One Health approach to characterize antimicrobial resistance in Morganella morganii in humans, freshwater fish and pigs

Abstract

Introduction

Antimicrobial resistance to carbapenems in Morganella morganii is emerging globally.

Materials and methods

Sixty isolates from freshwater fish, four from pig organs and 25 clinical patient isolates were included in the study. Freshwater fish and pork from wet markets in all districts in Hong Kong were obtained in April 2018 and January 2019. Food samples were inoculated to selective agars to look for Morganella morganii. Clinical isolates of M. morganii from three hospitals in Hong Kong were included in 2018–2022. Phenotypic antibiotic susceptibility testing was done by CLSI disc diffusion method. Whole-genome sequencing was performed to establish the antibiotic resistance gene (ARG) profile and phylogenetic relationship.

Results

Phenotypic resistance to fluoroquinolones was significantly higher for animal isolates than human isolates whereas that of aminoglycosides was significantly higher in human isolates. There was no significant difference in other tested antibiotics. There was no significant difference in ARG distribution between human and non-human isolates. Phylogenetically, 12 clinical isolates were clustered with corresponding freshwater fish isolates.

Conclusion

M. morganii isolates that caused human infections were phylogenetically related to those found in freshwater fish in wet markets. There was significant overlap between antimicrobial resistance genes in major antibiotic classes across isolates from humans and isolates from fish.

Introduction

Morganella morganii is a motile, non-lactose fermenting, facultative anaerobic Gram-negative bacillus that belongs to the Proteae tribe in the order Enterobacterales. It is characterized by the presence of inducible chromosomal AmpC and intrinsic resistance to multiple classes of antibiotics. The most common infections it causes are skin and soft tissue infections, genitourinary and intra-abdominal infections, affecting particularly patients with medical comorbidities.^1–3 Previous studies have shown a high 30-day mortality that ranged from 21.2% to 51.7% among bacteraemic patients.^1,3 There is also global concern on the emergence of extensively resistant M. morganii which could carry carbapenemases such as NDM-1, KPC-2, OXA-48 and IMP-1.^4–9

The incidence of M. morganii bloodstream infections was estimated at 9.2 cases per million population and rising in an Australian study.²

Apart from in humans, M. morganii is also found in the environment. It was shown to be residing within free-living amoeba in the environment, with a potential to carry antimicrobial resistance.¹⁰ From a One Health perspective, the convergence of antimicrobial resistance from food animals in human is of significant concern.¹¹ Given the emerging concern with multidrug-resistant M. morganii, a One Health approach would be useful in tracing the source of antimicrobial resistance in this species.

In this study, we examined the phenotypic and genotypic spectrum of antimicrobial resistance in M. morganii isolated from human and food chain animals.

Materials and methods

Bacterial strains

A total of 25 single patient isolates of M. morganii were collected for the period 2018 to 2022 from three public hospitals covering the New Territories East Region of Hong Kong. These isolates were cultured from specimens processed in the Department of Microbiology, Prince of Wales Hospital, Shatin, Hong Kong.

Blood culture was performed with a continuous monitoring blood culture system (BD BACTEC™ Blood Culture System, Becton Dickinson, NJ, USA). When flagged positive, the broth was sub-cultured onto horse blood agar (HBA) and chocolate blood agar (CBA) plates for 18–24 hours of incubation at 37°C in a 5% carbon dioxide environment. For sputum culture, the sample was inoculated to HBA and CBA for 18–24 hours of incubation at 37°C under atmospheric conditions. For urine culture, 10 µL of sample was spread on chromID CPS Elite agar (BioMérieux, Marcy-l’Étoile, France) for 18–24 hours of incubation at 37°C in atmospheric condition. For wound culture, the specimen was inoculated to HBA and CBA for 18–24 hours of incubation at 37°C in a 5% carbon dioxide environment.

The sampling method for food animals has been described before.¹² In brief, 1353 food animal samples, including 182 tilapia, 314 snakehead fish, 309 black carp, heads of 187 bighead carp fish and 361 pig organs, were obtained from wet markets from all 18 districts of Hong Kong from June 2016 to September 2019. All isolates were identified by MALDI-TOF (Bruker Daltonics, Bremen, Germany).

