., 2017), and can be mobilized by IncA/C conjugative plasmids (Carraro et al., 2014). In addition, some SGI1-related elements, such as Proteus genomic island 2 (PGI2), Acinetobacter genomic island 1 (AGI1) and their variants have been described in various bacteria including diverse serovars of S. enterica, Vibrio cholerae, P. mirabilis, and A. baumannii (Cummins et al., 2020). These GIs consist of a conserved backbone and a highly genetic variable multidrug resistant region derived from one or more complex class 1 integron carrying various resistance gene cassettes (Girlich et al., 2015). The backbone usually integrates into the chromosomes at 3 end of the trmE gene, and the multidrug resistant region often locates adjacent to the res gene (Lei et al., 2020). Noteworthy, SGI1 variant (SGI1-L) carrying resistance genes dfrA15, floR, tetA(G), bla CARB-2 and sul1 has also been identified in M. morganii (Schultz et al., 2017). Besides, carbapenemase gene bla NDM-1 has also been found in the MDR region among SGI1-like sequences (Girlich et al., 2015).
Antimicrobial resistance in M. morganii therefore can be introduced via both resistance plasmid acquisition and genomic island horizontal transfer. However, large-scale and long-term molecular investigations of clinical isolates of M. morganii has rarely been conducted. The aim of this study is to conduct a clinical and molecular epidemiological investigation of M. morganii isolated from three tertiary hospitals in China, and to unravel the molecular mechanisms underlying antimicrobial resistance of M. morganii in China.

Bacterial Isolates
All M. morganii strains isolated from clinical specimen were collected from three tertiary hospitals in China from June 2014 to June 2020 including The First Affiliated Hospital of Sun Yat-sen University, Guangzhou, China (Hospital ZY), Guangdong Provincial Hospital of Traditional Chinese Medicine, Guangzhou, China (Hospital SZY) and General Hospital of Ningxia Medical University, Yinchuan, China (Hospital NX). If more than one strain were isolated from the same patient, only the first isolated strains were included. All the M. morganii strains were identified by VITEK-2 automatic bacterial identification system (BioMérieux, France). Isolates were cultured on Columbia agar with 5% sheep blood (BioMérieux, France) at 37 • C in 5% CO 2 atmosphere for 16 to 18 h.

Conjugation Experiments
Conjugation experiment was performed to determine the transmissibility of carbapenem-resistant genes. The same amount (1 × 10 7 CFU/mL, counted using the Sysmex UF-1000i TM Automated Urine Particle Analyzer; Tokyo, Japan) of mid-logarithmic phase donor (strain zy_m28 and nx_m63) and recipient cells (E. coli C600) were mixed in 200 µL LB in 96-well plates. After mating for 6 h at 37 • C, 20 µL mixed cultures were spread on LB agar containing 1 µg/mL meropenem plus 100 µg/mL rifampin. The conjugation frequency was calculated as transconjugants divided by number of donors. All experiments were carried out three times. Species identification, antimicrobial susceptibility testing and resistance genes detection were further performed on the transconjugants.

Plasmids Assembly, Comparation and Phylogenetic Analysis
Plasmids were assembled using plasmidSPAdes (Antipov et al., 2016). The scaffolds were reordered using Ragout v2.3 (Kolmogorov et al., 2014) with reference to the most similar plasmid sequence recognized by BLASTN. The gaps were filled using PCR method (Primers and thermal conditions are presented in Supplementary Table 1). Plasmids or genetic elements visualization and comparison were conducted using gggenes 5 and Easyfig v2.2.2 (Sullivan et al., 2011). Single-copy orthogroups of assembled plasmid and other closely related plasmids were identified by OrthoFinder v2.3.7 (Emms and Kelly, 2019), and a maximum likelihood phylogenetic tree is then done from the concatenated alignment using RAxML v8.2.12 (Stamatakis, 2014). The phylogenetic tree is displayed and annotated using Evolview v3 (Subramanian et al., 2019).

Statistical Analysis
All the statistical analysis were performed by SPSS 19.0 (IBM Corp., Armonk, United States). Chi-square test or Fisher's exact test was applied to evaluate the differences of antibiotic resistance rates and resistance genes prevalence among the three hospitals. Mann-Whitney U rank sum test was applied to evaluate the differences of antimicrobial resistance genes distribution between different phylogenomic clades. P < 0.05 was considered statistically significant.

Ethical Considerations
This study was approved by Institutional Review Board of The First Affiliated Hospital of Sun Yat-sen University. The study was retrospective and all clinical data were anonymized.

