# Effectiveness of water system chemical disinfection against Pseudomonas aeruginosa infections, despite a not-so obvious connection

A Turban, A Morin-Le Bihan, L Derbier, C Piau-Couapel, N Nesseler, Vincent Cattoir, P. -Y. Donnio, G Menard

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## To cite this version:

A Turban, A Morin-Le Bihan, L Derbier, C Piau-Couapel, N Nesseler, et al.. Effectiveness of water system chemical disinfection against Pseudomonas aeruginosa infections, despite a not-so obvious connection. American Journal of Infection Control, 2024, 52 (12), pp.1432-1437. 10.1016/j.ajic.2024.08.028. hal-04720003

HAL Id: hal-04720003

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Submitted on 23 Jan 2026

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ELSEVIER

Contents lists available at [ScienceDirect](https://www.sciencedirect.com)

American Journal of Infection Control

journal homepage: [www.ajicjournal.org](https://www.ajicjournal.org)

AJIC
American Journal of
Infection Control

Major Article

Effectiveness of water system chemical disinfection against
*Pseudomonas aeruginosa* infections, despite a not-so-obvious
connection

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Adrien Turban PharmD<sup>a,c</sup>, Amélie Morin-Le Bihan PharmD<sup>a</sup>, Lucille Derbier<sup>a</sup>,
Caroline Piau-Couapel PharmD<sup>a</sup>, Nicolas Nesseler MD, PhD<sup>b</sup>,
Vincent Cattoir PharmD, PhD<sup>a,c</sup>, Pierre-Yves Donnio PharmD, PhD<sup>a,c</sup>,
Guillaume Ménard PharmD, PhD<sup>a,c,*</sup>

Key Words:

Pseudomonas aeruginosa

ICU

Hospital-acquired infection

Fourier transform infrared spectroscopy

Cross-transmission

Water system

**Background:** *Pseudomonas aeruginosa* is a well-recognized opportunistic pathogen frequently responsible for hospital-acquired infections. Acquisition routes of *P. aeruginosa* are both endogenous and exogenous, including transmission from a portion of the hospital water system.

**Methods:** The impact of disinfection procedures of the water system and description routes of *P. aeruginosa* transmission in a surgical intensive care unit over a 2-year period were investigated. Two distinct periods A and B were considered, respectively, before and after the disinfection. Fourier transform infrared spectroscopy was used to compare isolates recovered from patients and tap water.

**Results:** Overall, 21.3% of tap water samples were positive but with a significantly lower rate in period B. Concomitantly, the prevalence of patients positive for *P. aeruginosa* decreased from 2.6% to 1%, suggesting a correlation between the presence of environmental sources and patient contaminations. The results revealed that 18% of patients were involved in cross-transmission events not related to any isolate recovered from water, suggesting transmission through care practices. Conversely, only 1 environmental transmission event was suspected in a patient.

**Conclusions:** Although the link between the hospital environment and patients was unclear, HCW-associated care practices could be related to contaminated point-of-use waters and then indirect spreading to patients.

© 2024 The Author(s). Published by Elsevier Inc. on behalf of Association for Professionals in Infection Control and Epidemiology, Inc. This is an open access article under the CC BY license ([http://creativecommons.org/licenses/by/4.0/](http://creativecommons.org/licenses/by/4.0/)).

BACKGROUND

*Pseudomonas aeruginosa*, a nonfermenting gram-negative rod,¹ is a ubiquitous opportunistic pathogen that can be found in moist environments.² The severity of infections is related to the characteristics of patients and microbial factors contributing to bacterial pathogenicity. In addition, antibiotic treatment outcome is uncertain since *P. aeruginosa* clinical isolates are frequently resistant to many antibiotics. This species is often involved in infections among immunocompromised patients as

well as in hospital-acquired infections (HAIs) (such as bacteremia, ventilator-associated pneumonia [VAP], urinary tract infection, or surgical site infection) mainly in intensive care units (ICUs), where the risk of HAIs is higher than in other wards.³⁻⁶ *P. aeruginosa* has an estimated prevalence of 7% to 9% in all HAIs in Europe and in the United States⁴,⁷ and remains the first microorganism involved in ICU-acquired pneumonia.⁸ *P. aeruginosa* HAIs are associated with poor prognosis translated with high mortality and morbidity rates, especially in ICU patients.⁹