Antibiotic susceptibility testing (AST)

Disc diffusion was performed for the following antibiotics: cefotaxime, piperacillin-tazobactam, cefepime, ertapenem, ciprofloxacin, gentamicin and amikacin. This was performed and interpreted in accordance with CLSI M100 Ed35 (2025).¹³

Clinical data

Clinical details of human infection cases were retrieved from electronic health records. Baseline epidemiological information and the source of infection were reviewed with ethics approval from the Joint CUHK-NTEC Clinical Research Ethics Committee (ref. no. 2020.007). Declaration of Helsinki was adhered to during this study. The requirement on patient consent was waived by the ethics board.

Whole-genome sequencing (WGS)

Whole-genome sequencing was performed as previously described.¹³ Briefly, bacterial DNA was extracted with Tiangen TGuide Smart Universal DNA Kit (Tiangen, Beijing, China), followed by library preparation and sequencing using the Twist 96-plex Library Prep Kit (Twist Bioscience, CA, USA) and the Nextseq 500 high-output system, according to the manufacturer’s protocol (Illumina, CA, USA). Approximately 40× average coverage of 150 bp pair-end sequence data was generated. Genomes were assembled and analysed after performing quality control as previously described.¹⁴ Sequence reads were assessed via FastQC, while low quality reads and adaptors were removed while running de novo assembly via Shovill (v.1.0.9). Quality control and contamination was speculated using Kraken2 (v.2.0.8-beta) and QUAST (v.5.0.2). Detection of antibiotic resistance genes (ARGs) and plasmid replicons were performed using ABRicate (v.1.0.1), matching with the NCBI and PlasmidFinder databases. Sequence types were determined by submitting the genomes to Bigsdb (https://bigsdb.pasteur.fr/). The phylogenetic core SNP tree was generated using Parsnp (v.1.5.3) with default settings and visualized with iTOL (v.7.0). M. morganii ATCC25830 (GenBank no. CP034944.1) was used as the reference strain. Genome assemblies were deposited to NCBI database (BioProject accession no. PRJNA1290033). We compared our strains with a Chinese study involving 60 M. morganii strains from human and animals as most of the food products from the wet markets were imported from China.¹⁴ We downloaded seven genomes from human blood specimen and animals with GenBank accession numbers listed in Table S1 (available as Supplementary data at JAC-AMR Online).

Statistical analysis

Statistical analysis was performed with R v.4.4.1 (R Core Team, 2025). Summary statistics were used for exploratory analysis. Univariable analysis of categorical variables was conducted by Fisher’s exact test. P values <0.05 were considered statistically significant.

Results

Demographics of patients

The median age of the 25 patients was 76.4 years (range 53.0–86.0). Fifteen patients were male and 10 were female. Among the 25 isolates, 22 were obtained from blood cultures and the remaining 3 isolates from urine, wound and sputum respectively. Twelve cases (48%) were polymicrobial infections. Eight (32%) isolates were recovered from patients with urinary tract infections, six (24%) with biliary tract infections, four (16%) intra-abdominal infections, three (12%) with pneumonia, three (12%) with bacteraemia without other sources and one (4%) from a patient who had a soft tissue infection. As for food animal strains, 60 M. morganii isolates were obtained from freshwater fish, whereas four were recovered from pig offals.

Phenotypic antibiotic resistance in human and food animal isolates

The percentage resistance to the tested antibiotics on human, fish and pig isolates is shown in Table 1. Phenotypic resistance to fluoroquinolones (ciprofloxacin and levofloxacin) and aminoglycosides (amikacin and gentamicin) were significantly different in the groups, with higher resistance rate in animal isolates for fluoroquinolones(P < 0.01, P = 0.03) while that for aminoglycosides was significantly higher in human isolates (P = 0.046 and P = 0.01). For beta-lactams including cefotaxime, cefepime, piperacillin-tazobactam and ertapenem, there was no statistically significant difference between the resistance rates in animal or human isolates. Only one isolate was resistant to ertapenem (CUHK_MMOR13BC_18).

Table 1.