Antibiotic Resistance Rates of Morganella morganii
Common antibiotics had different MIC distributions. Ceftazidime, gentamicin, and trimethoprim-sulfamethoxazole showed classic bimodal distributions. Meropenem, ertapenem, amikacin, etc. showed clear distributions for the sensitive cases. Ciprofloxacin, levofloxacin and tobramycin showed more spread distributions around all the concentration ranges (Supplementary Figure 1). The susceptibility testing results showed that 130 (38.81%), 66 (19.70%), 61 (18.21%), 55 (16.42%), and 47 (14.0%) strains were resistant to trimethoprimsulfamethoxazole, ceftazidime, gentamicin, ciprofloxacin and ceftriaxone, respectively (Supplementary Table 4). Among them, strain zy_m28 from Hospital ZY and strain nx_m63 from Hospital NX showed intermediate resistant to meropenem (MIC = 2 µg/mL). There was no statistical difference of the resistance rates between the two hospitals in Guangzhou (Hospital ZY and Hospital SZY). However, the resistance rates of the ceftriaxone, ceftazidime, cefepime, ciprofloxacin, gentamicin, and trimethoprim-sulfamethoxazole were significantly higher in Hospital NX compared to Hospital ZY and SZY. Forty (11.9%) M. morganii isolates were recognized as multidrug resistance (MDR) strains as they were resistant to three or more classes of antibiotics, and the MDR M. morganii rates were also statistically different among the three hospitals (χ 2 = 10.5, P < 0.05). The MDR rate in Hospital NX was higher than in Hospital ZY. The strain zy_m28 was a MDR strain resistant to ceftriaxone, ciprofloxacin, gentamicin and trimethoprim-sulfamethoxazole.
Other PMQR genes qnrA1, qnrB1, oqxA, and oqxB were also detected. Sixty (17.9%) M. morganii strains carried at least one of the extended-spectrum β-lactamase (ESBL) resistance genes, and 103 (30.8%) carried at least one of the PMQR genes. There was no statistical difference in ESBL resistance genes prevalence among the three hospitals, while prevalence rates of PMQR genes among the three hospitals were statistically different (χ 2 = 9.3, P < 0.05). PMQR genes were more prevalent in Hospital NX than in Hospital ZY and SZY. Thirty-four (10.1%) M. morganii strains carried both ESBL and PMQR resistance genes, and bla TEM-104 coexistence with qnrD1 or aac(6 )Ib-cr were the most common combinations (Supplementary Table 5).

Phylogenomic Analysis and Population Structure of Morganella morganii
Two strains zy_m28 (GCA_014333515.1) and nx_m63 (GCA_014283905.1) harboring carbapenem-resistant genes were whole-genome sequenced. Other M. morganii genomes were accessed from NCBI assembly database and SRA database (Supplementary Figure 2). A total of 20663 filtered cgSNPs were generated by Roary v3.11.2 (Page et al., 2015). Phylogenomic analysis of the two and current public M. morganii genomes showed that these M. morganii clustered into multiple clades. It is interesting that clade 1 (showed in blue) associated with multiple resistance genes containing bla KPC-2 , bla IMP-10 , bla IMP-27 , bla OXA-48 , bla OXA-181 , bla NDM-1 , bla NDM-7 , bla GES-5 , mcr-1, and mcr-5, seemed to be widely spread in multiple global regions, including Asia, North America, South America, Europe, Australia and South Africa (Figure 1 and Supplementary  Table 7). Mann-Whitney U rank sum test showed that antimicrobial resistance genes (ARGs) in clade 1 was more than ARGs in non-clade 1 (P < 0.001) (Supplementary Figure 3A). Some noticeable MGEs, such as bla KPC-2 -carrying IncP-6 plasmid, bla NDM-1 -carrying IncN2 plasmid, bla NDM-7 -carrying IncX3 plasmid, bla OXA-48 -carrying IncL/M plasmid, bla KPC-2carrying IncR plasmid, mcr-1-carrying IncX4 plasmid, and MGIVflInd1 were identified in M. morganii for the first time in this interesting clade (Supplementary Tables 7, 8). rhierBAPS population structure analysis showed that M. morganii could be classified into ten sequence clusters (SCs) based on the cgSNPs. The interesting clade 1 associated with multiple resistance genes (Figure 1, blue clade) was classified as SC3 and SC5. PopPUNK population structure analysis showed that clade 1 could be classified as combined cluster 2, 7, 8, and 12 (Supplementary Table 7). PopPUNK offered more detailed population structure groups than rhierBAPS, which may be resulted from PopPUNK considered both the accessory distance and core distance (Supplementary Figure 3B). MDR strain zy_m28 was classified as SC3. Growth curve showed its greater fitness advantage in LB medium and M9CA minimal medium than strain nx_m63 classified as SC1 and larger maximum population capacity (Ym) than a plasmid-free control strain zy_m3 (Supplementary Figures 3C,D). There was no statistical relationship between population structure and geographical origin (χ 2 -test).
To compare the variations of current SGI1-related GIs, we then compared the genetic structure and conducted a phylogenetic analysis for SGI1-like sequences (Supplementary  Figure 4). In general, these GIs could be divided into three clades related to SGI1 family, AGI1 family and PGI2 family, respectively. The backbone regions in all GIs were relatively conservative, however, deletion, insertion and inversion events still happened (SGI1 family). Notably, most SGI1-like sequences were found in S. enterica and P. mirabilis, but it has now also been described in A. baumannii, E. coli and E. cloacae (AGI1 and PGI2 family). PGI2 family showed significant variations in MDR regions, bringing challenges to the control of antimicrobial resistance ( Figure 2B). PGI2-zym28 showed high identities with PGI2-C55 of animal origin (MK847915.1), suggesting  horizontal transfer of PGI2 variants facilitated the dissemination of antimicrobial resistance.
IncX3 plasmids are prevalent worldwide (Roer et al., 2018). Genetic structures of IncX3 plasmids in different species were compared, and a phylogenetic tree was also constructed. Phylogenetic tree of IncX3 plasmids showed minor genetic variabilities. There were three main types of transposons disseminated in IncX3 type plasmids, with one mainly associated with bla NDM , one with bla OXA-181 , and the other linked to bla KPC and ESBL-encoding genes (Figure 4). bla NDM genes were located on Tn125-like transposons (ISCR21-groL-groS-cutA-dsbD-trpF), while bla OXA-181 genes were located on the IS26-flanked transposons as described above. These transposons always integrated into the IncX3 backbone adjacent to umuD gene and flanked by Tn2 at the other side. IncX3 plasmids harboring carbapenemase genes were frequently described in different species, suggesting it is an important medium for the spread of carbapenemase genes. This poses a challenge for antimicrobial resistance control.