Acquisition routes of *P. aeruginosa* HAIs are both endogenous (from patient microbiota)¹⁰ and exogenous,³,⁹ including transmission from health care worker hands, through contaminated medical devices, or a portion of the hospital water system.¹¹⁻¹⁴ The hospital water system is conducive for the growth of *P. aeruginosa*, due to its favorable temperature and its complex design, that allows stagnation and biofilm

<footnote><sup>a</sup> CHU Rennes, Service de Bactériologie-Hygiène Hospitalière, Rennes, France</footnote>

<footnote><sup>b</sup> CHU Rennes, Service d'Anesthésie et de Soins Critiques, Rennes, France</footnote>

<footnote><sup>c</sup> UMR_S 1230 BRM, INSERM/University Rennes, Rennes, France</footnote>

<footnote>* Address correspondence to G. Ménard, PhD, CHU Rennes, Service de Bactériologie-Hygiène Hospitalière, F-35033 Rennes, France.</footnote>

<footnote>E-mail address: guillaume.menard@chu-rennes.fr (G. Ménard).</footnote>

<footnote>Conflicts of interest: None to report.</footnote>
development.¹² Those reservoirs can be associated with outbreaks in hospital settings, involving contaminated water outlets, taps, sinks, toilets, or drains.¹⁵ Preventing HAIs is therefore essential and requires implementation of preventive measures, regular water sampling, as well as suitable bacterial typing method.¹⁶⁻¹⁸

In hospital settings, bacterial typing methods are useful to monitor bacterial diffusion and changing epidemiologic trends and to investigate outbreaks. Pulsed-field gel electrophoresis (PFGE) or multilocus sequence typing (MLST) were considered as time- and cost-consuming molecular methods.¹⁹ Whole-genome sequencing (WGS) advent tends to replace them progressively but remains not applicable routinely by all microbiology laboratories due its cost and the need of bioinformatic expertise.²⁰ New fast-input technologies like Fourier transform infrared (FTIR) spectroscopy could be an interesting alternative since it seems sufficiently reliable for *P. aeruginosa* strain typing.²¹,²²

Confronted with a high number of *P. aeruginosa* HAIs occurring in a single ICU of our hospital, we evaluated in this setting the impact of different maintenance operations on the water system that occurred at the end of the first year and compared the results between the 2 periods. We also tried to decipher routes of transmission acquisition over a 2-year period by using FTIR typing results.

## METHODS

### Context

We conducted a single-center study from January 2021 to November 2022 in a surgical ICU at the University Hospital of Rennes, France. Two different periods were defined as follows: period A from January 2021 to November 2021 and period B from January 2022 to November 2022. The 2 periods were separated by an important water system maintenance procedure in December 2021, characterized first by the installation of point-of-use 0.22-µm filters on the taps and then by decontamination of the water system using electrochemically activated solutions (combination of hydrogen peroxide [H₂O₂] and silver ions), followed by removing of filters early January 2022. The biocide S15 (Sanosil) contains 7.5% H₂O₂ and 0.0075 g/100 g of silver. Briefly, the decontamination procedure was to introduce the product in the plumbing system until a

concentration of 2,000 ppm. Injection was retained 3 hours to comply with contact time followed by rinsing of the tapping points until the biocide used has disappeared. Recommendations in use at our hospital were to quarterly maintain the taps, consisting of disassembling faucet tips and disinfecting them, respectively, in white vinegar and then in a biocidal solution. These measures remained the same throughout the study. The plumbing system dates from 1997 and coincides with building development.

### Environmental samples

Water samples were collected from taps of the 17 rooms composing the ICU. Only tap water was sampled. During period A, tap waters were sampled on a quarterly basis (Fig. 1). At the beginning of period B, we performed a control in all rooms 1 month after the chemical disinfection procedure. After that, the frequency of tap samples was modified as follows: Rooms were separated into 3 groups (group 1: rooms 1, 3, 6, 9, 12, 15; group 2: rooms 2, 4, 7, 10, 13, 16; group 3: rooms 5, 8, 11, 14, 17), and each one was sampled alternatively on a quarterly basis.

