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Air dispersal of multidrug-resistant Acinetobacter baumannii: implications for nosocomial transmission during the COVID-19 pandemic

Aim To describe the nosocomial transmission of Air, multidrug-resistant, Acinetobacter baumannii, nosocomial, COVID-19 Acinetobacter baumannii (MRAB) in an open-cubicle neurology ward with low ceiling height, where MRAB isolates collected from air, commonly shared items, non-reachable high-level surfaces and patients were analysed epidemiologically and genetically by whole-genome sequencing. This is the first study to understand the genetic relatedness of air, environmental and clinical isolates of MRAB in the outbreak setting. Findings Of 11 highly care-dependent patients with 363 MRAB colonization days during COVID-19 pandemic, 10 (90.9%) and nine (81.8%) had cutaneous and gastrointestinal colonization, respectively. Of 160 environmental and air samples, 31 (19.4%) were MRAB-positive. The proportion of MRAB-contaminated commonly shared items was significantly lower in cohort than in non-cohort patient care (0/10, 0% vs 12/18, 66.7%; P<0.001). Air dispersal of MRAB was consistently detected during but not before diaper change in the cohort cubicle by 25-min air sampling (4/4,100% vs 0/4, 0%; P=0.029). The settle plate method revealed MRAB in two samples during diaper change. The proportion of MRAB-contaminated exhaust air grills was significantly higher when the cohort cubicle was occupied by six MRAB patients than when fewer than six patients were cared for in the cubicle (5/9, 55.6% vs 0/18, 0%; P=0.002). The proportion of MRAB-contaminated non-reachable high-level surfaces was also significantly higher when there were three or more MRAB patients in the cohort cubicle (8/31, 25.8% vs 0/24, 0%; P=0.016). Whole-genome sequencing revealed clonality of air, environment, and patients' isolates, suggestive of air dispersal of MRAB. Conclusions Our findings support the view that patient cohorting in enclosed cubicles with partitions and a closed door is preferred if single rooms are not available.

Air dispersal of multidrug-resistant Acinetobacter baumannii: implications for nosocomial transmission during the COVID-19 pandemic Introduction Nosocomial transmission of multidrug Acinetobacter baumannii (MRAB) has been increasingly recognized as a threat to healthcare facilities [1e6]. However, the outbreak of coronavirus disease 2019  has posed further challenge to the control of multidrug-resistant organisms (MDROs) in hospitals. Nosocomial outbreaks of MDROs such as carbapenemresistant A. baumannii have been reported during the COVID-19 pandemic [7e10]. In Hong Kong, we adopted a hospital-based approach to control COVID- 19. By admitting all newly diagnosed cases to airborne infection isolation rooms (AIIRs) in public hospitals, the risk of COVID-19 outbreak in the community as well as in hospital settings are both minimized [11,12]. However, because the priority use of isolation facilities was given to patients with clinical suspicion of COVID-19, the control of MDROs may be jeopardized. Here, we described two clusters of nosocomial MRAB transmission in a medical neurology ward, a conventionally designed general ward with open cubicles during the COVID-19 pandemic. We performed thorough environmental surveillance, including air sampling in patient cubicles and surface swabbing of frequently touched areas and non-reachable surfaces at high levels, such as the exhaust air grills in the ceiling. Whole-genome sequencing was performed to understand the epidemiological relatedness of patients, air and environmental isolates of MRAB, which has important implications for infection control and prevention.

Epidemiological investigation and control of the nosocomial transmission of MRAB
We described two clusters of nosocomial transmission of MRAB in a medical neurology ward, Queen Mary Hospital, a universityaffiliated hospital in which universal admission screening for multidrug-resistant organisms, including vancomycin-resistant Enterococci (VRE), carbapenem-resistant and carbapenemaseproducing Enterobacteriaceae (CRE/CPE), and MRAB are in place [13e15]. MRAB is defined as a strain of A. baumannii demonstrating in vitro resistance to all routinely tested antimicrobial agents (including ampicillin-sulbactam, piperacillin, piperacillin-tazobactam, ticarcillin-clavulanate, cefoperazonesulbactam, ceftazidime, ciprofloxacin, gentamicin, amikacin, tobramycin, cotrimoxazole and imipenem-cilastatin), as previously described [16]. Infection control nurses would screen microbiology laboratory reports for any new cases of MRAB and advise the wards regarding cohort nursing and environmental disinfection. Active surveillance culture (weekly plus at day 14 of hospitalization) was performed in all patients during and after the study period (until the discharge of the last patient). Contact tracing was performed for all hospitalized patients to identify any potential secondary cases. Screening specimens routinely included nasal swab, axillary and groin swabs, and rectal swab; sputum, tracheal aspirate, catheterized urine, drain fluid and wound swabs were collected if available. MRAB colonization days, defined as the number of MRAB-positive patient-days in the wards, was recorded. Cohort nursing with contact precautions and twice-daily environmental disinfection by chlorine dioxide solution 125 ppm (Tristel Solutions, NY, USA) were implemented in a designated cubicle of the ward. Staff put on personal protective equipment (PPE) outside the cubicle and removed the PPE inside the cubicle, and practised hand hygiene immediately after patient care.

