# Silent transmission of clonal Pseudomonas aeruginosa via duodenoscopes: microbiological and genomic investigation of a prolonged episode

A Cissé, A. Morin-Le Bihan, M. Pagenault, V. Cattoir, S. Reissier, G Menard

<figure><img src="image_1.png" /></figure>

## To cite this version:

A Cissé, A. Morin-Le Bihan, M. Pagenault, V. Cattoir, S. Reissier, et al., Silent transmission of clonal Pseudomonas aeruginosa via duodenoscopes: microbiological and genomic investigation of a prolonged episode. Journal of Hospital Infection, 2025, Online ahead of print. 10.1016/j.jhin.2025.11.025. hal-05419462

HAL Id: hal-05419462

https://hal.science/hal-05419462v1

Submitted on 7 Jan 2026

HAL is a multi-disciplinary open access archive for the deposit and dissemination of scientific research documents, whether they are published or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers.

L'archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

<figure><img src="image_2.png" /></figure>

Distributed under a Creative Commons CC BY 4.0 - Attribution - International License
Journal Pre-proof

Silent transmission of clonal *Pseudomonas aeruginosa* via duodenoscopes: microbiological and genomic investigation of a prolonged episode

A. Cissé, A. Morin-Le Bihan, M. Pagenault, V. Cattoir, S. Reissier, G. Ménard

PII: S0195-6701(25)00398-6

DOI: https://doi.org/10.1016/j.jhin.2025.11.025

Reference: YJHIN 7688

To appear in: *Journal of Hospital Infection*

Received Date: 3 October 2025

Revised Date: 29 October 2025

Accepted Date: 15 November 2025

<figure><img src="image_3.png" /></figure>

Please cite this article as: Cissé A, Morin-Le Bihan A, Pagenault M, Cattoir V, Reissier S, Ménard G, Silent transmission of clonal *Pseudomonas aeruginosa* via duodenoscopes: microbiological and genomic investigation of a prolonged episode, *Journal of Hospital Infection*, https://doi.org/10.1016/
j.jhin.2025.11.025.

This is a PDF of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability. This version will undergo additional copyediting, typesetting and review before it is published in its final form. As such, this version is no longer the Accepted Manuscript, but it is not yet the definitive Version of Record; we are providing this early version to give early visibility of the article. Please note that Elsevier's sharing policy for the Published Journal Article applies to this version, see: https://www.elsevier.com/about/policies-and-standards/sharing#4-published-journal-article. Please also note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

© 2025 Published by Elsevier Ltd on behalf of The Healthcare Infection Society.
# Silent transmission of clonal *Pseudomonas aeruginosa* via duodenoscopes: microbiological and genomic investigation of a prolonged episode

A. Cissé<fnref n="1" />, A. Morin-Le Bihan<fnref n="1" />, M. Pagenault<fnref n="2" />, V. Cattoir<fnref n="1" />,<fnref n="3" />, S. Reissier<fnref n="1" />,<fnref n="3" />, G. Ménard<fnref n="1" />,<fnref n="3" />*

<footnote n="1">¹CHU Rennes, Department of Bacteriology and Hospital Hygiene, Rennes, France</footnote>

<footnote n="2">²CHU Rennes, Department of Endoscopy and Gastroenterology, Rennes, France</footnote>

<footnote n="3">³UMR_S 1230 Inserm BRM, University of Rennes, Rennes, France</footnote>

<footnote>*Corresponding author: guillaume.menard@chu-rennes.fr</footnote>
**Abstract**

**Background:**

Duodenoscope-associated infections are typically linked to multidrug-resistant organisms and detected during outbreak investigations. However, the true burden of transmission involving non-MDRO pathogens remains largely underestimated. This study reports a prolonged episode of silent *Pseudomonas aeruginosa* transmission through duodenoscopes.

**Methods:**

A retrospective and prospective investigation was conducted at a French tertiary-care hospital from May 2021 to December 2022. Cases were defined as patients who underwent endoscopic retrograde cholangiopancreatography (ERCP) with duodenoscope D2 and developed *P. aeruginosa* infection or colonisation. Duodenoscope sampling was performed in accordance with national guidelines. Isolates were characterised using antibiotic susceptibility testing, quantitative antibiogram, Fourier-Transform infrared spectroscopy, and whole genome sequencing. Longitudinal surveillance of all duodenoscopes was implemented post-incident.

**Results:**

Eight patients were infected or colonised with *P. aeruginosa* after endoscopic retrograde cholangiopancreatography (ERCP) with duodenoscope D2, despite compliant routine microbiological testing in April and July 2021. D2 was declared non-compliant only in October 2021, revealing persistent contamination. Two patients developed non-biliary infections, suggesting overlooked transmission routes. During prospective surveillance, duodenoscope D4 was found to be contaminated after being used in two additional cases. WGS confirmed a clonal cluster (ST1320) comprising all patient isolates and those from D2 and D4 instruments. The similarity of isolates (0-6 SNPs) supports device persistence and delayed detection.

**Conclusion:**

This episode demonstrates that clonal *P. aeruginosa* contamination of duodenoscopes can persist undetected despite routine testing and strict reprocessing. Infections may arise outside typical outbreak settings and involve non-MDRO strains. Enhanced detection strategies, improved endoscope designs, and broader post-procedural surveillance are needed to prevent such silent transmission.

**Key words:** Duodenoscope-associated infection, *P. aeruginosa*, silent transmission, persistent contamination, whole genome sequencing,
**Introduction**

Millions of gastrointestinal endoscopy procedures are performed each year worldwide, including endoscopic retrograde cholangiopancreatography (ERCP), one of the most frequently used techniques in the management of biliary and pancreatic diseases [1]. While ERCP is a vital tool in modern gastroenterology, it carries intrinsic risks of healthcare-associated infections (HAIs) [2,3].

