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% CO₂ atmosphere for 16 to 18 h.

Minimum inhibitory concentrations (MICs) of piperacillin-tazobactam, ceftriaxone, ceftazidime, cefepime, aztreonam, ciprofloxacin, levofloxacin, gentamicin, tobramycin, amikacin and trimethoprim-sulfamethoxazole were analyzed by VITEK-2 drug sensitivity analysis system (BioMérieux, France). MICs of meropenem (ApexBio, United States) and ertapenem (Menlunbio, China) were tested using micro-broth dilution method according to the guidelines of 2019 Clinical and Laboratory Standards Institute (CLSI) criteria (CLSI, 2019). Strain Escherichia coli ATCC 25922 was used as the quality control strain.

DNA templates were extracted by the boiling method as previously described (Sun et al., 2014). Polymerase chain reaction (PCR) was performed to detect antimicrobial resistance genes, including β-lactam resistance genes (bla_KPC, bla_NDM, bla_OXA–48, bla_VIM, bla_IMP, bla_CTX–M, bla_TEM, and bla_SHV), and plasmid-mediated quinolone resistance (PMQR) genes [qnrA, qnrB, qnrC, qnrD, qnrS, aac(6′)-Ib-cr, qepA, and oqxAB] by using primers as described previously (Xiao-Min et al., 2014; Szabó et al., 2018; Cai et al., 2019). Primers synthesis and positive PCR products sequencing were conducted by Sangon company, Shanghai¹. BLASTN² was used to align the sequencing results. Primers and thermal conditions are presented in Supplementary Table 1.

Conjugation experiment was performed to determine the transmissibility of carbapenem-resistant genes. The same amount (1 × 10⁷ 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.

Two strains harboring carbapenem-resistant genes were whole-genome sequenced. Genomic DNA was extracted using a MiniBEST Bacteria Genomic DNA Extraction Kit (TaKaRa, Dalian, China). Chromosomal libraries with a 300 bp insert size were prepared as previously described (Cai et al., 2019) and all barcoded libraries were sequenced on a NextSeq 500 platform (Illumina Inc., San Diego, CA, United States). To construct a current phylogenomic tree, raw reads in fastq format or pre-assembled sequences in fasta format were downloaded from NCBI database for all publicly available M. morganii isolates (Supplementary Figure 2). Paired-end raw reads were filtered using FASTQ preprocessor Fastp v0.12.5 to exclude library adapter and low quality reads (Chen et al., 2018) and de novo assembled with Unicycler v0.4.9b (Wick et al., 2017). Then, the scaffolds were annotated with Prokka v1.14.6 (Seemann, 2014). Antimicrobial resistance genes and plasmid types were identified by ABRicate v0.8.13³. ICEs and MGIs were identified ICEberg 2.0 (Liu et al., 2018). Pan-genome analysis was done using Roary v3.11.2 (Page et al., 2015) and core genome single-nucleotide polymorphisms (cgSNPs) were extracted using SNP-sites (Page et al., 2016). cgSNPs were filtered using VCFtools v0.1.17 (Danecek et al., 2011). After filtering, 20663 cgSNPs out of a possible 32774 cgSNPs from 1425 core orthologous genes were kept. A maximum likelihood phylogenomic tree of 246 M. morganii genomes was constructed by RAxML v8.2.12 (Stamatakis, 2014) using the filtered cgSNPs. Population structure based on Bayesian analysis was then identified using rhierBAPS v1.0.1 (Tonkin-Hill et al., 2018) using the filtered cgSNPs. Besides, population structure analyzed by PopPUNK is also provided as a supplement (Lees et al., 2019). The phylogenetic tree is displayed and annotated using iTOL v5 (Letunic and Bork, 2021).

The GI was predicted by IslandViewer 4⁴. The scaffolds assembled by Unicycler v0.4.9b (Wick et al., 2017) were reordered using Ragout v2.3 (Kolmogorov et al., 2014) with reference to the most similar GI sequence recognized by BLASTN. The gaps were filled using PCR method (Primers and thermal conditions are presented in Supplementary Table 1). Then, the complete GI sequence was annotated with Prokka v1.14.6 (Seemann, 2014). To compare the variations of current SGI1-related GIs, single-copy orthogroups of GI sequence found in this study and other pre-assembled GIs from NCBI database 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 iTOL v5 (Letunic and Bork, 2021). Genetic elements of PGI2 family were visualized and compared using Easyfig v2.2.2 (Sullivan et al., 2011).

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 gggenes⁵ 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).

Newly sequenced genomic island and plasmids were deposited in GenBank under the accession numbers, MW080367.1, MW080368.1, and MW150990.1, respectively.

