On August 25, 2023, the hospital’s Xinglin Real-Time Infection Monitoring System detected four patients in the cardiovascular surgical ICU who tested positive for CRAB. Subsequent screening identified two additional patients with CRAB on August 26 and 29, 2023. Based on clinical manifestations, imaging findings, and laboratory test results, all cases were diagnosed as CRAB lower respiratory tract infections. The diagnosis of nosocomial infection followed the “Diagnostic Criteria for Hospital Infections (Trial)” issued by the Ministry of Health (10). The diagnostic criteria for CRAB lower respiratory tract infections should simultaneously meet the following three items. 1. The patient must meet at least one of the following: (1) Fever; (2) Elevated total white blood cell count and/or neutrophil percentage; (3) New or progressive imaging abnormalities (pulmonary infiltrates or cavities). 2. At least two of the following criteria must be met: (1) New emergence of purulent sputum, change in sputum characteristics, increased respiratory secretions, or increased suctioning frequency; (2) New onset or worsening of cough, dyspnea, or tachypnea. (3) Wet rales or bronchial breath sounds. (4) Decreased oxygenation index (PaO₂/FiO₂), increased oxygen therapy requirement. 3. Isolation of CRAB as the predominant organism from sputum or lower respiratory tract specimens.

By reviewing clinical data and conducting on-site epidemiological investigations, we obtained preliminary information on the number of infections, the three-dimensional distribution (time, place, and person), infection status in previous and concurrent periods, and possible transmission routes. We defined cases as patients in our ward whose submitted specimens tested positive for CRAB. To expand the case search, we screened all other patients in the ward from August 1 to August 31 by collecting sputum and other relevant specimens in combination with clinical symptoms. Case verification was conducted based on clinical symptoms, physical signs, and laboratory findings.

On the day of sampling, a sterile phenol red dextrose broth medium was prepared. Each glass test tube was filled with 2.5 mL of phenol red dextrose broth, into which one meropenem susceptibility test disc (10 μg) and one vancomycin susceptibility test disc (30 μg) were placed for the initial screening of CRAB. For sampling environmental surfaces and palms, sterile flocked swabs were moistened with sterile saline and used to swab extensively in both horizontal and vertical directions. Samples were promptly transferred to culture tubes and incubated at 35 °C for 48 h in a constant-temperature incubator. Test tubes in which the phenol red broth retained its red color were considered negative, while those exhibiting a yellow-turbid appearance were selected for quadrant streaking on blood agar plates. Following incubation at 35 °C for 24–48 h, bacterial identification and antimicrobial susceptibility testing were conducted. CRAB was defined as Acinetobacter baumannii resistant to any carbapenem antibiotic.

A comprehensive survey was conducted across 146 sampling sites, which included environmental surfaces within the ICU ward, the soiled utility room, and doctors’ offices, as well as the hands of medical personnel. The distribution of these sampling sites was as follows: 47 sites were associated with bed units (including bed rails, bedside tables, bed foot tables, call buttons, height adjustment panels, and curtains) and medical equipment surfaces (such as ventilator controls, electrocardiogram monitors, infusion pump knobs, sphygmomanometers, and stethoscopes). Additionally, 7 sites were sampled from work attire, 7 sites from doctors’ offices and nurse station areas (including mouse and keyboard, desk, telephone, and medical record folders), and 5 sites from the soiled utility room (including washing machine handles and interior, bedpans, urinals, and mops). Furthermore, 28 sites were sampled from sink areas located in the ICU, soiled utility room, and doctors’ offices, while 12 sites were from the environment surrounding infected patients (including isolation gown surfaces, skin around endotracheal tubes, flooring, and air conditioning supply and return vents). Moreover, 21 sites were sampled from hand surfaces, 13 sites from urine-bag valve switch surfaces, and 6 sites from public items within the ward. Concurrently, oropharyngeal and nasopharyngeal swabs were collected from ward staff using the same culture medium for microbial screening.

The VITEK2-Compact automated microbial identification and antimicrobial susceptibility testing system (Bio-Merieux, France) and corresponding detection cards were used for the experiments. For drugs where the MIC range determined by the instrument did not meet the CLSI breakpoint criteria, disk diffusion or E-test strips were used. Antimicrobial susceptibility test results were interpreted according to the 2022 CLSI standards (11).

After pure culture, colonies were resuspended in buffer solution (20 mmol/L Tris–HCl, 5 mmol/L EDTA) and nucleic acids were extracted using the MagNA Pure 24 System. DNA concentration was quantified using the Qubit 3.0 Fluorometer. Genomic DNA was extracted, and libraries were constructed using the Illumina DNA Prep Tagmentation Kit. Sequencing was performed on the Illumina Miseq platform with a depth of ≥100 ×.

Conduct quality control on raw reads using fastp to eliminate low-quality bases, resulting in clean reads. Subsequently, perform de novo assembly utilizing the assembly module of the EToKi tool, assigning strain numbers to the assembled contigs. Using the sequence from SAMN 40446890 as a reference, a maximum likelihood phylogenetic tree was constructed based on 2,691 core genome SNPs using EToKi (available at https://github.com/zheminzhou/EToKi). The resulting output was uploaded to the iTOL online platform (https://itol.embl.de) for graphical enhancement and annotation. Plasmid sequences were identified from the assembled genomes using the PlasT module of KleTy (DOI: 10.1186/s13073-024-01399-0; accessible at https://github.com/Zhou-lab-SUDA/KleTy). The ISFinder tool was employed to predict insertion sequences and transposons. Within R (version 4.4.3), the heatmap package was utilized to visualize the core SNP frequency matrix, while iTOL was used to visualize the phylogenetic tree and annotate sample information.