The sampling technique and microbiological analysis were standardized and conducted by the technical staff of our infection prevention team. Briefly, 500 mL of tap water were collected after preflush water, filtered by membrane filtration, and then plated on Plate Count Agar and *Pseudomonas* Isolation Agar (BD Diagnostic Systems). After a 2-day incubation at 30°C, colony-forming units were counted, and bacterial identification was performed by Matrix Assisted Laser Desorption Ionization - Time of Flight mass spectrometry (Microflex; Bruker Daltonics). A noncompliant result was considered when *P. aeruginosa* concentration was > 0 colony-forming unit/mL, as recommended by the French guidelines.²³ Antimicrobial susceptibility testing was performed using the agar disc diffusion method on Mueller-Hinton agar (Thermo Fisher Scientific) and interpreted following 2022 CA-SFM/EUCAST guidelines.

During both periods, if *P. aeruginosa* was initially detected, additional measures were implemented as follows: no use of water point supply, maintenance of fittings, and regular rinsing. They were followed by another tap water sample until availability of a compliant microbiological result.

<figure><img src="image_5.png" /><figcaption>**Fig. 1.** Overview of the methodology used in the surgical intensive care unit for tap water sampling frequency. For period A, tap water of each room was collected on a quarterly basis. For period B, 3 groups were constituted (1, 2, 3), each of 5 or 6 rooms, and tap water was sampled for each one alternatively on a quarterly basis. (ª for group 1, ᵇ for group 2 and ᶜ for group 3).</figcaption></figure>
## Clinical samples

For period A, analysis was conducted retrospectively, and all patients with at least 1 *P. aeruginosa*-positive sample (carriage or infection) were included, whereas during period B, samples were collected prospectively (carriage or infection). Infected or colonized status was defined by the clinical presentation, since no screening sample (respiratory, digestive, or cutaneous) was realized. All isolates were identified by Matrix Assisted Laser Desorption Ionization - Time of Flight mass spectrometry (Microflex). Antimicrobial susceptibility testing was performed as described above, and isolates were stored at -80 °C.

## FTIR spectroscopy

All environmental *P. aeruginosa* strains and the first clinical isolate recovered from each patient were typed using FTIR spectroscopy (IR Biotyper; Bruker Daltonics). Isolates were cultured on Columbia blood agars (Thermo Fisher Scientific) and incubated for 24 ± 2 hours at 35 ± 2 °C as recommended.²² Isolates were prepared in a room with controlled temperature (22 ± 2 °C) and hygrometry (29% ± 2%). Each sample was treated with 50 µL of 70% ethanol and 50 µL of sterile water in a tube containing metal rods and then vortexed to obtain a uniform suspension. A volume of 15 µL of suspension was spotted in triplicates on the IRBT silicon plate (Bruker Daltonics). All plates were validated using 2 triplicates of the *P. aeruginosa* ATCC 27853 reference strain. Quality control was performed according to the manufacturer's recommendations. Average mean spectra were analyzed using the OPUS 7.5 software (Bruker). To define groups, that is, related isolates, we used the cutoff value provided by the manufacturer (0.2-0.25) and the results obtained with the ATCC 27853 strain to consider both intra- and interplate variations. The cutoff value referred to which distance the spectra were considered to be in the same group.

## Determination of sequence type

Sequence type (ST) of 1 isolate per group identified through FTIR typing method was determined. Briefly, genomic deoxyribonucleic acid was extracted using the kit Quick deoxyribonucleic acid Fungal/Bacterial miniprep kit (ZymoResearch). Concentration was measured using the nanodrop-automated system (Thermo Fisher Scientific). Extracts were sent to the BGI platform for library preparation and sequencing. MLST was performed using Qiagen CLC Genomics Workbench v24.0.1 (Qiagen).

## Transmission evaluation

Since no screening for *P. aeruginosa* was performed at the admission in the ICU, it was not possible to distinguish colonized from non-colonized patients at the admission. We defined HAIs as infections occurring after more than 48 hours of hospitalization, and in other cases, they were classified as imported. Interhuman cross-transmission event (CTE) was suspected between 2 patients if (1) they were hospitalized in the ICU over the same time, (2) at least one of them had an HAI, (3) the 2 isolates were similar when compared by FTIR, and (4) no similar isolate (by FTIR) was recovered from the environment. Environmental transmission event (ETE) was suspected if (1) similar isolates were found in both environmental and patient samples, (2) there was temporal and spatial correlation, that is, if a patient was hospitalized in a contaminated room. In other cases, the relation between patient and environment isolates was considered as undefined.