The incidence of post-ERCP sepsis is reported to range from 0.3% to 5.4% [4], although this is likely an underestimation [5]. Most endoscopy-related HAIs are endogenous, but a minority are exogenous and associated with contaminated duodenoscopes, largely due to their complex design and the challenges of effective reprocessing [6]. The presence of sealed working channels and elevator mechanisms facilitates biofilm formation and protects embedded organisms, rendering them resistant to high-level disinfection despite strict adherence to reprocessing guidelines.

The true rate of exogenous infection linked to contaminated endoscopes remains unknown, although numerous studies have documented contamination and related outbreaks. Nationwide studies reported high duodenoscope contamination confirming thus the ongoing risk in non-outbreak settings [6-8]. Recent modelling suggests that the real-world risk of duodenoscope-associated infections (DAI) may be up to 180 times higher than previously estimated, implying that many events go undetected [9]. Most documented DAI outbreaks have involved multidrug-resistant organisms (MDROs) [10], particularly carbapenemase-producing *Enterobacterales* (CPE) [11-13]. However, cases of DAI caused by non-MDRO pathogens have also been described, although they are likely underreported in the absence of systematic microbiological screening [5].

Here, we report a prolonged episode of *Pseudomonas aeruginosa* contamination—outside the context of typical MDRO outbreaks—affecting multiple patients and duodenoscopes in a French tertiary-care hospital. Our investigation combined epidemiological reconstruction, advanced microbiological and molecular analyses, and longitudinal surveillance to assess the persistence of contamination and its role in patient-to-patient transmission.
# Materials and methods

## Context

This investigation was conducted at the Centre Hospitalier Universitaire (CHU) of Rennes, a 1,800-bed tertiary-care teaching hospital in western France. The endoscopy unit performs several hundred ERCP procedures annually using reusable video duodenoscopes.

Throughout the study period (May 2021 – December 2022), the duodenoscope fleet consistently comprised four reusable duodénoscopes (Olympus®). However, the composition of the fleet evolved over time due to scheduled decommissioning and replacement of older devices. Full details of the device acquisition and withdrawal dates are shown in Table 1.

<table><caption>Table 1: Characteristics and life cycle of duodenoscopes used at Rennes University Hospital (2014–2022)</caption><thead><tr><th>Label</th><th>Model</th><th>Internal ID</th><th>Brand</th><th>Date of commissioning</th><th>Date of disposal</th></tr></thead><tbody><tr><td rowspan="7">Video duodenoscope</td><td>TJF-160VR</td><td>D2</td><td rowspan="7">OLYMPUS</td><td>21/07/2014</td><td>07/02/2022</td></tr><tr><td>TJF-160VR</td><td>D3</td><td>10/03/2015</td><td>07/02/2022</td></tr><tr><td>TJF-160VR</td><td>D4</td><td>27/11/2015</td><td>01/12/2022</td></tr><tr><td>TJF-Q190V</td><td>D5</td><td>16/11/2020</td><td>-</td></tr><tr><td>TJF-Q190V</td><td>D6</td><td>11/01/2022</td><td>-</td></tr><tr><td>TJF-Q190V</td><td>D7</td><td>11/01/2022</td><td>-</td></tr><tr><td>TJF-Q190V</td><td>D8</td><td>08/12/2022</td><td>-</td></tr></tbody></table>

Devices were reprocessed after each use using high-level disinfection (HLD) in automated endoscope repressor (AER) followed by drying and storage in the PlasmaTYPHOON® system. Reprocessing data were logged electronically to ensure traceability.

## Case Definition

A case was defined as any patient who underwent ERCP with the Olympus TJF-160VR duodenoscope (hereafter referred to as D2) between May and October 2021 and subsequently developed an infection or colonisation with *P. aeruginosa* resistant to both ceftazidime and meropenem. Cases included both symptomatic infections (e.g. cholangitis, bacteraemia,
peritonitis) and asymptomatic colonisation, as determined by clinical assessment and the type of specimen collected.

## Epidemiological investigations

A retrospective analysis was conducted to identify all patients who had undergone ERCP using the D2 duodenoscope during the exposure period. Clinical and microbiological data were reviewed to assess the onset of symptoms, microbiological findings, and possible epidemiological links. Infection prevention team (IPT) collaborated with gastroenterology staff throughout the investigation to ensure case validation and contact tracing. Each endoscope was traceable through electronic reprocessing logs, and reprocessing cycle completion was verified prior to clinical reuse.

Prospective surveillance was also implemented from November 2021 to December 2022. This included the active monitoring of *P. aeruginosa* infections in post-ERCP patients and the monthly microbiological screening of the entire duodenoscope fleet.

## Duodenoscope sampling

Microbiological surveillance of duodenoscopes was carried out in accordance with the French national guidelines [14] (Instruction DGOS/PF2/DGS/VSS1/PP3/2018/195 du 2 août 2018), which define both the sampling methodology and the interpretation thresholds for contamination. Sampling was performed aseptically after completion of the reprocessing cycle, as part of routine surveillance or in response to suspected contamination. First, the distal end (including elevator mechanism) was sampled after immersion in the neutralizing buffer solution (DNP 97 thiuosulfate, Thermoscientific) following sampled using a sterile brush. Secondly, for each device, internal channels were sampled by flushing with 200 ml of recovering solution; the effluent was collected from the distal end into sterile container. Each sample was filtered through 0,45 µm cellulose membrane filters and plated onto trypticase soy agar (TSA) for 5 days at 30°C. Colonies isolated on both membranes were added and results were expressed as a total number of colony-forming units (CFU)/endoscope and interpreted using the criteria defined by distinguishing high-concern microorganisms (HCM) and low-concern microorganisms (LCM). Following any non-compliant result, retesting was performed after full reprocessing, and contamination levels were expressed in CFU per channel. Duodenoscopes with two successive non-compliant results were referred for technical maintenance.

