# Spatio-temporal analysis of infra-specific genetic variations among a Pseudomonas aeruginosawater network hospital population: invasion and selection of clonal complexes

Rachel Lavenir, M. Sanroma, S. Gibert, Olivier Crouzet, F. Laurent, J.

Kravtsoff, M.-A. Mazoyer, Benoît Cournoyer

► To cite this version:

Rachel Lavenir, M. Sanroma, S. Gibert, Olivier Crouzet, F. Laurent, et al. Spatio-temporal analysis of infra-specific genetic variations among a Pseudomonas aeruginosawater network hospital population: invasion and selection of clonal complexes. Journal of Applied Microbiology, 2008, 105 (5), pp.1491-1501. 10.1111/j.1365-2672.2008.03907.x. hal-01604717

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ORIGINAL ARTICLE

# Spatio-temporal analysis of infra-specific genetic variations among a *Pseudomonas aeruginosa* water network hospital population: invasion and selection of clonal complexes

R. Lavenir¹,², M. Sanroma¹,², S. Gibert¹,², O. Crouzet¹,², F. Laurent¹, J. Kravtsoff¹,³, M.-A. Mazoyer¹,³ and B. Cournoyer¹,²

1 Université de Lyon, Lyon, France

2 Research group on 'Bacterial Opportunistic Pathogens and Environment', UMR5557 Ecologie Microbienne, Université Lyon 1, CNRS, and Ecole Nationale Vétérinaire de Lyon, France

3 Laboratoire de Microbiologie, ISPB, Université Lyon 1, France

**Keywords**

cross-contamination, faecal carriage, hospital,
*Pseudomonas aeruginosa*, water.

**Correspondence**

Benoit Cournoyer, UMR CNRS 5557 Ecologie Microbienne, Mendel Bldg., 5th floor, Université Lyon 1, 69 622 Villeurbanne Cedex, France.

E-mail: cournoye@biomserv.univ-lyon1.fr

2008/0311: received 22 February 2008,
revised 7 April 2008 and accepted 9 April
2008

doi:10.1111/j.1365-2672.2008.03907.x

**Abstract**

**Aims:** To investigate infra-specific spatio-temporal dynamics of a hospital water network *Pseudomonas aeruginosa* population. To infer the origin of water network isolates and assess their potential health hazard.

**Methods and Results:** 168 *P. aeruginosa* strains were isolated from tap waters and swabs of tap nozzle aerators of a hospital unit, over 2 years, and from rectal swabs and nosocomial infections. Genetic diversity among this collection was assessed by pulsed field gel electrophoresis of *SpeI* restricted genomic DNA. Virulence gene sets, biofilm properties, and hypochlorite resistance were analysed. Exactly 68% of the water samples and 74% of the tap nozzle aerators harboured *P. aeruginosa*. The strains were divided into 22 clonal lineages, with one dominant clone shown to have been involved in a nosocomial infection.

**Conclusions:** An important turnover among the *P. aeruginosa* hospital population was observed. Some clonal lineages were found to persist, spread in the unit, and diversify into clonal complexes. Rectal carriage appeared an important source of contamination of the water network.

**Significance and Impact of the Study:** High *P. aeruginosa* infra-specific population diversity suggested a broad ability in colonizing water networks but persistence analysis indicated a strong selection leading to the emergence of dominant clones.

**Introduction**

Nosocomial infections are of increasing concern.
A European 1-day point prevalence study highlighted
that 31% of intensive care unit (ICU) patients can
acquire nosocomial infections (Vincent 2000). The mor-
tality associated with these infections was estimated to
be between 10% and 35% (Vincent 2000; Garcia-Martin
et al. 2001). The aetiological agents involved are mainly
bacterial opportunistic pathogens, and *Pseudomonas*
*aeruginosa* was considered one of the most predominant
species (Vincent 2000). *Pseudomonas aeruginosa* can
cause wound infections, septicemia, pneumonia, endo-

carditis, otitis, and keratitis (McManus *et al.* 1985; Rich-
ard *et al.* 1994). It is associated with high morbidity and
mortality in patients with cystic fibrosis (CF; Saiman
and Siegel 2004).