## Statistical analysis

To evaluate the impact of water system maintenance procedures, we compared data from the 2 periods of inclusion (before and after maintenance). For each one, proportions of contaminated taps and infected patients were compared using $\chi^2$ test and Fisher exact test. The difference was considered statistically significant if $P < .05$. The prevalence of *P. aeruginosa*-positive patients, either colonized or infected, was estimated using the number of hospitalized patients in this ICU during the study period.

## RESULTS

### Environmental samples

During the survey, 174 tap water samples were analyzed, 92 during period A and 82 during period B, and 20.7% (36/174) of them were positive for *P. aeruginosa* (Fig. 2). For period A, 27/92 (29.3%) taps were positive for *P. aeruginosa*, corresponding to 11 different rooms that were contaminated at least once. The distribution of positive samples was as follows: 3/17 (17.6%) in May, 7/17 (41.2%) in August, and 7/17 (41.2%) in November 2021. Tap waters were free of *P. aeruginosa* after, respectively, 1 and 2 additional measures in May and August 2021. However, in November 2021, 3 tap waters still yield positive cultures for *P. aeruginosa*, after 2 additional measures, requiring more in-depth treatment characterized by the installation of point-of-use filters and a chemical disinfection. In period B, 9/82

<figure><img src="image_6.png" /><figcaption>**Fig. 2.** Schematic workflow of environmental and clinical samples analysis during both periods. *Three samples were not typed because strains were not stored.</figcaption></figure>
<table><caption>**Table 1**
Main characteristics of patients, clinical samples, and antibiotic susceptibility</caption><thead><tr><th rowspan="2">Age, median (y)</th><th colspan="2">Patients positive with <i>Pseudomonas aeruginosa</i><br>(n = 42)</th><th rowspan="2"></th></tr><tr><th>Period A</th><th>Period B</th></tr></thead><tbody><tr><td>Sex, n (%)</td><td>30</td><td>12</td><td></td></tr><tr><td>Male</td><td>26 (86.6)</td><td>10 (83.3)</td><td>P > .5</td></tr><tr><td>Female</td><td>4 (13.3)</td><td>2 (16.6)</td><td>P > .5</td></tr><tr><td>Sample origins, n (%)<br>(n = 98)</td><td>63</td><td>36</td><td></td></tr><tr><td>Respiratory</td><td>36/63 (57.1)</td><td>19/36 (52.7)</td><td></td></tr><tr><td>Blood</td><td>19/63 (30.1)</td><td>9/36 (25)</td><td></td></tr><tr><td>Other<sup>a</sup></td><td>8/63 (12.7)</td><td>8/36 (22.2)</td><td></td></tr><tr><td>Antibiotic susceptibility<br>Piperacillin-tazobactam</td><td></td><td></td><td></td></tr><tr><td>Resistant (%)</td><td>32</td><td></td><td></td></tr><tr><td>Cefepime</td><td></td><td></td><td></td></tr><tr><td>Resistant (%)</td><td>16.6</td><td></td><td></td></tr><tr><td>Ceftazidime</td><td></td><td></td><td></td></tr><tr><td>Resistant (%)</td><td>21</td><td></td><td></td></tr><tr><td>Ciprofloxacin</td><td></td><td></td><td></td></tr><tr><td>Resistant (%)</td><td>2.8</td><td></td><td></td></tr><tr><td>Imipenem</td><td></td><td></td><td></td></tr><tr><td>Resistant (%)</td><td>32</td><td></td><td></td></tr><tr><td>Meropenem</td><td></td><td></td><td></td></tr><tr><td>Resistant (%)</td><td>11</td><td></td><td></td></tr></tbody></table>

(10.9%) taps were positive, corresponding to 4 different rooms, of which 3 were not contaminated in period A. For all water samples, no multidrug-resistant isolates were identified.