When necessary, bacteriologically controlled rinsing water of the AER was also controlled.
## Microbiological identification and characterization

All *P. aeruginosa* isolates from patients and duodenoscopes were identified by matrix-assisted laser desorption/ionization-time-of flight mass spectrometry (MALDI-TOF MS, Biotyper Brucker). Antibiotic susceptibility was assessed using the agar disk-diffusion method and interpreted following the Comité de l'Antibiogramme de la Société Française de Microbiologie/European Committee on Antimicrobial Susceptibility Testing (CA-SFM/EUCAST) current guidelines.

Identification of *P. aeruginosa* related-strains was first realized based on the quantitative antibiogram (QA) method [15]. Briefly, ten antibiotics were selected and inhibition zone diameters were collected to calculate Euclidian distance as already described method [15]. Two strains were considered as related when the calculate ED was ≤ 4.5mm.

All suspected isolates were further typed using two different approaches: Fourier transform infrared spectroscopy (FTIR) (IR Biotyper; Bruker Daltonics) and the whole genome sequencing (WGS). FTIR was performed as recently described [16]. Quality control was performed according to the manufacturer's recommendations. Average mean spectra were analysed using the OPUS 7.5 software. To define groups, i.e. related isolates, we used the cut-off value provided by the manufacturer (0.2-0.25) and the results obtained with the *P. aeruginosa* ATCC 27853 strain to consider both intra- and inter-plate variations. The cut-off value referred up to which distance the spectra were considered to be in the same group. WGS was performed at the French National Reference Center for *Pseudomonas* spp (CNR Résistance aux antibiotiques - Expertise des souches). Genomic analysis of the strains was based on reads and nucleotide sequences obtained by sequencing (NextSeq500, Illumina). Phylogeny was determined according to the maximum likelihood Inference of Single Nucleotide Polymorphism (SNPs) determined against the genome of *P. aeruginosa* reference strain PAO1, using SNIPPY-core (v4.6.0). The number of SNPs between strains was mapped on a neighbor-joining phylogenetic tree.

## Other investigations

A structured root-cause analysis (RCA) was performed jointly by the IPT and gastroenterology teams following the outbreak alert in October 2021. This included a full chronology of clinical and technical events, identification of deviations from standard procedures, and contributing factors related to human behaviour, equipment use, and process gaps.
# Results

## Outbreak description

In October 2021, a patient (P3) was admitted to the emergency department with clinical sepsis. Blood cultures revealed *P. aeruginosa* isolate resistant to both ceftazidime and meropenem. As the patient had undergone an ERCP procedure with biliary stent placement four days earlier, a diagnosis of post-ERCP cholangitis was made. The D2 duodenoscope used during the procedure was immediately quarantined pending microbiological investigations.

A few days later, a second patient (P4), who had undergone an ERCP with the same D2 duodenoscope in August 2021, was identified with *P. aeruginosa* peritonitis presenting the same resistance profile. As a result, a DAI outbreak was declared in collaboration with the IPT and gastroenterology teams. National health authorities were promptly notified.

Initial microbiological investigations revealed that the *P. aeruginosa* strains isolated from both D2 and the first two patients (P3 and P4) shared an identical antibiotic resistance profile. Although multiple *P. aeruginosa* strains were isolated from D2, at least one matched the clinical isolates. Rinsing water from the AER was compliant with national standards and tested negative for *P. aeruginosa*.

A retrospective review identified 57 patients who had undergone ERCP with D2 between May and October 2021. Among them, five additional cases of *P. aeruginosa* infection or colonization were identified (P1, P2, P5, P6, and P7). QA analysis confirmed that one environmental strain from D2 and all clinical isolates were related (Figure 1). Patient P3 was the first detected case, but P1 was retrospectively identified as the index case (Figure 2, Table 2). Interestingly, the incriminated D2 duodenoscope was microbiologically compliant during routine testing conducted in both April and July 2021, and remained so until the outbreak investigation in October 2021.

<table><caption>**Table 2: Characteristics of patients with *Pseudomonas aeruginosa* infection or colonisation, exposure to duodenoscope D2, and corresponding microbiological results (May–November 2021).***P1: index case. **P3: first case detected. Multiple ERCP and detections were listed chronologically.</caption><thead><tr><td>Case</td><td>ERCP dates</td><td>Detection dates</td><td>Delay (days)</td><td>Diagnosis</td></tr></thead><tbody><tr><td>P1*</td><td>31/05, 08/06, 26/08</td><td>08/06, 25/06, 26/08</td><td>9</td><td>Cholangitis</td></tr></tbody></table>
<table><tr><td>P2</td><td>03/09/21</td><td>12/09, 15/09</td><td>9</td><td>Cholangitis with bacteraemia</td></tr><tr><td>P3**</td><td>30/09/21</td><td>03/10/21</td><td>3</td><td>Bacteraemia</td></tr><tr><td>P4</td><td>19/08/21</td><td>14/10/21</td><td>56</td><td>Peritonitis</td></tr><tr><td>P5</td><td>08/07/21</td><td>16/11/21</td><td>131</td><td>Biliary colonization</td></tr><tr><td>P6</td><td>01/10/21</td><td>11/10, 08/03, 09/03/22</td><td>10</td><td>Sinusitis</td></tr><tr><td>P7</td><td>27/07/21</td><td>26/11/21</td><td>122</td><td>Testicular necrosis</td></tr></table>

Most patients developed clinical infections, while one was found to be colonized only (P5). Two cases were unrelated to biliary pathology: one patient presented with sinusitis (P6), and another with testicular necrosis (P7) both requiring surgical intervention. The median time from ERCP to identification of *P. aeruginosa* was 10 days [3-131].