Epidemiological studies carried out in hospital ICU
showed that 50% of *P. aeruginosa* nosocomial infections
are due to cross-contamination between patients or
patients and hospital staff. The other 50% was suggested
to involve other sources like water networks (Bergmans
et al. 1998; Bertrand et al. 2001). Rectal carriage of
*P. aeruginosa* might explain some of these cross-contami-
nations between individuals. Around 20% of the human
population shows rectal carriage of these bacteria, and
this proportion can increase dramatically in a hospital situation (Yoshioka *et al.* 1983; Bonten *et al.* 1999; Bertrand *et al.* 2001). However, *P. aeruginosa* also shows a predilection for wet environments. At the hospital, it can be frequently isolated from water networks (Trautmann *et al.* 2005), including drains of sinks and showers. From 16% (Lasheras *et al.* 2006) to 60% (Reuter *et al.* 2002) of hospital water networks can be contaminated by *P. aeruginosa*. Analyses making use of molecular typing methods highlighted that 35% (Reuter *et al.* 2002) to 50% (Petignat *et al.* 2006) of *P. aeruginosa* nosocomial infections were related to strains found in these networks. The use of tap disposable sterile filters was thus recommended to reduce such infections (Martino *et al.* 1985; Vianelli *et al.* 2006). Additionally, the use of thermal shocks was proposed to prevent *P. aeruginosa* outlets contamination (van der Mee-Marquet *et al.* 2005). Nevertheless, some authors consider these practices not sufficiently reliable, and recommended the use of bottled water and hand disinfection to limit the spread of *P. aeruginosa* (Petignat *et al.* 2006). It is noteworthy that most of the studies concerning the spread of *P. aeruginosa* in hospital units were performed in intensive care ones, and were rarely exhaustive and performed for a prolonged period of time. They mainly concerned nosocomial outbreaks.

In fact, little is known about the natural abundance, diversity, persistence, and spread of *P. aeruginosa* in hospital units. There are actually few data about the spatiotemporal dynamics of *P. aeruginosa* hospital populations, and about the processes that can allow these bacteria to live in water pipes and other hospital niches. It is not known if the turnover among these populations is important or if there are resident clones that might not necessarily cause infections. Understanding the organization of a *P. aeruginosa* hospital population could thus reveal particular evolutionary trends that might play a role in the emergence of epidemic clones.

Here, we present the first report of a long-term study aiming at investigating the fate of a natural hospital water network population of *P. aeruginosa*. This study was performed in a unit undergoing classical hygienic practices, with disinfection and replacement of tap nozzle aerators according to weekly microbiological quality analyses of the water network. The unit welcomes, for long stays, medullary, cranial traumatized, polytraumatized wounded patients, or patients with important after-effects of cerebral vascular accidents. This report presents the data collected over a 2-year period. *Pseudomonas aeruginosa* population of this network appeared highly diverse, with an important turnover but some persistent clones. A persistent clone was found to dominate the network, and be a health hazard.

# Materials and Methods

## Description of the hospital unit

A long-stay care unit of the Hospices Civils de Lyon (HCL - Université Lyon 1) was selected for this study. This unit has 22 beds located in 14 rooms. Each room contains a sink, with the exception of rooms 5 and 12 that have two. Two external taps, one located outdoor, on a wall of the building, and one located in the maintenance cabinet (after room 14) of the unit, were included as controls. The unit is supplied by a single water pipe that goes from room 1 to 14, and stops at the maintenance cabinet.

## Sampling strategy

Samplings of the cold water network were performed from September 2004 to May 2006, at the following dates: September, October, November and December 2004, January, February and March 2005, and May 2006. A total of 91 water samples were analysed. Water (100 ml) was collected in sterile flasks containing 10 mg of thiosulfate, after opening each tap for 1 min. Fifty-three swabs of tap nozzle aerators were collected in November 2004, February and March 2005, and May 2006. The taps were routinely disinfected throughout the study, by the clinical staff of the unit. Eight months before the last sampling time, a general descaling of the network was performed, and tap nozzle aerators were changed. In October 2004, February 2005, and March 2005, 50 rectal swabs from 43 patients were collected by the medical staff. *Pseudomonas aeruginosa* strains from nosocomial infections (16 patients) that occurred in the unit were collected from June 2005 to May 2006 (Table 1). These infections are considered as being part of the normal background of this long-stay care unit.