## Patients and clinical isolates

Overall, 42 patients were infected or colonized with *P aeruginosa*, providing a total of 99 clinical samples (Fig. 2). Thirty patients were identified during period A and 12 during period B. The majority of isolates were recovered from the respiratory tract (55/99, 55.5%) or blood cultures (28/99, 28.3%) (Table 1). Out of the 42 cases, 36 (85.7%) were suspected to be HAIs, including 9 bacteremia (including 4 related to VAP) and 19 VAP alone. Antibiotic susceptibility testing was performed on 72 isolates (Table 1).

<table><caption>**Table 2**
Distribution of grouped isolates from FTIR in periods A and B</caption><thead><tr><th rowspan="2">Groups<br>Period</th><th rowspan="2">ST</th><th colspan="2">Environmental samples</th><th colspan="2">Clinical samples</th><th colspan="4">Mixed<sup>a</sup></th></tr><tr><th>A</th><th>B</th><th>A</th><th>B</th><th colspan="2">Environmental samples</th><th colspan="2">Clinical samples</th></tr><tr><th></th><th></th><th></th><th></th><th></th><th></th><th>A</th><th>B</th><th>A</th><th>B</th></tr></thead><tbody><tr><td>G1</td><td>ST111</td><td></td><td></td><td></td><td></td><td>1</td><td></td><td>1</td><td></td></tr><tr><td>G2</td><td>ST111</td><td>3</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>G3</td><td>ST111</td><td>5</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>G4</td><td>ST298</td><td></td><td></td><td></td><td></td><td></td><td>2</td><td></td><td>2</td></tr><tr><td>G5</td><td>ST446</td><td></td><td></td><td></td><td></td><td>2</td><td></td><td>1</td><td></td></tr><tr><td>G6</td><td>†</td><td></td><td></td><td>2</td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>G7</td><td>ST253</td><td></td><td></td><td></td><td>2</td><td></td><td></td><td></td><td></td></tr><tr><td>G8</td><td>ST179</td><td></td><td></td><td></td><td></td><td>9</td><td>1</td><td>2</td><td>1</td></tr><tr><td>G9</td><td>ST179</td><td></td><td>4</td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>G10</td><td>ST1320</td><td></td><td></td><td>5</td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>G11</td><td>ST298</td><td></td><td></td><td></td><td></td><td></td><td>1</td><td>1</td><td>1</td></tr><tr><td>G12</td><td>†</td><td></td><td></td><td>1</td><td>1</td><td></td><td></td><td></td><td></td></tr><tr><td>G13</td><td>ST1228</td><td></td><td></td><td>1</td><td>1</td><td></td><td></td><td></td><td></td></tr></tbody></table>

NOTE. Distinctions were drawn according to the sample origin (environmental, clinical, or mixed).

FTIR, Fourier transform infrared; ST, sequence type.

a Clusters composed of environmental and clinical samples.

† Strains were not available for sequencing.

## Impact of the water network disinfection procedures

During period A, 29.3% (27/92) of tap water samples were positive, whereas only 10.9% (9/82) were positive in period B, revealing a significant decrease (P = .0028). Without additional measures, including another tap water sample, the rate decreased from 25% (17/68) to 8% (6/74), there again, the decrease was significant (P = .0109). Interestingly, 5/9 of tap water-positive samples during period B concerned only a single room. In addition, the prevalence of *P aeruginosa*-positive patients decreased from 2.6% to 1% (P = .0046), suggesting a link between environmental contamination and colonization/infection in patients.

## IR data and transmission evaluation

To determine acquisition routes of *P aeruginosa* infections, FTIR was employed to identify related isolates. To validate IRBT as a routine typing method, *P aeruginosa* ATCC 27853 reference strain was used as an internal standard and has been tested repeatedly, enabled us to calculate a cutoff value of 0.18, lower than the value provided by the manufacturer under similar conditions.