Collection of environmental and air samples for MRAB culture
Nine frequently touched and commonly shared items or surfaces including trolley for intravenous medication administration set, cart for changing diapers, blood pressure monitoring machine and cuff, computer on wheels A and B, tablet for inpatient medication order entry, table/desk, computer keyboard, and mouse in nurse station were sampled ( Figure 1). Three high-level non-reachable surfaces including the top surface of the vital sign monitoring screen hanging at the upper wall, walls (area of A3-paper size in landscape orientation) just below the ceiling, and exhaust air grills in the ceiling were also sampled. Flexible premoistened sterile polywipe sponge swabs of 5 Â 10 cm in size (Medical Wire & Equipment, Corsham, UK) were used for environmental sampling as previously described [17e19].
Air samples were collected by the Sartorius MD8 airscan sampling device (Sartorius AG, Germany) with sterile gelatin filters: 80 mm in diameter and 3-mm pore size (type 17528-80-ACD) (Sartorius AG, Germany) as described previously with minor modifications [19,20]. Briefly, the air sampler was placed at the centre of the cohort cubicle for patients colonized or infected with MRAB. One thousand litres of air at a rate of 40 L/ min were collected by a gelatin filter for the first 25 min before changing a diaper, and another 1000 L of air were collected by another gelatin filter for the next 25 min during changing of the diaper. In addition, MacConkey agar with meropenem (2 mg/ mL) was used as settle plates [21] for 25 min and 4-h exposure, according to different manoeuvers of air sample collection (Table 1).

Laboratory investigation and whole-genome sequencing
Patients and environmental specimens were incubated in 2 mL of brain heart infusion enrichment broth with 10 mg/mL vancomycin (Sigma-Aldrich, St Louis, MO, USA) and 0.5 mg/mL meropenem (Hospira, Melbourne, Australia) at 35 C for 18 h. Ten microlitres of the enriched broth were subcultured on to MacConkey agar with 2 mg/mL meropenem for further incubation at 35 C for 48 h in air. Acinetobacter species were identified by matrix-assisted laser desorption/ionization time-offlight mass spectrometry (Bruker Daltonics, Bremen, Germany). Antimicrobial susceptibility tests were performed using the KirbyeBauer disk diffusion method according to the Clinical and Laboratory Standards Institute recommendations, or manufacturer's instructions. Whole-genome sequencing of 10 patient isolates, 11 environmental isolates and two clinical reference strains was performed. DNA extraction was performed using the Qiagen DNeasy Blood and Tissue kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. Library was prepared by the Nanopore native barcoding kit (SQK-LSK109) with a modified protocol [22]. Sequencing was performed on a Nanopore MinION device using an R9.4 flow cell. De novo assembly of raw data reads was performed using Canu version 1.9 and polished with racon version 1.4.10 and medaka 1.3.2. Whole-genome sequence alignment and maximum-likelihood whole-genome phylogenetic tree were performed by the CLC Genomics Workbench version 21.3 (Qiagen, Hilden, Germany) and the IQ-Tree 1.6.12. This study was approved by the Institutional Review Board of The University of Hong Kong/Hospital Authority Hong Kong West Hospital Cluster.
Statistical analysis c 2 -test, Fisher's exact test, and t-test were used as appropriate. A P-value of <0.05 was considered statistically significant.

Epidemiological investigation and control of the nosocomial transmission of MRAB
A total of 11 patients were involved in two clusters of nosocomial transmission of MRAB in the mixed-gender medical neurology ward between 29 th June 2020 and 26 th September 2020 (study period) (Supplementary Table S1). Cases 1 and 2 were epidemiologically linked, whereas cases 3e11 belonged to another cluster. All patients were highly care-dependent with prolonged hospitalization (median: 51 days, range: 18e158). The total and median (range) MRAB colonization days was 363 and 21 (range, 4e102), respectively. Among 11 MRAB patients, colonization rate of axilla and groin was 90.9%, whereas colonization rates in rectal and respiratory specimens (sputum or endotracheal aspirate) were both 81.8%. Seven (63.6%) of 11 MRAB patients had MRAB colonization in three or more body sites. Patients with MRAB colonization of three or more body sites had significantly higher numbers of MRAB colonization days (mean AE standard deviation) than those with two or fewer body sites (48.1 AE 29.1 vs 6.5 AE 2.3, P¼0.029). A total of 158 patients were actively screened for MRAB until the discharge of the last patient on 11 th February 2021. The MRAB attack rate was 7.0% (11/158 patients).

