Peking University Third Hospital is a 1400-bed tertiary-care teaching hospital. There are six wards in the orthopaedic department with a total of 200 hospital beds. Approximately 10,000 patients received orthopaedic surgeries annually, including spinal, articular and traumatic surgeries.
Background of the outbreak
In April 2017, four patients in orthopaedic ward 2 experienced postoperative SSIs, showing fever, pain, and purulent drainage from the surgical site. Magnetic Resonance Imaging (MRI) showed a large amount of effusion deep in the wound. Wound drainage and/or peripheral blood samples were collected and sent for microbiological analysis. The following day, A. hydrophila was recovered from the pus or drainage samples. One patient also tested positive for A. hydrophila in blood samples.
An outbreak of nosocomial infection due to A. hydrophila was suspected. All patients in the same ward were checked carefully for possible SSIs. To obtain baseline prevalence data, hospital microbiological laboratory databases were reviewed for A. hydrophila isolates identified from 2008 to 2017, which was the period for which complete records were available. Two similar cases were identified in June and July 2016 in orthopaedic ward 2. Physicians reviewed the 6 cases for clinical manifestations and risk factors for the acquisition of A. hydrophila SSIs in detail. The phenotypes and drug resistance patterns of the A. hydrophila isolates were compared by microbiologists. After discussion and combination of the data, infection control measures were taken, and the first environmental investigation was initiated but failed to identify the A. hydrophila isolate. Unexpectedly, in May 2017, a seventh patient developed a postoperative SSI of A. hydrophila in the same ward, and in July 2017, an eighth patient was identified as having a postoperative A. hydrophila SSI and bloodstream infection in orthopaedic ward 6. He was previously hospitalized in orthopaedic ward 2 for two days after operation.
A case was defined as any patient with symptoms and signs consistent with SSIs and drainage or blood culture positive for A. hydrophila since 2008. SSI was defined as erythema, induration, pain, and septic drainage from the surgical site. Postoperative A. hydrophila infection was defined as being nosocomial if the infection was acquired within 30 days after a surgical procedure .
Infection control strategies implemented
An infection control team consisting of surgeons, microbiologists, nosocomial infection experts and head nurses in the hospital was quickly set up to stop the occurrence of similar infections. All healthcare workers in the ward were informed of the possibility of an A. hydrophila SSI outbreak. Heightened infection control measures were enforced, especially contact precautions for all infected patients. The team initiated the first environmental and healthcare worker screening cultures in April 2017, the time of occurrence of the fourth case. The environmental cultures included samples from taps, sinks, drains, toilets, showers, door handles in wards, doctors’ offices, treatment rooms and changing rooms. Work countertops, keyboards, computer mice, stethoscopes, and telephones were also included. The surfaces of pillows, sheets, callers, tables, drainage tubes, drainage bottles, and measuring cylinders in the rooms of patients were also sampled for culture. Blood agar plates (5% Sheep Blood) and China blue agar plates were used for environmental surveillance. However, there were no positive results in the first screening culture. A second environmental screening culture was performed in July 2017. A fish tank in the nurses station caught our attention. The second culture included samples from the decorative stones, filter gauze, inner walls and water in the fish tank.
Identification of isolates and antimicrobial susceptibility testing
The organisms in all the patient samples (n = 10) and tank samples (n = 2) were identified as A. hydrophila/caviae using a biochemical phenotypic identification system (Vitek 2 Compact, bioMérieux, Marcy l’Etoile, France). The organisms were identified as A. hydrophila using matrix-assisted laser desorption ionization mass spectrometry-time of flight (MALDI-TOF MS). DNA sequencing analysis was performed using three pairs of primers, including 16S rRNA, gyrB and rpoB, as described previously [14, 15] to further confirm the identity of the isolates. Antibiotic susceptibility tests were conducted for 12 isolates using the Vitek 2 Compact system and the disk diffusion method. The Clinical and Laboratory Standards Institute (CLSI) M45-A3 criteria were used to define susceptibility and resistance to the antibiotics tested .
Pulsed-field gel electrophoresis (PFGE)
Pulsed-field gel electrophoresis (PFGE) was conducted according to the standardized PulseNet PFGE protocol for pathogenic gram-negative enteric bacteria  with a slight modification. Briefly, whole-cell genomic DNA from lysed cell cultures of each isolate embedded in 1% agarose plugs (Bio-Rad, Richmond, CA, USA) were digested with the restriction enzyme XbaI (New England Biolabs, Ipswich, MA) and separated by electrophoresis through 1% pulsed-field-certified agarose (Bio-Rad) using a CHEF-Mapper instrument. Electrophoretic switch times of 4–40 s were used with a 6 V/cm current and a switch angle of 120° at a constant temperature of 14 °C. PFGE patterns were interpreted using the criteria proposed by Tenover et al . An isolate is designated genetically indistinguishable if its restriction pattern has the same numbers of bands and the corresponding bands are the same apparent size; An isolate is considered unrelated to the outbreak strain if there are seven or more band differences between the outbreak pattern and that of the test isolate.
Detection of virulence genes and multilocus sequence typing (MLST)
Polymerase chain reactions (PCRs) using previously described primers and conditions were conducted to detect the virulence genes encoding heat-stable enterotoxin (ast), haemolysin (ahh1), hemolysin (asa1), cytotoxic enterotoxin (act), enolase (eno) and components of the type III secretion system (ascV and aexT) . Six housekeeping genes (gyrB, groL, gltA, metG, ppsA and recA) were chosen for the MLST analysis according to the method previously reported by Martino. Amplified PCR products corresponding to the expected sizes were sequenced as previously described . Each unique allelic profile, as defined by the allele numbers of the 6 loci, was assigned a sequence type (ST) number. New nucleotide sequences were deposited in the Aeromonas MLST database (http://pubmlst.org/aeromonas).
Whole genome sequencing (WGS) and genome assembly
Resistance to aztreonam and ceftazidime of one A. hydrophila isolate collected from patient 4 developed after one week of empirical therapy with ceftriaxone. WGS of the latter resistant A. hydrophila isolate 5 (AE456) was performed using an Illumina NovaSeq PE150. Illumina PCR adapter reads and low quality reads from the paired-end were filtered by the step of quality control using Readfq (vision 10). All good quality paired reads were assembled using the SOAP denovo (http://soap.genomics.org.cn/soapdenovo.html) and ABySS into a number of scaffolds. We used the ARDB (Antibiotic Resistance Genes Database) to perform the antibiotic resistant genes analyses.