Genetic Context of Mobile Genetic Elements in Morganella morganii Strain nx_m63
Strain nx_m63 was recovered from the urine of a 57-yearold Chinese patient with deep vein thrombosis and urinary tract infection, and a MDR E. coli strain was also cultured from the same sample. WGS analysis showed that nx_m63 harbored a bla IMP-1 -carrying IncP-1β type plasmid pNXM63-IMP (MW150990.1).
pNXM63-IMP carried both Tn402-like integron and Tn21like integron that were inserted into the downstream of traC2 and trfA, respectively. bla IMP-1 was located on the Tn402-like type 1 integron without accompanying other gene cassettes ( Figure 5A). We then compared the genetic structure of current bla IMP -carrying Tn402-like integrons and found this type of integrons could both integrate into chromosomes and plasmids in various species (Figure 5B). In comparison to a similar bla IMP-1 -carrying IncP-1β plasmid pA22732 (KJ588780.1), the tni operon of the Tn402-like type 1 integron in pNXM63-IMP was complete. Except for bla IMP-1 , other resistance genes, including ESBL gene bla CTX-M-14 (CP031122.1) and carbapenemase gene bla VIM-1 (CP040126.1) have also been found in Tn402-like type 1 integrons. We should closely monitor these flexible carriers of resistance genes.