Percentage Resistance to eight antibiotics in M. morganii from fish, pigs and human (N= 89 isolates)

	Total (n = 89) n (%)	Fish/pig (n = 64) n (%)	Human (n = 25) n (%)	P value
Piperacillin-tazobactam	11 (12.4%)	5 (7.8%)	6 (24%)	0.07
Cefotaxime	12 (13.5%)	9 (14.1%)	3 (12%)	1
Cefepime	4 (4.5%)	2 (3.1%)	2 (8%)	0.31
Ertapenem	1 (1.1%)	0 (0%)	1 (4%)	0.28
Amikacin	9 (10.1%)	3 (4.7%)	6 (24%)	0.01
Gentamicin	19 (21.3%)	10 (15.6%)	9 (36%)	0.046
Ciprofloxacin	71 (79.8%)	59 (92.2%)	12 (48%)	<0.01
Levofloxacin	46 (51.7%)	38 (59.4%)	8 (32%)	0.03

P value was performed using Fisher’s exact test.

Genome analysis in human and food animal isolates

Table 2 summarizes the molecular characteristics of the 89 M. morganii strains showing their STs, plasmids and ARGs and conferring antibiotic resistance classes. The main ST was ST14 (7.9%, 7/89), while ST8, ST22, ST94, ST112 and ST116, each had five isolates, contributing to 5.6% of all strains).

Table 2.

Molecular characteristics of M. morganii

Molecular characteristics	Total (n = 89) n (%)	Animal (n = 64) n (%)	Human (n = 25) n (%)	P value
Sequence types
ST14	7 (7.9%)	6 (9.4%)	1 (4%)	0.668
ST8	5 (5.6%)	4 (6.3%)	1 (4%)	1
ST22	5 (5.6%)	2 (3.1%)	3 (12%)	0.13
ST94	5 (5.6%)	5 (7.8%)	0 (0%)	0.32
ST96	5 (5.6%)	5 (7.8%)	0 (0%)	0.32
ST112	5 (5.6%)	5 (7.8%)	0 (0%)	0.32
ST116	5 (5.6%)	5 (7.8%)	0 (0%)	0.32
ST21	4 (4.5%)	3 (4.7%)	1 (4%)	1
ST26	4 (4.5%)	1 (1.6%)	3 (12%)	0.06
ST118	4 (4.5%)	4 (6.3%)	0 (0%)	0.57
Other ST (n < 3)	41 (46.1%)	27 (42.2%)	14 (56%)	—
Unknown	3 (3.4%)	1 (1.6%)	2 (8%)	—
Plasmid present	36 (40.4%)	28 (43.8%)	8 (32%)	0.35
Col3M	33 (37.1%)	27 (42.2%)	6 (24%)	0.14
Col156	2 (2.2%)	2 (3.1%)	0 (0%)	1
Col(MG828)	1 (1.1%)	1 (1.6%)	0 (0%)	1
ColpVC	1 (1.1%)	0 (0%)	1 (4%)	1
IncQ1	1 (1.1%)	0 (0%)	1 (4%)	1
ARGs
Aminoglycoside resistance
aadA gene family	40 (44.9%)	32 (50%)	8 (32%)	0.16
aph(3″) gene family	27 (30.3%)	21 (32.8%)	6 (24%)	0.46
aac(6′) gene family	19 (21.3%)	16 (25%)	3 (12%)	0.25
aac(3) gene family	18 (20.2%)	13 (20.3%)	5 (20%)	1
ant(2″)-Ia	11 (12.4%)	9 (14.1%)	1 (4%)	0.27
Trimethoprim resistance
dfrA gene family	41 (46.1%)	32 (50%)	9 (36%)	0.25
Beta-lactamases
DHA gene family	81 (91%)	59 (92.2%)	22 (88%)	0.68
MOR-2	3 (3.4%)	2 (3.1%)	1 (4%)	1
blaOXA-1	11 (12.4%)	8 (12.5%)	3 (12%)	1
blaOXA-10	8 (9%)	8 (12.5%)	0 (0%)	0.1
blaOXA-4	1 (1.1%)	1 (1.6%)	0 (0%)	1
blaCARB2	1 (1.1%)	0 (0%)	1 (4%)	0.28
blaVEB-1	1 (1.1%)	1 (1.6%)	0 (0%)	1
blaTEM-1	10 (11.2%)	7 (10.9%)	3 (12%)	1
blaCTX-M-3	3 (3.4%)	3 (4.7%)	0 (0%)	0.56

P value was performed using Fisher’s exact test.