During August 2023, the cardiovascular surgical ICU admitted 105 patients, among whom six developed CRAB lower respiratory tract infections, with an incidence rate of 5.71%. All six patients had undergone surgery and mechanical ventilation. Patient A was the first to test positive for CRAB in endotracheal aspirate (ETA) specimens on August 4. On August 22, three additional patients tested positive for CRAB in the ETA. Subsequent screening detected CRAB in two more patients on August 26 and 29. Of these six patients, four were male and two were female, aged between 34 and 69 years, with hospital stays ranging from 29 to 67 days (Table 1).

In 2022, among 96 patients, there was 1 case of CRAB lower respiratory tract infection, with an incidence rate of 1.04%. From July 1 to July 31, 2023, among 89 hospitalized patients, 2 cases of CRAB lower respiratory tract infection occurred, with an incidence rate of 2.25% (Figure 1). The six patients had overlapping hospitalization periods and were detected within a short time frame. The cardiovascular surgical ICU has 14 beds, including one single room, one double room, and one 11 - bed ward. The six patients were located in beds 2, 5, 8, 11, 12, and 14, with some beds in proximity. Five of the six patients were in the 11 - bed ward, while the remaining patient was in the double room (Figure 2).

A total of 146 environmental and hand hygiene samples were collected, with 21 CRAB isolates detected, yielding a positive rate of 14.38%. During active patient care, 19 isolates were detected on bed units, medical equipment surfaces, ICU sink areas, and staff hands. Specifically, 2 isolates were found on the surfaces of urine bag drainage valve switches of infected patients, 3 on medical equipment surfaces, 1 on sphygmomanometers and stethoscopes, and 1 on isolation gowns. After environmental disinfection, 1 isolate was detected on the surfaces of bed units and medical equipment of non - infected patients, and another on the surface of a urine bag drainage valve switch. Notably, CRAB was not detected in any of the 38 oropharyngeal or nasopharyngeal swabs collected from 12 nurses, 12 doctors, and 2 orderlies within the ward.

CRAB strains from the environment showed resistance to most antibiotics. Only two strains had intermediate susceptibility to amikacin, and two were sensitive. In sputum samples, the strain from patient B had intermediate susceptibility to levofloxacin and was tigecycline - susceptible. The other five patients’ strains had consistent drug - susceptibility results and showed intermediate susceptibility to tigecycline (Table 2).

Two main phylogenetic clades were identified: one clade included the isolate from patient B and the simple resuscitation bag, and the other clade included all other isolates, indicating a close genetic relationship between the two groups (Figure 3). Multilocus sequence typing (MLST) revealed that all isolates belonged to sequence type ST2. All isolates were identified as lipopolysaccharide outer core locus 1 (OCL 1), with only the isolates from patient B and the simple resuscitation bag identified as capsular polysaccharide locus 7 (KL 7), while all other isolates were KL 196. All strains harbor the antibiotic resistance genes blaOXA-66, blaOXA-23, blaADC-30, blaTEM-1, aadA1, aph(3″)-Ib, aph(6)-Id, armA, ant(3″)-IIa, aph(3″)-Ia, adeC, amvA, sul1 and tet(B). Most strains carried plasmids of types PT_2181, PT_6395, and PT_1900, along with transposons Tn6166, Tn6080, and Tn6206, as well as insertion sequences ISEc29 and ISEc28.

High-quality SNPs (with a frequency and base quality of over 20%) were detected between every pair of strains. SNP differences of < 14 indicated homology (12). Patient B and the simple resuscitation bag had 43 core genome SNP differences, 2024 ~ 2063 differences from other environmental isolates, and 2030 ~ 209 differences from other patients. Differences in SNPs among other patients ranged from 20 to 103. Five patients exhibited SNP differences ranging from 12 to 126 when compared to environmental isolates. Specifically, Patient F (CDC320) and the instrument surface (CDC301) showed 12 SNP differences. Patient E (CDC315) demonstrated 13 SNP differences with the faucet outlet (CDC304), the urine bag drainage valve switch from another patient’s urine collection bag (CDC327), and the hand of a support staff member (CDC329). With the exception of the simple resuscitation bag, all other CRAB strains detected in the environment exhibited SNP differences ranging from 9 to 98 per pair. Among these, seven pairs of isolates had SNP differences of less than 14 (Figure 4).

During the investigation, we promptly guided the implementation of the following hospital infection control measures: (1) assigning dedicated caregivers and specialized medical teams; (2) promoting hand hygiene through hand disinfection and enhancing hand hygiene training and monitoring for orderly and medical staff; (3) replacing ICU sink faucets with touch-free ones and strengthening pipeline disinfection; (4) intensifying cleaning and disinfection of environmental surfaces and medical equipment, increasing the frequency to three times daily; and (5) centrally washing orderlies’ clothes daily, increasing the reserve of isolation gowns, and providing training and assessment on the proper use of protective gear. Following these enhanced interventions, no new CRAB cases were identified in September, and the CRAB cluster was successfully controlled.