## *Pseudomonas aeruginosa* platings, identification and numerations

To isolate *P. aeruginosa* from the water samples, 100 ml was filtered through sterile 0·22-µm filters (Millipore, Molsheim, France). These filters were then transferred onto plates containing a medium named PAB-CN, and which is a *Pseudomonas* agar base medium (Oxoid, Dardilly, France) supplemented with *cetrimide* (200 mg l⁻¹) and *nalidixic* acid (15 mg l⁻¹). The plates were incubated for 48 h at 37°C. An enrichment step was used to isolate *P. aeruginosa* from tap nozzle aerators and patients’ rectal swabs. This step required an inoculation, using the swabs, of 5 ml of Luria-Bertani (LB) broths supplemented with Irgasan, as described previously (Fonseca *et al.* 1986). These broths were incubated for 18 h at 37°C with shaking at 180 rev min⁻¹, before plating serial dilutions on PAB-CN.
<table><caption>**Table 1** Selection of *Pseudomonas aeruginosa* strains isolated from background cases of nosocomial infections among the investigated hospital unit</caption><thead><tr><td>Strain</td><td>Origin</td><td>Date</td></tr></thead><tbody><tr><td>poe115</td><td>Sputum</td><td>2006 March</td></tr><tr><td>poe116</td><td>Bronchial aspiration</td><td>2006 March</td></tr><tr><td>poe117</td><td>Sputum</td><td>2006 March</td></tr><tr><td>poe118</td><td>Bedsore</td><td>2006 March</td></tr><tr><td>poe119</td><td>Sputum</td><td>2006 March</td></tr><tr><td>poe120</td><td>Bronchial aspiration</td><td>2006 March</td></tr><tr><td>poe121</td><td>Bronchial aspiration</td><td>2006 April</td></tr><tr><td>poe122</td><td>Bronchial aspiration</td><td>2006 April</td></tr><tr><td>poe123</td><td>Urine</td><td>2006 May</td></tr><tr><td>poe124</td><td>Urine</td><td>2006 March</td></tr><tr><td>poe125</td><td>Urine</td><td>2006 January</td></tr><tr><td>poe126</td><td>Urine</td><td>2006 February</td></tr><tr><td>poe127</td><td>Urine</td><td>2006 January</td></tr><tr><td>poe128</td><td>Blood culture</td><td>2005 June</td></tr><tr><td>poe129</td><td>Blood culture</td><td>2006 February</td></tr><tr><td>poe130</td><td>Sputum</td><td>2006 May</td></tr></tbody></table>

Colony-forming units (CFU) obtained on PAB-CN were divided into morphotypes. From 2 to 10 CFU per morphotype and sampling times were analysed using biochemical tests (oxidase assay and pyocyanine detection under ultraviolet light), and the *P. aeruginosa* *ecfX* polymerase chain reaction (PCR) assay (Lavenir *et al.* 2007) that is described in the next section. *Pseudomonas aeruginosa* plate counts were estimated according to the results of this identification scheme.

*Pseudomonas aeruginosa* resistance to sodium hypochlorite was tested according to previous studies (Mazzola *et al.* 2006; Small *et al.* 2007), by plating strains on LB containing 1·5% (w/v) agar and 1% (v/v) sodium hypochlorite (Sigma Aldrich, Saint-Quentin Fallavier, France). Strains showing growth within 7 days at 37°C were considered resistant.