Environmental and air samples for MRAB culture
A total of 160 environmental and air samples were prospectively collected from 3 rd July 2020 to 24 th November 2020 (eight weeks after the diagnosis of the last MRAB patient) (

Whole-genome sequencing
Among the 11 MRAB patients, whole-genome chromosomal DNA sequence of MRAB could be retrieved from 10 patient isolates. The maximum likelihood tree included 10 patient MRAB strains, eight environmental isolates and three air isolates. We included two other clinical MRAB reference strains isolated in 2019 and two MRAB genome sequences from Gen-Bank as the outgroup of the tree (Figure 2). From the maximum likelihood tree, the sequences of the MRAB strains isolated from patients, environmental and air samples clustered    together, suggesting that all patient and environmental isolates shared the same origin with epidemiological linkage.

Discussion
Air dispersal of A. baummanii was previously investigated in clinical settings (Table II) [22e34], mainly focusing on the dissemination of carbapenem-susceptible and carbapenemresistant A. baummanii in intensive care units. In contrast to the previous studies using pulse field gel electrophoresis, repetitive extragenic palindromic sequence polymerase chain reaction, and multi-locus sequence typing, this is the first study using whole-genome sequencing to understand the genetic relatedness of air, environmental and clinical isolates of MRAB in the outbreak setting.
Our findings have important implications in MRAB infection control. The clonal relationship between the air, environmental and clinical isolates not only confirmed the extensive contamination of MRAB during outbreak [18,35], but the presence of air dispersal of MRAB during change of diaper deserves further investigation. 90% of our patients had gastrointestinal colonization of MRAB, thus it was not unexpected to detect MRAB in air samples when diapers were being changed sequentially within a short time. Alternatively, dispersal of MRAB in air may be related to the manipulation of the bed linen, as 80% of patients also carry MRAB on the skin and 1 million skin squamous cells containing viable micro-organisms are shed daily from normal skin [36]. In addition to the detection of MRAB in the air samples, environmental contamination of non-reachable surfaces further supports air dispersal of MRAB. Therefore, to prevent extensive environmental contamination and reduce the transmission of MDROs such as MRAB [37], single-room isolation is strongly recommended.
However, the availability of single-room isolation facilities may be limited in acute hospitals, especially during the COVID-19 pandemic. In Hong Kong, patients with suspected or confirmed COVID-19 were given priority of admission to isolation facilities [11,12]. As illustrated in our case, nosocomial transmission of MRAB occurred readily in a conventionally designed general ward with low ceiling height and open cubicles, where the direction of airflow was from the patient cubicle to the corridor. The same ward design has been shown to contribute to the nosocomial outbreak of COVID-19 by possible airborne transmission from an unrecognized index patient [38]. When we performed environmental sampling of the exhaust air grills, all three exhaust air grills in the corridor were positive for MRAB. This may explain the extensive contamination of the commonly shared items and surfaces in the ward at the onset of this outbreak. Although hand hygiene of healthcare workers remained an important confounding factor for environmental contamination and nosocomial transmission of MRAB, this outbreak occurred during the COVID-19 pandemic, where hand hygiene compliance among healthcare workers increased [39].
When the MRAB patients were cohorted in a designated cubicle, the extent of air dispersal and environmental contamination of the non-reachable surfaces inside the cubicle was significantly increased with the increasing number of MRAB patients during the cohort period. Increasing the number of patients will increase the bacterial burden in the confined clinical area, as illustrated in patients with VRE colonization, where the extent of environmental contamination was associated with the number of positive body sites [40]. Chlorhexidine gluconate bathing may reduce the cutaneous colonization of MRAB [41]. In our patient cohort, the detection rates of MRAB in air and non-reachable surfaces gradually decreased after the initiation of daily chlorhexidine gluconate bathing during hospitalization for the MRAB patients.
There are several limitations to this study. We did not perform air sampling continuously during the cohort period and did not assess the extent of air dispersal of MRAB throughout the day. However, we believe diaper change is a high-risk procedure for air dispersal of MRAB. In addition, the different methods of air and environmental sampling were performed sequentially instead of simultaneously, which makes it difficult to compare the yield of different methods, especially when the number of MRAB patients decreased with time. Nevertheless, our findings emphasized the presence of extensive air dispersal of MRAB in a conventionally designed ward with low ceiling height. The lessons learned are that we may consider adding a portable high-efficiency particulate air filter to the conventionally designed open cubicle, especially when the cubicle is fully occupied by MRAB patients. More importantly, the setting of an air ventilation system with a self-contained air inflow and exhaust within a cubicle, and preferably with the door closed, should be included in the hospital renovation and redevelopment programme.