## Longitudinal surveillance

From November 2021 to December 2022, a prospective surveillance program was implemented. This included monthly microbiological testing of all duodenoscopes in use (instead of the previous quarterly schedule) and active clinical surveillance for *P. aeruginosa* infections following ERCP procedures.

A total of 60 microbiological samples were collected during this period, including routine testing (n = 35), post-maintenance testing (n = 10), retesting following previous non-compliance (n = 12), and pre-use testing of newly acquired duodenoscopes (n = 3) (Table S1). Overall, 75% were microbiologically compliant, and 80% of routine tests met the conformity criteria. Among the seven non-compliant routine tests, five yielded high-concern microorganisms (HCMs), including *P. aeruginosa* (n = 2; duodenoscopes D4 and D6), *Enterobacterales* (n = 2), and one fungal isolate. Notably, the duodenoscope D2 remained free of *P. aeruginosa* throughout this surveillance period.

No statistically significant difference was observed between TJF-160VR and TJF-Q190 duodenoscope models in terms of compliance rates for routine or retesting controls. There was also no evidence of correlation between microbial contamination and duration of storage in the PlasmaTYPHOON® drying system.

Two duodenoscopes (D4 and D6) were of particular concern due to high levels of microbiological non-compliance, repeated manufacturer maintenance, and the presence of *P. aeruginosa* despite the absence of contamination in rinse water from the AER.
Data from the initial outbreak phase were retrospectively cross-referenced, revealing notable findings. Patients P6 and P7, previously involved in the outbreak, were readmitted in 2022 and underwent ERCP procedures with duodenoscopes D6 and D4, respectively. In addition, a new case (P8) was identified: this patient had undergone ERCP with duodenoscope D2 in September 2021 and was readmitted in March 2022 with *P. aeruginosa* bacteraemia and cholangitis. He benefited another ERCP with D4.

Antibiotic resistance profiles of *P. aeruginosa* isolates from D4 and P8 were identical to those observed during the first outbreak episode. QA analysis suggested that strains from D4 and P8 were related to the previous cluster, whereas strains from D6 were not (data not shown).

Despite the implementation of monthly microbiological testing, D4 was not identified as contaminated with *P. aeruginosa* until May 2022—after both P8 and P7 had undergone ERCP with this device in March and April 2022, respectively (**Figure 3**). No additional suspected cases were identified, although the absence of systematic patient screening may have limited detection.

Microbiological testing revealed that both the internal channels and the distal end were contaminated in duodenoscope D4 (as D2), while only the channels were affected in D6. The estimated bacterial load ranged from 48 to uncountable CFUs per endoscope. Each of these endoscopes underwent two additional reprocessing cycles followed by retesting. Despite this, *P. aeruginosa* contamination persisted. The first round of retesting showed lower levels of contamination than the second, suggesting possible biofilm accumulation and regrowth over time. These findings were particularly marked in duodenoscope D4, and mainly involved the instrument and suction channels.

## Microbiological characterization

A total of 23 *P. aeruginosa* isolates were recovered between October 2021 and December 2022, including 8 from ERCP patients and 15 from three different duodenoscopes (D2, D4, and D6). Clinical isolates displayed a consistent antibiotic resistance profile, while environmental isolates showed greater phenotypic diversity: three resistance phenotypes were identified from D2, one from D4, and two from D6 (Table S2). One strain from D2 and all strains from D4 were presumed identical to patient isolates based on antibiotic susceptibility profiles and QA analysis.

Two complementary strain typing methods were used to assess clonality: IRTB and WGS. Both methods confirmed the preliminary findings (**Figure 4, Figure 5**). Using a similarity threshold
of 0.087 for IRTB and a maximum of 6 single nucleotide polymorphisms (SNPs) in WGS, a
clonal cluster of *P. aeruginosa* sequence type ST1320 was identified. This cluster included 15
isolates: all 8 patient strains, 1 strain from D2, and 6 from D4, and was characterized by a AmpC
β-lactamase overproduction attributable to mutations in *ampD* (F104L) and *dacB* (frameshift
205_206insT). In addition, repression of *oprD* expression was linked to mutations in the CzcSR
two-component system sensor *czcS* (E44K) and in *mexS* (E54G), which regulates the RND
efflux pump MexEF-OprN. Other strains from D2 were identified as ST309 (n=2) and ST348
(n=3), with no genetic mutations detected in genes involved in antibiotic resistance (wild-type
phenotype). The strain from D6 was identified as ST252, showing AmpC β-lactamase
overproduction due to a mutation in *sltB1* (Y158stop).