Thirty-six strains (40.4%) showed the presence of plasmids in their genome and were largely plasmid col3M (33/89, 37.1%). Col3M was reported to carry the qnrD1 gene, conferring resistance to quinolone.¹⁴ Most isolates from human and animals were positive for class C beta-lactamases, namely the bla_DHA family (81/89, 91%), which is intrinsically resistant in M. morganii. Other types of beta-lactamase genes found were bla_OXA-4 and bla_CARB2 genes as well as bla_OXA-1 and bla_OXA-10 that confer cephalosporin resistance (11/89, 12.4% and 8/89, 9%). ARGs related to ESBL were observed for bla_VEB-1 (1/89, 1.1%), bla_TEM-1 (10/89, 11.2%) and bla_CTX-M-3 genes (3/89, 3.4%). All three strains with bla_CTX-M-3 genes were isolated from fish. Aminoglycoside-modifying enzymes, in particular the aph(3″) gene family, was found in 32.8% (n = 21/64) of fish/pig isolates and 24% (6/25) of human isolates. There was no statistical difference in ARG prevalence between human isolates and food animal isolates (Table 2, Table S2). Carbapenemase was uncommon with only one harboured bla_NDM (CUHK_MMOR13BC_18), which corresponded to the ertapenem-resistant isolate in Table 1. The strain also contained ble-MBL gene, which is often noted to be in the vicinity of bla_NDM conferring resistance not only to carbapenems, but also bleomycin.¹⁵

Our core-genome SNP tree showed that one strain (CUHK_MMOR13BC_18) was more distant from the other M. morganii strains (Figure 1). Five clades with a mix of human and animal strains (Clades 1–5) (Figure S1) were identified. Clade 1 belonged to ST21 (4/89, 4.5%), containing one human (4% of 25 strains), one pig and two fish strains (collectively 4.7% of 64 strains). Clade 2 was ST22 (5/89, 5.6%), which consisted of three human and two fish strains and a human strain with unknown ST. The third clade (Clade 3) was ST26 (4/89, 4.5%) and contained a mix of three human and one fish strain. ARGs was less observed in this clade compared with Clades 1 and 2. Clade 4 was largely ST14 (7/89, 7.9%), containing one human and six fish strains with a signature of aminoglycoside resistance genes [aac(3)-IId, aadA5, aph(3″)-Ib, aph(6)-Id]. The last clade, Clade 5, contained the fewest ARGs and was ST8 (four fish strains and a human strain making up to 5.6% of the 89 strains).

Figure 1.

Phylogenetic core SNP tree of 89 M. morganii from human and food animals. Coloured labels are defined in the legend. The isolation year and the presence or absence of antimicrobial resistance genes/plasmid replicons are shown.

While comparing seven human blood isolates and food animal strains from a Chinese study, we noted only three out of seven strains of M. morganii were similar and related to our strains in Clades 1 and 4.¹⁴ The human blood isolate from Guangdong (ZJF894) clustered with our study strains in Clade 1. Another pig strain from stool (171016) was closely related to Clade 4, while the human blood isolates ZJC25 and 229813 clustered with our strains in Clade 4. For a phylogenetic tree incorporating strains from our study and that of the Chinese study, please refer to Figure S2.