### PCR analyses

PCR (*Taq* polymerase chain reaction) DNA amplifications of *ecfX* (Lavenir *et al.* 2007), *lasI*, *lasR*, *rhlI* and *rhlR* (Schaber *et al.* 2004), *lasA* (Lomholt *et al.* 2001), *lasB*, *aprA* and *rhlAB* (Zhu *et al.* 2004), *algD* (Beatrice *et al.* 2005), *exoS*, *exoT*, *exoU* and *exoY* (Ajayi *et al.* 2003), and *exoA* (Khan and Cerniglia 1994) were performed as previously described (see Table 2). These PCR were carried out in 50 µl reaction mixture containing 2·5 U *Taq* polymerase (Invitrogen), 200 µmol l⁻¹ dNTP, 1 × PCR reaction buffer (Invitrogen), 2 mmol l⁻¹ MgCl₂, 10% dimethyl sulfoxide (DMSO), and 0.5 µmol l⁻¹ of each primer. PCR were performed directly on bacterial cells, using a Rapid-Cycler (MJ Research Inc. PTC-100, USA) that was set with an initial DNA denaturation step of 5 min at 95°C,

35 cycles of 45 s at 94°C, 90 s at the appropriate annealing temperature (see Table 2), and 45 s at 72°C, and a final elongation for 5 min at 72°C. The PCR products were visualized by electrophoresis using a 2% agarose gel, staining with ethidium bromide, and exposure to UV light. Each PCR was at least performed twice.

### Pulsed field gel electrophoresis (PFGE) of *SpeI* DNA typings

PFGE analyses of *SpeI* restricted genomic DNA (PFGE-*SpeI*) were performed as described previously (Romling *et al.* 1994). PFGE was performed using a CHEF-DR III apparatus (Bio-Rad), set at 5.0 V cm⁻¹, with a linear ramping from 5 to 25 s for 11 h and 5 to 60 s for 15 h. DNA patterns were transformed into a 0/1 discrete character matrix. The matrix was analysed by the maximum parsimony (MP) method (using the Wagner model that assumes that all changes between all states are allowed and count equally) and the consensus program of the *PHYLIP* 3·5 package (Retief 2000). The consensus dendrogram of all the computed trees was visualized using *MACCLADE* 3·04 (Maddison and Maddison 1989). A cut off, set at less than seven band changes between pairs of profiles (following the recommendations of Romling *et al.* 1994), was introduced in the analysis of the PFGE data set, and used to identify reliable PFGE *SpeI* groupings. Other groups were not considered.

### Biofilm assay

Biofilm formation was measured as described previously (O'Toole and Kolter 1998) with the following modifications. Bacterial strains were grown overnight without agitation in LB medium, resuspended in M63 medium supplemented with 0·05% casamino acids and 0·2% glucose to an OD₆₀₀ of 1, and incubated in 96-well microtiter plates (Becton Dickinson, Le-Pont-de-Claix, France) at 37°C for 24 h. Biofilm formation was measured by crystal violet (0·1% w/v) staining and spectrophotometric analysis at OD₆₀₀. The strains were considered as very good biofilm (B+) producers when giving a biofilm formation value equivalent to 120% (or more) of one of the PAO1 reference strains. Otherwise, the strains were considered normal or weak biofilm producers.