Root-cause analysis

A RCA was conducted to investigate systemic and contextual factors that may have contributed to the episode. The implicated duodenoscope (D2) had undergone routine reprocessing according to protocol, and its last routine microbiological test in July 2021 was compliant. However, several operational vulnerabilities were identified. Between August and September 2021, the endoscopy unit was experiencing equipment shortages, with up to 50% of the duodenoscope fleet under repair (2/4). As a result, D2 had been used intensively (up to three procedures per day). Additionally, delays and administrative errors in the repair chain led to prolonged unavailability of one duodenoscope, increasing pressure on the remaining devices. Between July and September, at least one AER was unavailable on 35 out of 65 working days, significantly impacting reprocessing capacity. Human resource constraints were also reported: staff shortages, high turnover of healthcare assistants (with over 169 cumulative days of sick leave), and replacement by undertrained personnel affected adherence to optimal reprocessing timelines and handling procedures.
Discussion

This study describes a prolonged episode of *P. aeruginosa* contamination involving duodenoscopes in a tertiary-care hospital, leading to multiple patient infections over several months. Unlike most documented DAIs, this outbreak was caused by a non-MDRO strain and was only detected after retrospective analysis of clinical cases—despite adherence to reprocessing protocols and enhanced surveillance. The event illustrates the persistent and under-estimated risk of exogenous transmission linked to flexible endoscopes.

Our data illustrate how both routine and post-incident surveillance failed to detect contamination in a timely manner. In the first phase, duodenoscope D2 tested microbiologically compliant in April and July 2021, while several patients—beginning with P1—developed infections during the same period. Patient P1, who underwent multiple ERCPs and tested positive as early as June, is the most plausible origin of contamination. However, this remains hypothetical given the absence of systematic microbiological screening of all exposed patients. Other investigations have shown that endoscope contamination may also originate from environmental reservoirs, including *Pseudomonas* spp. contamination of AERs [17] highlighting that both patient and environmental sources can contribute to their transmission.

In the longitudinal follow-up phase, duodenoscope D4 remained compliant throughout early 2022 but was later declared non-compliant in May—after being used for ERCP procedures on patients P8 and P7. These findings highlight the limitations of culture-based time-point testing and underscore the need for more sensitive or continuous detection strategies [18]. Monthly testing alone was insufficient to prevent delayed transmission, in line with evidence showing that culture-based surveillance has limited sensitivity and delayed turnaround. Optimised sampling protocols [19], and molecular approaches [20] have been proposed to improve detection, yet variability in methods and frequency continues to undermine reliability. Importantly, culture-negative results do not reliably indicate absence of viable bacteria [21], a finding echoed in experimental studies where duodenoscopes intermittently tested negative before later yielding *P. aeruginosa*, reflecting biofilm persistence [9,22]. In line with these, Cimen et al. further demonstrated that a duodenoscope that had repeatedly tested negative with routine cultures, was the source of transmission of ESBL-producing *Enterobacterales* after dismantling [10]. These findings highlight that endoscopes deemed “patient-ready” may still harbour HCM.

A RCA revealed several vulnerabilities and confirmed that contextual and organisational factors can also amplify the risk of contamination, that was already confirmed [23]. These
systemic constraints likely facilitated the persistence and unnoticed transmission of *P. aeruginosa*, despite compliance with reprocessing protocols

This challenge is further complicated by infections presenting in non-biliary sites. In our cohort, *P. aeruginosa* was also isolated in cases of sinusitis and testicular necrosis—diagnoses that do not typically raise suspicion for endoscope-related transmission. This highlights the difficulty of recognizing low-grade or atypical DAIs, especially when surveillance focuses solely on cholangitis or bacteraemia.

Older duodenoscope models with sealed elevator mechanisms, such as the TJF-160VR, have often been considered at higher risk for persistent colonization. Design innovations—including disposable elevator caps or detachable distal ends—have been associated with lower contamination rates, although not consistently reaching statistical significance [6,24,25]. In our investigation, however, both sealed models (TJF-160VR) and more recent non-sealed models (TJFQ-190V) were in use, and we did not observe a clear difference in contamination patterns between them. This suggests that, while redesign may contribute to risk reduction, device architecture alone is unlikely to fully prevent persistence, reinforcing the need for multifaceted prevention strategies.

Our investigation benefited from a robust microbiological approach. By combining phenotypic and complementary typing methods, we identified a clonal ST1320 population linking clinical and device isolates. This integrated strategy not only supported outbreak attribution but also enabled targeted feedback to IPT. Recent studies confirm the added value of high-resolution typing in endoscope-related investigations: Tan et al. showed that WGS revealed the persistence of genomically linked *P. aeruginosa* isolates within a single endoscope despite repeated reprocessing, pointing to biofilm-associated contamination [26]. Similarly, it was demonstrated that WGS was essential to confirm DAIs, including those caused by non-MDRO pathogens that would otherwise have remained unrecognised with conventional culture and antibiogram analysis [5]. Together, these findings underscore the pivotal role of WGS in distinguishing transient contamination from true persistent transmission, fully consistent with the approach adopted in our investigation.

However, this study has limitations. The lack of systematic screening may have led to underdiagnosis of colonized patients. Environmental sampling was limited to duodenoscopes and rinse water, other potential reservoirs were not assessed. Finally, while epidemiological links are strong, transmission remains inferred in the absence of real-time contamination detection.
To sum up, our findings highlight the vulnerability of current reprocessing and surveillance
systems in detecting persistent duodenoscope contamination by *P. aeruginosa*. Even in non-
MDRO contexts, silent transmission may occur over extended periods. Beyond outbreak
settings, residual contamination after reprocessing has also been documented with occasional
detection of environmental Gram-negative bacilli including *Pseudomonas* spp [27]. Although
these events were not linked to clinical cases, they illustrate the persistence of low-level
contamination even in well-controlled settings.