Discussion

To our knowledge, most of the literature focuses either on the clinical epidemiology of M. morganii or the prevalence of the species and subspecies in the environment, but has not yet brought both aspects together from a One Health perspective. Our study brings forth the issue of potential cross-host transmission by comparing genome content and AMR phenotypes in clinical and food animal strains that were isolated in a similar time period. The ability of M. morganii to acquire ARGs through mobile genetic elements when it is already intrinsically resistant to a certain number of antibiotics (AmpC) renders grave concern in One Health and AMR, especially when it is also regarded as part of the human normal flora and is ubiquitous in the environment. Despite our low number of human isolates, we selected strains with more severe infection for comparison as these strains were the more worrisome ones when compared with the non-invasive strains. Furthermore, the number of invasive infections has been almost static in Hong Kong with an average of 24–25 cases in our tertiary hospital per annum from 2018 to 2022. This number is also similar to a recent epidemiology study in Beijing where 218 clinical M. morganii isolates were assessed for the period 2016–2023 (i.e. an 8-year period).¹⁶

We noted five clades of our strains contained genomes from both animal and human strains. This indicates their similarities in genomic content where most of them belonged to certain STs (ST21, ST22, ST26, ST14 and ST8). Therefore, cross-host transmission is possible for these clades. M. morganii is conventionally considered a nosocomial pathogen arising from a healthy gut microbiome.¹⁷ This study hence calls for further investigation to understand potential routes of M. morganii infection in humans from its niche. Zoonosis has been demonstrated in non-typhoidal Salmonella, and potential zoonosis via consumption of handling of food animals was also noted in Streptococcus agalactiae ST283 and ESBL-producing Enterobacterales.^12,18–20 Therefore, AMR and source of M. morganii warrants active surveillance to prevent similar infection or outbreaks in the community.

Intrinsic resistance to β-lactams, including ampicillin, penicillin, first- and second-generation cephalosporin, fosfomycin and colistin are known to M. morganii. They are generally susceptible to fourth-generation cephalosporins, carbapenems, quinolones,and trimethoprim/sulfamethoxazole. However, the carriage of ESBL genes and NDM gene in our collection of human and food animal isolates is concerning, as transmission of AMR via the food chain is well-documented across various bacteria. Fortunately, we did not observe the extensive carriage of carbapenemase genes as in the previous studies.^4,9

Recently, the mobile colistin resistance (mcr) gene has also been reported in sewage and food products, highlighting the escalation of AMR in M. morganii.²¹ Fortunately, the mcr gene was absent in our food animal strains.

The Hong Kong Special Administrative Region, China, is a small yet densely populated area and relies on imported food products for daily consumption. As mainland China is the main import supplier for Hong Kong wet markets, from vegetables to seafood and farm animals, it made sense to compare corresponding human and animal strains reported from mainland in this regard. Whereas the mainland China animal strains were distinct from our human cohort isolates, the human strains clustered with our clades, underpinning the significance of cross-border microbial surveillance.

Further study is warranted to characterize the link between human and food animals in the setting of M. morganii. An epidemiological study on the link between food intake and M. morganii invasive infection can be done to establish the potential link between food intake and subsequent infection. Enhanced surveillance on other food, such as beef, mutton, seawater fish or vegetables, should be considered to obtain a more comprehensive view from a One Health perspective.

Conclusion

Similar to all Gram-negative bacteria, M. morganii is more than capable of acquiring antibiotic resistance via horizontal transfer of mobile genetic elements. It is ubiquitous in the environment and is part of the normal human flora. Our study has noted genome similarities in terms of AMR and other molecular characteristics of M. morganii in both food animals and human bacteraemia strains. Thus, its potential in zoonosis and role in AMR necessitates active surveillance of the bacteria before the issue escalates.

Funding

We acknowledge the funding bodies for supporting this project. This project was supported by the Food and Health Bureau, Government of Hong Kong Special Administrative Region: Commissioned Programme for Control of Infectious Diseases (no. CID-CUHK-C, PI:MI) and the Health and Medical Research Fund (HMRF no. 17106212, PI:MI). The funding bodies were not involved in the study’s design, data collection, analysis or interpretation of data or in manuscript writing.

Author contributions

Conceptualization, supervision and administration, M.I.; methodology, data retrieval and genomic analyses, A.L.H.L., C.L., W.S.L. and D.S.; laboratory sample preparation, W.S.L. and D.S. and first draft of the manuscript, A.L.H.L. and C.L. All authors contributed to critical input and editing of manuscript.

Transparency declarations

None to declare.

Supplementary data

Figures S1 and S2 and Tables S1 and S2 are available as Supplementary data at JAC-AMR Online.