### Results

#### Detection and plate count numbers of *Pseudomonas aeruginosa* in the water network

Ninety-one cold tap waters and 53 tap nozzle aerators were screened for the presence of *P. aeruginosa* using the
<table><caption>**Table 2** Polymerase chain reaction (PCR) primers and their respective annealing temperatures. Targeted genes and PCR size products are indicated</caption><thead><tr><th>Primer</th><th>Oligonucleotide sequence (5'-3')</th><th>Annealing temp. (°C)</th><th>Targeted gene, product size (pb)</th><th>Reference</th></tr></thead><tbody><tr><td>ECF1</td><td>ATGGATGAGCGCTTCCGTG (19)</td><td>58</td><td>ecfX, 528</td><td>Lavenir et al. 2007</td></tr><tr><td>ECF2</td><td>TCATCCTTCGCCTCCCTG (18)</td><td>58</td><td>ecfX, 528</td><td>Lavenir et al. 2007</td></tr><tr><td>lasI-fp</td><td>TCGACGAGATGGAAATCGATG (21)</td><td>59</td><td>lasI, 363</td><td>Schaber et al. 2004</td></tr><tr><td>lasI-rp</td><td>GCTCGATGCCGATCTTCAG (19)</td><td>59</td><td>lasI, 363</td><td>Schaber et al. 2004</td></tr><tr><td>lasR-fp</td><td>ATGGCCTTGGTTGACGGTT (19)</td><td>50</td><td>lasR, 725</td><td>Schaber et al. 2004</td></tr><tr><td>lasR-rp</td><td>GCAAGATCAGAGAGTAATAAGACCCA (26)</td><td>50</td><td>lasR, 725</td><td>Schaber et al. 2004</td></tr><tr><td>rhll-fp</td><td>CGAATTGCTCTCTGAATCGCT (21)</td><td>59</td><td>rhll, 143</td><td>Schaber et al. 2004</td></tr><tr><td>rhll-rp</td><td>GGCTCATGGCGACGATGTA (19)</td><td>59</td><td>rhll, 143</td><td>Schaber et al. 2004</td></tr><tr><td>rhIR-fp</td><td>TCGATTACTACGCCTATGGCG (21)</td><td>59</td><td>rhIR, 207</td><td>Schaber et al. 2004</td></tr><tr><td>rhIR-rp</td><td>TTCCAGAGCATCCGGCTCT (19)</td><td>59</td><td>rhIR, 207</td><td>Schaber et al. 2004</td></tr><tr><td>lasA-f</td><td>CGCCATCCAACCTGATGCAAT (21)</td><td>56</td><td>lasA, 514</td><td>Lomholt et al. 2001</td></tr><tr><td>lasA-r</td><td>AGGCCGGGGTTGTACAACGGA (21)</td><td>56</td><td>lasA, 514</td><td>Lomholt et al. 2001</td></tr><tr><td>lasB-f</td><td>TTCTACCCGAAGGACTGATAC (21)</td><td>52</td><td>lasB, 153</td><td>Zhu et al. 2004</td></tr><tr><td>lasB-r</td><td>AACACCCATGATCGCAAC (18)</td><td>52</td><td>lasB, 153</td><td>Zhu et al. 2004</td></tr><tr><td>aprA-f</td><td>ACCCTGTCCTATTCGTTC (19)</td><td>52</td><td>aprA, 140</td><td>Zhu et al. 2004</td></tr><tr><td>aprA-r</td><td>GATTGCAGCGACAACTTGG (19)</td><td>52</td><td>aprA, 140</td><td>Zhu et al. 2004</td></tr><tr><td>rhlAB-f</td><td>TCATGGAATTGTCACAACCGC (21)</td><td>52</td><td>rhlAB, 151</td><td>Zhu et al. 2004</td></tr><tr><td>rhlAB-r</td><td>ATACGGCAAAATCATGGCAAC (21)</td><td>52</td><td>rhlAB, 151</td><td>Zhu et al. 2004</td></tr><tr><td>algD-f</td><td>GCGACCTGGACCTGGGCT (18)</td><td>56</td><td>algD, 180</td><td>Beatrice et al. 2005</td></tr><tr><td>algD-r</td><td>TCCTCGATCAGCGGGATC (18)</td><td>56</td><td>algD, 180</td><td>Beatrice et al. 2005</td></tr><tr><td>exoS-f</td><td>GCGAGGTCAGCAGAGTATCG (20)</td><td>58</td><td>exoS, 118</td><td>Ajayi et al. 2003</td></tr><tr><td>exoS-r</td><td>TTCGGCGTCACTGTGGATGC (20)</td><td>58</td><td>exoS, 118</td><td>Ajayi et al. 2003</td></tr><tr><td>exoU-f</td><td>CCGTTGTGGTGCCGTTGAAG (20)</td><td>58</td><td>exoU, 134</td><td>Ajayi et al. 2003</td></tr><tr><td>exoU-r</td><td>CCAGATGTTCACCGACTCGC (20)</td><td>58</td><td>exoU, 134</td><td>Ajayi et al. 2003</td></tr><tr><td>exoT-f</td><td>AATCGCCGTCCAACTGCATGCG (22)</td><td>58</td><td>exoT, 152</td><td>Ajayi et al. 2003</td></tr><tr><td>exoT-r</td><td>TGTTCGCCGAGGTACTGCTC (20)</td><td>58</td><td>exoT, 152</td><td>Ajayi et al. 2003</td></tr><tr><td>exoY-f</td><td>CGGATTCTATGGCAGGGAGG (20)</td><td>58</td><td>exoY, 289</td><td>Ajayi et al. 2003</td></tr><tr><td>exoY-r</td><td>GCCCTTGATGCACTCGACCA (20)</td><td>58</td><td>exoY, 289</td><td>Ajayi et al. 2003</td></tr><tr><td>eta1B</td><td>AACCAGCTCAGCCACATGTC (20)</td><td>65</td><td>exoA, 207</td><td>Khan and Cerniglia 1994</td></tr><tr><td>eta2</td><td>CGCTGGCCCATTCGCTCCAGCGCT (24)</td><td>65</td><td>exoA, 207</td><td>Khan and Cerniglia 1994</td></tr></tbody></table>