Improved device design, more sensitive surveillance strategies, and expanded microbiological
monitoring are needed to better prevent and detect DAIs.
**Conflict of interest:** none

**Ethics:** not required

**Funding statement:** none

<figure><img src="image_4.png" /></figure>
References

[1] Balan GG, Sfarti CV, Chiriac SA, Stanciu C, Trifan A. Duodenoscope-associated infections: a review. Eur J Clin Microbiol Infect Dis 2019;38:2205-13.
https://doi.org/10.1007/s10096-019-03671-3.

[2] Hutfless S, Shiratori Y, Chu D, Liu S, Kalloo A. Risk factors for infections after endoscopic retrograde cholangiopancreatography (ERCP): a retrospective cohort analysis of US Medicare Fee-For-Service claims, 2015-2021. BMJ Open 2022;12:e065077. https://doi.org/10.1136/bmjopen-2022-065077.

[3] Leem G, Sung MJ, Park JH, Kim SJ, Jo JH, Lee HS, et al. Randomized Trial of Prophylactic Antibiotics for Endoscopic Retrograde Cholangiopancreatography in Patients With Biliary Obstruction. Am J Gastroenterol 2024;119:183-90.
https://doi.org/10.14309/ajg.0000000000002495.

[4] Kovaleva J, Peters FTM, van der Mei HC, Degener JE. Transmission of infection by flexible gastrointestinal endoscopy and bronchoscopy. Clin Microbiol Rev 2013;26:231-54. https://doi.org/10.1128/CMR.00085-12.

[5] van der Ploeg K, Klaassen CHW, Vos MC, Severin JA, Mason-Slingerland BCGC, Bruno MJ. A search strategy for detecting duodenoscope-associated infections: a retrospective observational study. J Hosp Infect 2024;147:56-62.
https://doi.org/10.1016/j.jhin.2024.02.015.

[6] Okamoto N, Schzaniecka A, Hirano M, Benedict M, Baba S, Horino Y, et al. A prospective, multicenter, clinical study of duodenoscope contamination after reprocessing. Infect Control Hosp Epidemiol 2022;43:1901-9.
https://doi.org/10.1017/ice.2021.525.

[7] Pineau L. Endoscope reprocessing: Retrospective analysis of 90,311 samples. Endosc Int Open 2023;11:E247-57. https://doi.org/10.1055/a-1991-1391.

[8] Casini B, Spagnolo AM, Sartini M, Tuvo B, Scarpaci M, Barchitta M, et al. Microbiological surveillance post-reprocessing of flexible endoscopes used in digestive endoscopy: a national study. J Hosp Infect 2023;131:139-47.
https://doi.org/10.1016/j.jhin.2022.09.024.

[9] Kwakman JA, Erler NS, Vos MC, Bruno MJ. Risk evaluation of duodenoscope-associated infections in the Netherlands calls for a heightened awareness of device-related infections: a systematic review. Endoscopy 2022;54:148-55.
https://doi.org/10.1055/a-1467-6294.

[10] Cimen C, Bathoorn E, Loeve AJ, Fliss M, Berends MS, Nagengast WB, et al. Uncovering the spread of drug-resistant bacteria through next-generation sequencing based surveillance: transmission of extended-spectrum β-lactamase-producing Enterobacterales by a contaminated duodenoscope. Antimicrob Resist Infect Control 2024;13:31. https://doi.org/10.1186/s13756-024-01386-5.

[11] Kim S, Russell D, Mohamadnejad M, Makker J, Sedarat A, Watson RR, et al. Risk factors associated with the transmission of carbapenem-resistant Enterobacteriaceae via contaminated duodenoscopes. Gastrointest Endosc 2016;83:1121-9.
https://doi.org/10.1016/j.gie.2016.03.790.

[12] Bourigault C, Le Gallou F, Bodet N, Musquer N, Juvin M-E, Corvec S, et al. Duodenoscopy: an amplifier of cross-transmission during a carbapenemase-producing Enterobacteriaceae outbreak in a gastroenterology pathway. J Hosp Infect 2018;99:422-6.
https://doi.org/10.1016/j.jhin.2018.04.015.

[13] Yang S, Hemarajata P, Hindler J, Li F, Adisetiyo H, Aldrovandi G, et al. Evolution and Transmission of Carbapenem-Resistant Klebsiella pneumoniae Expressing the blaOXA-232 Gene During an Institutional Outbreak Associated With Endoscopic Retrograde
Cholangiopancreatography. Clin Infect Dis 2017;64:894-901.
https://doi.org/10.1093/cid/ciw876.

[14] Légifrance - Droit national en vigueur - Circulaires et instructions - INSTRUCTION N° DGOS/PF2/DGS/VVS1/PP3/2018/195 du 2 août 2018 relative à l'actualisation du traitement des endoscopes souples thermosensibles à canaux de type duodénoscope au sein des structures de soins n.d.
https://www.legifrance.gouv.fr/download/pdf/circ?id=43880 (accessed September 8, 2025).

[15] Morin-Le Bihan A, Le Neindre K, Dejoies L, Piau C, Donnio P-Y, Ménard G. Use of the quantitative antibiogram method for assessing nosocomial transmission of ESBL-producing Enterobacterales in a French hospital. J Hosp Infect 2023;135:132-8.
https://doi.org/10.1016/j.jhin.2023.01.023.

[16] Turban A, Morin-Le Bihan A, Derbier L, Piau-Couapel C, Nesseler N, Cattoir V, et al. Effectiveness of water system chemical disinfection against Pseudomonas aeruginosa infections, despite a not-so-obvious connection. Am J Infect Control 2024;52:1432-7.
https://doi.org/10.1016/j.ajic.2024.08.028.