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.

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.

A total of 335 M. morganii clinical isolates were collected from two hospitals in Guangzhou, Southeast China and one hospital in Yinchuan, Northwest China during June 2014 to June 2020. In general, urine (26.6%, 89/335), wound secretion (14.0%, 47/335), sputum (11.9%, 40/335) and shunt fluid (10.2%, 34/335) were the top four most frequent sample types of M. morganii clinical isolates, while the sample-type distributions among the three hospitals were statistically different (χ² = 85.0, P < 0.001) (Supplementary Table 2). These strains were mainly isolated from hepatic-biliary-pancreatic surgery (14.0%, 47/335), intensive care unit (11.0%, 37/335), general surgery (9.6%, 32/335), and burn center (6.0%, 20/335) (Supplementary Table 3).

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 trimethoprim-sulfamethoxazole, 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 (χ² = 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.

All isolates were detected by PCR for β-lactam resistance genes and PMQR genes. PCR results showed that 41 (12.2%), 27 (8.1%), 80 (23.9%), and 40 (12.0%) M. morganii strains carried bla_TEM, bla_CTX–M, aac(6′)Ib-cr and qnrD1, respectively (Table 1). Among bla_TEM and bla_CTX–M, bla_TEM–104 and bla_{CTX–M–162} were the most popular subtypes. Two carbapenem-resistant genes bla_OXA–181 and bla_IMP–1 were detected in strain zy_m28 and nx_m63, respectively. MDR strain zy_m28 carried bla_OXA–181, bla_TEM–1, bla_CTX–M–3, qnrS1 and aac(6′)Ib-cr at the same time. 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 (χ² = 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).

Carbapenem-resistant genes bla_OXA–181 and bla_IMP–1 could be successfully transferred from strain zy_m28 and nx_m63 to the E. coli C600. Conjugation frequencies of bla_OXA–181 and bla_IMP–1 plasmids were (2.4 ± 7.9) × 10^–4 per donor cell and (1.2 ± 3.8) × 10^–4 per donor cell, respectively. Susceptibility testing results showed that transconjugants zy_m28-E. coli C600 were resistant to meropenem, ciprofloxacin, and transconjugants nx_m63-E. coli C600 were resistant to meropenem (Supplementary Table 6). Transconjugants zy_m28-E. coli C600 were found to contain bla_OXA–181, qnrS1 but without bla_TEM–1, bla_CTX–M–3 and aac(6′)Ib-cr. Transconjugants nx_m63-E. coli C600 were found to contain bla_IMP–1 as expected. Conjugative transfer of carbapenem-resistant genes bla_OXA–181 and bla_IMP–1 could be a threat to public health worldwide.

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–2-carrying 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 (χ²-test).

Strain zy_m28 was isolated from the right pelvic drainage fluid of a 28-year-old Korea patient with Crohn’s disease and intestinal infection, and Enterococcus raffinosus strain was also cultured from the same sample. WGS analysis showed that two MGEs, PGI2 variant PGI2-zym28 (MW080367.1) and bla_OXA–181-carrying IncX3 plasmid pZYM28-OXA-181 (MW080368.1) coexisted in zy_m28.

Bioinformatics analysis showed that PGI2-zym28 integrated into the chromosome between the trmE and EamA-like transporter family genes. The MDR region in PGI2-zym28 contained one complete class 1 integron carrying dfrA16, bla_PSE–1, aadA2, cmlA1 and aadA1 gene cassettes at the left side, and one partial class 1 integron missing the 5′-CS carrying aac(6′)-Ib-cr, bla_OXA–1, catB3 and arr-3 gene cassettes at the right side, as well as various additional resistance genes including bla_TEM–1, bla_CTX–M–3, fosA3, tet(A), floR, mph(E) and msr(E) connected by IS26 (Figure 2A).

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.

pZYM28-OXA-181 was an IncX3 type plasmid carrying bla_OXA–181 and qnrS1. Resistance genes bla_OXA–181 and qnrS1 were flanked by two same oriented IS26. The same structure was also found in IncF and IncN plasmids (Figure 3). Though the fragment (IS26-IS3000-bla_OXA–181-ISKpn19-Tn3-qnrS-IS26) was relatively conservative, variations still happened. In plasmid pOXA-484_EC-JS316 (CP058621.1), bla_OXA–181 was replaced with bla_OXA–484. While in plasmid pEC2-1 (CP041956.1), the transposon was flanked by IS6 on the 3′ side, joining aac(6′)-Ib-cr, bla_CTX–M–3 and bla_TEM–1 with the help of some transposons.

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.

Strain nx_m63 was recovered from the urine of a 57-year-old 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 Tn21-like 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.