PAB-CN medium. Seven morphotypes, listed from the highest to lowest population size number, could be revealed using this medium: (i) small (<2 mm of diameter) pink circular colonies, (ii) medium size (around 4 mm in diameter) blue/green viscous circular colonies, (iii) large (more than 5 mm) dark pink circular colonies, (iv) medium size yellow viscous colonies, (v) very small (less than 1 mm) yellow colonies, (vi) medium size dark yellow colonies, and (vii) large white viscous colonies. For each sample, two to ten isolates per morphotype were kept for further analysis. These isolates were grown on the King's B medium to check their ability at producing a fluorescein. Their ability to produce an oxidase was also tested. The *P. aeruginosa* *ecfX* PCR screening was used to finalize the identification scheme. Only morphotypes 2, 3, and 4 were confirmed, using these tests, to belong to the *P. aeruginosa* species. *Pseudomonas aeruginosa* plate counts were thus limited to these three morphotypes, and are given in Table 3. Plate counts are not given for tap nozzle aerators because an Irgasan enrichment step was required.

Using this procedure, cold tap water samples or swabs of tap nozzle aerators of the sink of each room, including the one dedicated to maintenance, were found to have contained *P. aeruginosa* cells (Table 3). Overall, 68% (61/91) of the water samples, and 74% (39/53) of the tap nozzle aerators harboured *P. aeruginosa*. The abundance of *P. aeruginosa* in water was highly variable according to the room and sampling time. It could vary from undetectable *P. aeruginosa* cells in 100 ml of cold water to 1000 cells per millilitre, as observed in room 13 (Table 3). The cold water samples of rooms 2, 5, 7, 8, 9, and 10 were found positive for *P. aeruginosa* on all sampling dates (Table 3).

## Diversity, persistence and spread of *Pseudomonas aeruginosa* water network isolates

One hundred and twenty-seven strains of the water network were kept for further analyses. Seventy-six of these strains were isolated from water samples, and 51 from tap nozzle aerators. PFGE-SpeI analysis of these strains was
<table><caption>**Table 3** *Pseudomonas aeruginosa* tap water plate count numbers and detection from swabs of tap nozzle aerators, from September 2004 to May 2006. Platings were performed on a *Pseudomonas* agar base supplemented with cetrimide and nalidixic acid</caption><thead><tr><th>Room</th><th>Tap waters<br>(CFU ml<sup>-1</sup>)</th><th>Tap nozzle<br>aerators*</th></tr></thead><tbody><tr><td>Outdoor building tap</td><td>0</td><td>-</td></tr><tr><td>1</td><td>0</td><td>- to +</td></tr><tr><td>2</td><td>15-20</td><td>+</td></tr><tr><td>3</td><td>0-20</td><td>- to +</td></tr><tr><td>4</td><td>0-15</td><td>- to +</td></tr><tr><td>5</td><td>9-182</td><td>+</td></tr><tr><td>6</td><td>0-206</td><td>+</td></tr><tr><td>7</td><td>101-208</td><td>+</td></tr><tr><td>8</td><td>7-152</td><td>+</td></tr><tr><td>9</td><td>20-82</td><td>+</td></tr><tr><td>10</td><td>1-141</td><td>+</td></tr><tr><td>11</td><td>0-52</td><td>+</td></tr><tr><td>12</td><td>0-312</td><td>- to +</td></tr><tr><td>13</td><td>0-1000</td><td>+</td></tr><tr><td>14</td><td>0-52</td><td>- to +</td></tr><tr><td>Maintenance cabinet</td><td>0-2</td><td>-</td></tr></tbody></table>