[17] Botana-Rial M, Leiro-Fernández V, Núñez-Delgado M, Álvarez-Fernández M, Otero-Fernández S, Bello-Rodríguez H, et al. A Pseudo-Outbreak of Pseudomonas putida and Stenotrophomonas maltophilia in a Bronchoscopy Unit. Respiration 2016;92:274-8.
https://doi.org/10.1159/000449137.

[18] van der Ploeg K, Severin JA, Klaassen CHW, Vos MC, Bruno MJ, Mason-Slingerland BCGC. Contaminated duodenoscopes in ERCP: sensitivity of detection and risk of underdetection. Gastrointest Endosc 2025;101:141-8.
https://doi.org/10.1016/j.gie.2024.08.004.

[19] Gazdik MA, Coombs J, Burke JP, Lopansri BK. Comparison of Two Culture Methods for Use in Assessing Microbial Contamination of Duodenoscopes. J Clin Microbiol 2016;54:312-6. https://doi.org/10.1128/JCM.02754-15.

[20] Valeriani F, Agodi A, Casini B, Cristina ML, D'Errico MM, Gianfranceschi G, et al. Potential testing of reprocessing procedures by real-time polymerase chain reaction: A multicenter study of colonoscopy devices. Am J Infect Control 2018;46:159-64.
https://doi.org/10.1016/j.ajic.2017.08.008.

[21] Thornhill G, David M. Endoscope-associated infections: A microbiologist's perspective on current technologies. Techniques in Gastrointestinal Endoscopy 2019;21:150625.
https://doi.org/10.1016/j.tgie.2019.150625.

[22] Kwakman JA, Vos MC, Bruno MJ. Investigation of the efficacy of an innovative endoscope drying and storage method in a simulated ERCP setting. Endosc Int Open 2023;11:E419-25. https://doi.org/10.1055/a-2017-3872.

[23] Rauwers AW, Troelstra A, Fluit AC, Wissink C, Loeve AJ, Vleggaar FP, et al. Independent root-cause analysis of contributing factors, including dismantling of 2 duodenoscopes, to investigate an outbreak of multidrug-resistant Klebsiella pneumoniae. Gastrointest Endosc 2019;90:793-804. https://doi.org/10.1016/j.gie.2019.05.016.

[24] Forbes N, Elmunzer BJ, Allain T, Parkins MD, Sheth PM, Waddell BJ, et al. Effect of Disposable Elevator Cap Duodenoscopes on Persistent Microbial Contamination and Technical Performance of Endoscopic Retrograde Cholangiopancreatography: The ICECAP Randomized Clinical Trial. JAMA Intern Med 2023;183:191-200.
https://doi.org/10.1001/jamainternmed.2022.6394.

[25] Ridtitid W, Buathong J, Chatsuwan T, Angsuwatcharakon P, Luangsukrerk T, Mekaroonkamol P, et al. Contamination of Disposable Distal Cap Duodenoscopes and Detachable Elevator Duodenoscopes After Reprocessing: A Randomized Trial. J Gastroenterol Hepatol 2025;40:520-7. https://doi.org/10.1111/jgh.16827.
[26] Tan GSE, Chia GJM, Thevasagayam NM, Loy SQD, Prakki SRS, Lim ZQ, et al. Whole-genome sequencing establishes persistence of biofilm-associated Pseudomonas aeruginosa detected from microbiological surveillance of gastrointestinal endoscopes. J Hosp Infect 2024;152:73–80. https://doi.org/10.1016/j.jhin.2024.07.007.

[27] Marchese V, Di Carlo D, Fazio G, Gioè SM, Luca A, Aldduino R, et al. Microbiological Surveillance of Endoscopes in a Southern Italian Transplantation Hospital: A Retrospective Study from 2016 to 2019. Int J Environ Res Public Health 2021;18:3057. https://doi.org/10.3390/ijerph18063057.

## Captions

**Figure 1.** Identification of *P. aeruginosa* related-strains using the quantitative antibiogram method (January to November 2021). All *P. aeruginosa* isolates detected at our hospital during the study period—including both patient and environmental samples—were analysed. A similarity cut-off value was defined to assess relatedness. The cluster highlighted in purple includes one isolate from duodenoscope D2, the isolates from patients P3 and P4, and five additional patients who had undergone ERCP procedures with duodenoscope D2.

**Figure 2.** Timeline of the *Pseudomonas aeruginosa* outbreak associated with duodenoscope D2 (May–November 2021). Each line represents either duodenoscope D2 or patients (P1–P7) exposed during endoscopic retrograde cholangiopancreatography (ERCP). Blue circles indicate ERCP procedures, red circles represent *P. aeruginosa* isolation dates, and green or red squares correspond to compliant and non-compliant microbiological tests of D2, respectively.

**Figure 3.** Timeline of endoscopic retrograde cholangiopancreatography (ERCP) procedures and microbiological testing for duodenoscope D4 (January–May 2022). Each line represents duodenoscope D4 or patients (P7 and P8) exposed during the study period. Blue circles indicate ERCP procedures, while green and red squares correspond to compliant and non-compliant microbiological tests of D4, respectively.

**Figure 4.** Infrared spectroscopy clustering of *Pseudomonas aeruginosa* isolates from patients and duodenoscopes. Dendrogram based on Fourier-transform infrared spectroscopy (FTIR) analysis, showing relatedness between *P. aeruginosa* isolates collected from clinical
cases (P1–P8) and from various sampling sites of duodenoscopes D2, D4, and D6. A similarity
cut-off of 0.087 was applied to define related clusters.