DISCUSSION
In this study, we collected all the M. morganii strains isolated from June 2014 to June 2020 in three tertiary hospitals in China, regardless of sample types and department sources, to provide an overview of molecular epidemiology of M. morganii. The antibiotic resistance rates and prevalence of resistance genes FIGURE 4 | Phylogenetic analysis of IncX3 plasmids combined with the comparisons of their resistance regions. Branch length represents genetic variabilities. Genes and ORFs are shown as arrows without orientations. The colors of symbols beside leaf labels indicate bacterial hosts of these plasmids and leaf label colors represent geographic origins. pZYM28-OXA-181 in this study is noted with a green hollow rectangle. Cointegrate plasmids consisting of IncX3 and other type of plasmid backbones are noted with red *. The resistance regions comparisons were drawn with gggenes aligned with bla NDM .
from Hospital NX in Northwest China were generally higher than those of Hospital ZY and SZY in Southeast China, which may be resulted from the differences in economic and medical conditions, prescribing behaviors and differences in knowledge of antimicrobial clinicians (Zhen et al., 2019). We also found that PMQR and ESBL-encoding genes usually coexisted, causing multidrug resistance, consistent with the research of Briales et al. (2012). Conjugation experiment confirmed the co-transfer of bla OXA-181 and qnrS1 in our study.
Phylogenomic and population structure analysis of public M. morganii genomes showed that clade 1 (rhierBAPS SC3 and SC5) associated with multiple resistance genes seemed to be widely spread in multiple global regions. The resistance genes pattern of this clade was largely divergent from other clades. Various putative IMEs or ICEs, and noticeable carbapenem and polymyxin -resistant plasmids were identified in the clade. Growth curve showed that strain zy_m28 belonging to rhierBAPS SC3, had greater fitness advantage than strain nx_m63 belonging to rhierBAPS SC1 and even had larger maximum population capacity (Ym) than a plasmid-free control strain zy_m3. A reasonable guess is that these subclusters may be more suitable hosts to hold acquired resistance genes than other clusters because of low fitness cost and may cause resistance issue in the future. Further studies and more samples are needed to demonstrate this hypothesis.
Mobile genetic elements are essential in horizontal transfer of resistance genes. In this study, we identified a PGI2 variant PGI2-zym28 carrying bla CTX-M-3 and aac(6 )-Ib-cr. To the best of our knowledge, this is the first report of PGI2 variant in M. morganii. PGI2-zym28 showed high identities with PGI2-C55 initially found in a P. mirabilis strain isolated from a Chicken in 2018 from Shandong, China (Lei et al., 2020). The result suggests that Other information, such as accession number, strain, sequence type, host, country, year and sequences with the same genetic pattern are also shown. The information of the Tn402-like type 1 integron found in pNXM63-IMP in this study is shown in green. Genes and ORFs are shown as arrows, which indicate their orientations of transcription. Shared regions with 75-100% identity are indicated by gradual shading. The picture was drawn with Easyfig v2.2.2.
PGI2-like sequences may be transferred horizontally between P. mirabilis and M. morganii among humans and animal sources. Further studies are needed to monitor the spread of PGI2-like sequences in clinical settings and understand their effects on clinical antimicrobial resistance.
An IncX3 plasmid pZYM28-OXA-181 harboring bla OXA-181 and qnrS1 was found to coexist with PGI2-zym28 in strain zy_m28. bla OXA-181 , a variant of bla OXA-48 family, is showing an increasing prevalence since first reported in India in 2007 (Qin et al., 2018). While bla OXA-181 has been found in different plasmid types, such as IncN (McGann et al., 2015), IncT (Villa et al., 2013) and IncX3 (Qin et al., 2018), bla OXA-181 -harboring IncX3 plasmids are the most prevalent (Roer et al., 2018;Feng et al., 2019). There are two main epidemic types of IncX3 plasmids, one spreading bla OXA-181 and qnrS together on an IS26-flanked composite transposon and the other spreading bla NDM and bla SHV-12 together on a Tn125-like transposon. To the best of our knowledge, this is the first report of IncX3 type plasmid carrying bla OXA-181 in M. morganii. The spread of bla OXA-181 -harboring IncX3 plasmid in clinical M. morganii strains may further limit clinical therapeutic solutions.
Another noticeable plasmid, an IncP-1β type plasmid pNXM63-IMP carrying bla IMP-1 on a Tn402-like class 1 integron, was found in the M. morganii strain nx_m63 for the first time. The Tn402-like integrons carrying bla IMP were found in multiple species including P. aeruginosa (Xiong et al., 2013), E. cloacae (Wang et al., 2015), and K. pneumoniae (Jiang et al., 2017). Interestingly, they were often reported in Asia especially in China. Although the bla IMP-1 -carrying Tn402-like integron found in this study did not carry other resistance gene cassettes, it is worth noting that with the help of various transposases, more complex multidrug resistant Tn402-like integrons carrying both bla IMP-45 and bla VIM-1 are emerging (unpublished data, CP040126.1).
This study demonstrates that a clade of M. morganii is prone to acquire resistance genes, and multidrug resistant M. morganii are increasing by harboring a variety of MGEs including two newly discovered ones in the species. The variation and evolution of these MGEs may bring more extensive and challenging antimicrobial resistance issue. Horizontal infection control strategies should be considered in tackling antimicrobial resistance in M. morganii and other pathogens.

DATA AVAILABILITY STATEMENT
The original contributions presented in the study are publicly available. This data can be found here: https://www.ncbi.nlm. nih.gov/genbank/, under the accession numbers, MW080367.1, MW080368.1, and MW150990.1.

FUNDING
This study was funded by the National Natural Science Foundation of China (81772249 and 81871703) and the Guangdong Science and Technology Program (No. 2019A030317003). The funders did not participate in study design, data collection and interpretation, or the decision to submit the work for publication.