For both periods, 39 clinical and 36 environmental isolates were typed (Fig. 2). Among all the isolates, 50 were distributed in 13 different groups (from G1 to G13) (Table 2), and 25 were singletons. Grouped isolates were recovered from 22 clinical and 28 environmental samples, respectively. MLST typing was performed on 1 strain from 11 groups (strains from G6 and G12 were not available for sequencing), and no sequence type appears to be significantly predominant (see Table 2). Three groups were composed of only environmental isolates, 5 were composed of only clinical isolates, and 5 were mixed. Six rooms were contaminated by different *P aeruginosa* clones over the time of study. Four clones were found during both periods. Seventeen patients (43.6%) had singleton isolates. CTE was suspected in 2 cases involving, respectively, 2 patients (G6) and 5 patients (G10), representing 17.9% of the typed patients. Interestingly, these 2 clusters occurred in period A. Direct ETE was suspected in only 1 case (2.6%), since a combination of epidemiologic and typing data showed that the same *P aeruginosa* clone (G4) was first found in water, then from a patient hospitalized in the same room during the same period. This clone was susceptible to all tested antibiotics except for cefotaxime, imipenem, and meropenem. Another case (G1) was suspected to be an ETE, but the timing did not match because

<footnote>a Including urinary, abdominal, pus, and intravascular device samples.</footnote>
the patient was first positive and then secondly tap water. Fur-
thermore, it was difficult to correctly identify other environ-
mental-patient transmissions because patients and water samples
were grouped without any geographic or temporal match.

DISCUSSION

In this study, we evaluated the impact of maintenance proce-
dures comprising mechanical and chemical methods since routine
procedures (purge, maintenance of the faucet) were poorly effective
on long-term eradication of *P aeruginosa*.¹⁶ Second, we also tried to
describe the *P aeruginosa* routes of transmission in a surgical ICU
using a new fast-input bacterial typing technology.

Outbreak fine-tuned analysis and identification of several sources of *P. aeruginosa* constitute a difficult task but remain a key point in hospital-epidemic control. WGS represents an undeniable asset to resolve fine-scale relationships either in outbreak settings or in a nonepidemic context.<sup>16,24,15</sup> PFGE was also used in many studies as a reliable typing method.<sup>9,25,26</sup> As previously mentioned, these methods are time-consuming (PFGE) or not yet widely available (WGS) for real-time bacterial typing. FTIR is a quick and robust alternative typing method, mainly for gram-negative bacteria,<sup>22</sup> which showed reliable results for monitoring and comparing *P. aeruginosa* epidemiologic features.<sup>21,22</sup> Manufacturer's recommendations advocate a FTIR cutoff value of 0.20 to 0.25, but studies highlighted lower cutoff values by comparison to PFGE and WGS, respectively, of 0.184 to 0.374 and 0.132 to 173.<sup>21,22</sup> We then used FTIR to analyze *P. aeruginosa* route of transmissions without referring to other reference typing methods. Hence, our internal standard allowed to calculate a cutoff value of 0.18, a result similar to data already described, enabling consistent results to evaluate acquisition sources.<sup>21,22</sup>

At the end of 2021, an important disinfection procedure was con-
ducted because the routine procedure was not sufficient. Positive tap
water samples implied a proximal contamination of the water system in
the case of one-off contamination, but if the results were persistent, a
distal water system contamination was to be suspected. Despite several
additional measures between August and November, the contamination
was still evident, emphasizing a defective water system with probably
biofilms, thus requiring more in-depth treatment. First, point-of-use
filters were introduced on each outlet ICU room. This well-recognized
intervention showed successful results to limit contamination, but
paradoxically, it is the most expensive intervention<sup>17,18,27</sup> because filters
should be maintained and changed regularly. In addition, those filters
can also be contaminated.<sup>28</sup> As a result, prevention by filter introduction
is not a perennial option. Second, the water system was disinfected using
a combination of H<sub>2</sub>O<sub>2</sub> and silver ions. Silver-stabilized H<sub>2</sub>O<sub>2</sub>, a recent
extension of H<sub>2</sub>O<sub>2</sub>, is notably designed for water disinfection and was
shown to be highly effective in dental unit water treatment against
various microorganisms, including *P. aeruginosa*.<sup>29,30</sup> The precise me-
chanism of action is not yet fully elucidated, but the 2 components may
act synergistically, silver promoting interaction with bacterial surfaces,
thereby resulting in higher concentrations of H<sub>2</sub>O<sub>2</sub>.<sup>29</sup> Controls carried
out in all rooms at 7 days and 1 month were free of *P. aeruginosa* but for 1
room, demonstrating the successful procedure disinfection. Although the
methodology to collect environmental samples was slightly different
between both periods, which can underestimate the number of con-
taminated samples, a significant decrease was observed, sustainable over
time. *P. aeruginosa* strains in drinking water often exist in the viable but
not culturable state, making them undetectable by standard culture-
based methods,<sup>31</sup> which can also lead to an underestimation of their
presence. However, several *P. aeruginosa* clones were still found in tap
water after the decontamination, highlighting the difficulty to eradicate
environmental contamination of *P. aeruginosa* by the probable im-
portance of biofilm formation into hospital water system.<sup>16,32,33</sup>