**Figure 5.** Whole-genome sequencing–based phylogenetic tree and pairwise SNP distance matrix of *P. aeruginosa* isolates from patients and duodenoscopes. Maximum likelihood phylogenetic tree (left) and matrix of pairwise single nucleotide polymorphism (SNP) distances (right) among *P. aeruginosa* isolates collected from infected patients (P1–P8)* and environmental samples from duodenoscopes D2 and D4 (S1_D2 to S12_D4). All isolates belong to the same sequence type (ST1320).* P7 strain was not sequenced as it was no longer available in storage.
<figure><img src="image_5.png" /></figure>

<figure><img src="image_6.png" /></figure>
<figure><img src="image_7.png" /></figure>

Spectrum Name

S14_D6 - Suction channel

S15_D6 - Suction channel

S13_D6 - All channels

<figure><img src="image_8.png" /></figure>

S4-D2 - Elevator wire channel

S6_D2 - All channels

S3_D2 - Distal end

P5

P2

P1

P7

P4

P3

P8

<figure><img src="image_9.png" /></figure>

S9_D4 - Instrument channel

P6

S10_D4 - Elevator wire channel

S12_D4 - Suction channel

S1_D2 - All channels

S7_D4 - All channels

S8_D4 - Distal end

S11_D4 - Distal end

S2_D2 - All channels

S5_D2 - Suction channel

<figure><img src="image_10.png" /></figure>
<figure><img src="image_11.png" /></figure>

<table><thead><tr><th></th><th>ST</th><th>P8</th><th>P6</th><th>P5</th><th>P4</th><th>P2</th><th>P1</th><th>P3</th><th>S1_D2</th><th>S7_D4</th><th>S8_D4</th><th>S9_D4</th><th>S10_D4</th><th>S11_D4</th><th>S12_D4</th></tr></thead><tbody><tr><td>P8</td><td>1320</td><td>0</td><td>4</td><td>4</td><td>3</td><td>5</td><td>3</td><td>6</td><td>3</td><td>4</td><td>3</td><td>5</td><td>3</td><td>3</td><td>4</td></tr><tr><td>P6</td><td>1320</td><td>4</td><td>0</td><td>2</td><td>1</td><td>3</td><td>1</td><td>4</td><td>1</td><td>2</td><td>1</td><td>3</td><td>1</td><td>1</td><td>2</td></tr><tr><td>P5</td><td>1320</td><td>4</td><td>2</td><td>0</td><td>1</td><td>3</td><td>1</td><td>4</td><td>1</td><td>2</td><td>1</td><td>3</td><td>1</td><td>1</td><td>2</td></tr><tr><td>P4</td><td>1320</td><td>3</td><td>1</td><td>1</td><td>0</td><td>2</td><td>0</td><td>3</td><td>0</td><td>1</td><td>0</td><td>2</td><td>0</td><td>0</td><td>1</td></tr><tr><td>P2</td><td>1320</td><td>5</td><td>3</td><td>3</td><td>2</td><td>0</td><td>2</td><td>5</td><td>2</td><td>3</td><td>2</td><td>4</td><td>2</td><td>2</td><td>3</td></tr><tr><td>P1</td><td>1320</td><td>3</td><td>1</td><td>1</td><td>0</td><td>2</td><td>0</td><td>3</td><td>0</td><td>1</td><td>0</td><td>2</td><td>0</td><td>0</td><td>1</td></tr><tr><td>P3</td><td>1320</td><td>6</td><td>4</td><td>4</td><td>3</td><td>5</td><td>3</td><td>0</td><td>3</td><td>4</td><td>3</td><td>5</td><td>3</td><td>3</td><td>4</td></tr><tr><td>S1_D2</td><td>1320</td><td>3</td><td>1</td><td>1</td><td>0</td><td>2</td><td>0</td><td>3</td><td>0</td><td>1</td><td>0</td><td>2</td><td>0</td><td>0</td><td>1</td></tr><tr><td>S7_D4</td><td>1320</td><td>4</td><td>2</td><td>2</td><td>1</td><td>3</td><td>1</td><td>4</td><td>1</td><td>0</td><td>1</td><td>3</td><td>1</td><td>1</td><td>2</td></tr><tr><td>S8_D4</td><td>1320</td><td>3</td><td>1</td><td>1</td><td>0</td><td>2</td><td>0</td><td>3</td><td>0</td><td>1</td><td>0</td><td>2</td><td>0</td><td>0</td><td>1</td></tr><tr><td>S9_D4</td><td>1320</td><td>5</td><td>3</td><td>3</td><td>2</td><td>4</td><td>2</td><td>5</td><td>2</td><td>3</td><td>2</td><td>0</td><td>2</td><td>2</td><td>3</td></tr><tr><td>S10_D4</td><td>1320</td><td>3</td><td>1</td><td>1</td><td>0</td><td>2</td><td>0</td><td>3</td><td>0</td><td>1</td><td>0</td><td>2</td><td>0</td><td>0</td><td>1</td></tr><tr><td>S11_D4</td><td>1320</td><td>3</td><td>1</td><td>1</td><td>0</td><td>2</td><td>0</td><td>3</td><td>0</td><td>1</td><td>0</td><td>2</td><td>0</td><td>0</td><td>1</td></tr><tr><td>S12_D4</td><td>1320</td><td>4</td><td>2</td><td>2</td><td>1</td><td>3</td><td>1</td><td>4</td><td>1</td><td>2</td><td>1</td><td>3</td><td>1</td><td>1</td><td>0</td></tr></tbody></table>
<figure><img src="image_12.png" /></figure>

<figure><img src="image_13.png" /></figure>
<figure><img src="image_14.png" /></figure>

<figure><img src="image_15.png" /></figure>