Clinical history

Four patients aged 36–77 years were admitted to the orthopedic ward of Peking Union Medical College Hospital between July and August, 2019, and presented with cervical spondylosis, lumbar spinal stenosis, lumbar disc herniation, and thoracolumbar kyphosis. The patients underwent spine surgery with internal fixation (Figs. 1 and 2; Table 1). Three patients developed a fever after surgery between August 6 and 15, 2019, with surgery-fever intervals of 4–12 d. Despite the initial absence of fever in the fourth patient, A. hydrophila was isolated from their drainage fluid 6 d-post surgery. A. hydrophila alone was isolated from the blood or drainage fluid samples of all patients (Fig. 1), suggesting that A. hydrophila might have been the causative agent of the outbreak. Antibiotic treatments administered to all patients mainly included meropenem and vancomycin. The fever of the patient 1 and patient 2 subsided with the meropenem and vancomycin treatments. Despite repeated changes in antibiotic treatment for the third patient, the patient’s fever persisted and the drainage fluid cultures consistently contained A. hydrophila. To control the infection, a reoperation was performed to remove internal fixation and debridement, after which the patient’s temperature was eventually restored. During their stay in the hospital, the patients presented complications including cerebrospinal fluid (CSF) leak, poor wound healing, acute kidney injury, or acute heart failure. All four patients were eventually discharged, with the hospitalization duration being 18–53 d (Table 1).

Identification of isolates and setting cultures

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and SpeciesFinder 2.0 identified all three isolates as A. hydrophila. The ANI of all three strains was > 95% compared to that of A. hydrophila ATCC 7966, which satisfies previously established criteria for assigning the same species. In addition, the evolutionary tree of the core genes of Aeromonas indicated that the three clinical isolates were closely clustered with A. hydrophila (Fig. S2).

A total of 50 environmental samples were collected from the disinfectants, sterile cotton swabs, bone grain, infusion packs, surgical instruments, and handrails of the resident beds in five rooms. None of the samples were culture-positive. Fecal cultures for the four hospitalized patients were negative for A. hydrophila.

Fig. 1

Epidemiology of the A. hydrophila outbreak. Colored text and bars represent the isolate sources

Fig. 2

Locations of patients infected with A. hydrophila in the hospital

Table 1 Clinical records of patients in the outbreakSequencing and genomic analysis

We performed sequencing of the 19B23009, 19W05620, and 19W06265 genomes, and determined that their total genome sizes were all 4.88 Mb. The three de novo assemblies resulted in draft genomes composed of few scaffolds (140, 163, and 146) with high N50 values (387,668 bp, 387,667 bp, and 387,668 bp, respectively). The three draft genomes had an average GC content of 61%. Variation in the GC content of the genome is shown in the inner circle of Supplement Figure S1.

MLST of isolates and pairwise single nucleotide polymorphisms in the core genome

MLST analysis of all A. hydrophila isolates revealed that they belonged to a novel sequence type (ST1172, deposited in the PubMLST database: https://pubmlst.org/aeromonas/). We compared the loci of housekeeping genes of ST4523 (gyrB, groL, gltA, metG, ppsA, and recA: loci 801, 331, 334, 337, 361, and 355, respectively) with ST466 (loci 338, 331, 334, 337, 361, and 355) and found that gyrB was mutated from sequence 1 to 2. In the phylogenetic tree, ST1172 formed clusters with ST251 and ST516 (Fig. 3). The multiple sequence alignment of strains ST251, ST516, and ST1172 is shown in Supplementary Figure S3.

Fig. 3

Maximum likelihood trees constructed from concatenated nucleotide sequences (gltA-groL‐gyrB‐metG‐ppsA‐recA) using MEGA X. ST1172 formed clusters with ST516 and ST251

Antibiotic resistance profile and phenotypic ESBL detection

All three isolates were susceptible to amikacin and levofloxacin but resistant to imipenem, piperacillin/tazobactam, ceftriaxone, cefuroxime, and cefoxitin (Table 2). Isolate 19W05620 had increased ceftazidime resistance (minimum inhibitory concentration ≥ 64 µg/mL). All three isolates possessed the same chromosomally encoded β-lactamases, including blaOXA-724 (β-lactamase), imiH (metallo-β-lactamase [MBL]), and blaMOX-13 (AmpC) in plasmids (Table 3).

Table 2 Antimicrobial susceptibility patterns of three A. hydrophila isolatesTable 3 Antimicrobial genotype prediction of the three A. hydrophila isolatesDistribution of virulence determinants

We screened virulence factors related to the pathogenicity of A. hydrophila using the VFDB and detected the toxin factors aerA/act, ahh1, ast, hlyA, hemolysin III, and thermostable hemolysin along with the T3SS secretion system and many other adherence factors, such as flgC and flaB (Supplement Table S1). The expression of the secretion system (86 items), toxin (12 items), immune evasion (1 item), serum resistance (1 item), and lps rfb locus (Klebsiella) were compared in Supplement Table S1.