Over the 2-year period, 42 patients were identified as positive for *P. aeruginosa*, most of whom were infected (88.1%), with an estimated

prevalence of 1.8% of hospitalized patients in this ICU. The majority of
infections were categorized as HAIs, suggesting a significant in-ICU ac-
quisition rate, but probably overestimated because there was no initial
screening on admission. Several studies attest various carriage rates at
ICU admission: 11.6%,<sup>34</sup> 17%,<sup>26</sup> and 55.4%.<sup>24</sup> Such differences can be ex-
plained by sampling technique (rectal, cutaneous, and respiratory) as
well as by their repeatability<sup>24,35</sup> or local ecology. In our study, no
screening was performed at ICU admission, but FTIR revealed that
43.60% of positive patients were colonized or infected by unique isolates,
suggesting an endogenous origin. This hypothesis was also supported by
others, establishing an endogenous origin if patients had a unique
*P. aeruginosa* genotype without any concordance with other patients or
faucet samples.<sup>25</sup> The relevance of a *P. aeruginosa* screening could thus be
raised, not necessarily for multidrug-resistant *P. aeruginosa* detection but
rather to identify colonized ICU patients that are more frequently at risk
of subsequent infections.<sup>34</sup>

Endogenous origins were not predominant for *P. aeruginosa* HAIs, highlighting a significant part of exogenous contamination mediated through health care workers (HCWs) or the hospital environment. We tried to identify these situations by distinguishing CTE and ETE transmission routes. Through FTIR and our defined criteria, we found that 17.9% of patients were involved in CTE, whereas ETE concerned only 2.6%. These results support that exogenous acquisition is mediated by HCW care practices rather than by the environment, but these findings must be interpreted with caution. Indeed, the water system seems to be continually contaminated by *P. aeruginosa*, but the participation of environment to cross-transmission is unclear as the majority of environmental-patient situations were not concordant. Identical clones were found in tap water and then in patients without any temporal or localization matches, so the implication of the environment as a source of the infection was not clearly proven but may suggest connections between different transmission sources. This assumption is based on differences between the 2 periods in both tap water and clinical samples. After the important disinfection procedure, a significant decrease of contaminated tap water samples was observed, and the proportion of positive *P. aeruginosa* patients was significantly lower in period B. HCWs represent an interface between contaminated environments and patients. During the 2 periods, care practices did not change, but hand hygiene with alcohol-based hand rub was not optimal, patient waste waters were disposed of down handwash basins, and faucet maintenance was not entirely effective. Thus, we speculate that environmental water is a source of contamination, but transmission to patients is rather induced by HCWs and their associated care practices. These findings are also consistent with the important number of CTEs and the large number of relation patient-environment unclassified that we observed. Our results are similar with those described recently,<sup>24</sup> reported higher proportions of CTEs through HCWs and endogenous acquisition than acquisition from water-mediated environment. In contrast, other studies<sup>9,25,17,18</sup> reported important rates of water exogenous acquisition, without necessarily considering epidemiologic spatiotemporal data, and the hub that HCWs can represent.

CONCLUSION

Above all, this study demonstrates (1) the relative successfulness of the water system chemical disinfection and (2) the utility of the IRBT as a quick, low-cost, real-time typing tool for the monitoring of *P. aeruginosa* strains. Indubitably, *P. aeruginosa* HAIs are a recurrent threat in the ICUs, but the origins of infection are still under debate. Monitoring simultaneously water samples and patients, including screening samples, could be a sophisticated option but essential to get more about the origins of *P. aeruginosa* infections. A key point is also HCW practices in alcohol-based hand rub, disposal of wastewater, and faucet maintenance to limit *P. aeruginosa* cross-transmission but also biofilm formation in proximal water system.
Acknowledgments

We acknowledge the technical staff of the Service de Bactériologie-Hygienne Hospitalière of Centre Hospitalier Universitaire de Rennes for excellent work.

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