performed. The PFGE-SpeI profiles were converted into a matrix of discrete characters and analysed by a parsimony method that allows tracing the number of band changes between pairs of profiles. This analysis allowed to infer a dendrogram grouping strains according to the number of differences observed between each pair of PFGE-SpeI profiles, and to generate branch lengths matching the number of changes. This dendrogram is shown in Fig. 1. Strains showing identical SpeI PFGE profiles were defined as being individuals of a same clone. Only PFGE-SpeI groups showing up to six differences were considered to be reliable (as demonstrated by Romling *et al.* 1994). These groups were hereafter termed clonal complexes. Exactly 86% (19 over 22) of the SpeI PFGE groupings showed four or less DNA band changes. Figure 1 shows the relatedness between the 133 PFGE-SpeI profiles (available upon request) observed among the 168 strains analysed in this study (127 strains from the water network, and 25 rectal and 16 nosocomial strains that are presented in the next section). Figure 2 shows PFGE-SpeI profiles of a selection of clones and clonal complexes revealed during this study.

Thirty-two PFGE-SpeI profiles were recorded among the water network (Fig. 1). Groups 1, 8, 9, 13, 14, 15, 19, 20, and 22 were made of strictly identical PFGE-SpeI profiles and were thus defined as clones (e.g. see Fig. 2 to visualize the PFGE-SpeI profiles of clones 13, 14, 19, and 22). The other groups (2–4, 6, 11, 16–18, and 21) were considered to reveal clonal complexes of strains (e.g. see Fig. 2 to

visualize the PFGE-SpeI profiles of complexes 16 and 18). The most frequent PFGE-SpeI profile, observed at various sampling times and in several rooms, was the one of clone 22 (Fig. 2). This clone represented 24% (41/171) of the water network isolates detected in this study. Complexes or clones 1, 2, 3, 4, 6, 8, 9, 11, 15, 16, 17, 19, 20, 21, and 22 included two or more strains isolated from distinct samples of the water network. Figure 3a and b illustrate the ability of these groups of strains to persist and spread in the medical unit. Over the 2-year period of this study, some groups (1, 9, 16, 17, 19, 21, and 22) were detected at least thrice (Fig. 3a), and some (groups 1, 16, 21, and 22) were detected among at least three rooms (Fig. 3b).

## PFGE-SpeI analysis of nosocomial and rectal

### *Pseudomonas aeruginosa*

Some nosocomial *P. aeruginosa* isolates were collected and analysed during this study (Table 1). Moreover, 25 rectal carriage strains, coming from 25 out of 43 sampled patients (58%), were isolated using an enrichment procedure for *P. aeruginosa* (see Fig. 1 for the sampling dates). The nosocomial (n = 16) and rectal (n = 25) strains were analysed by the PFGE-SpeI approach (Fig. 1). These strains showed a relatively greater genetic diversity than the one observed for the water network isolates. Thirty-eight distinct PFGE-SpeI profiles were observed while 32 out of 171 were recorded for the water network isolates. PFGE-SpeI profiles of clones 13 and 14, and of clonal complex 18, were obtained from strains isolated from tap nozzle aerators and rectal swabs (Figs 2 and 3c). The most frequent PFGE-SpeI profile recorded among the water network isolates, i.e. clone 22 (see previous section), was recorded among the nosocomial isolates (Fig. 3c). Groups 1, 2, 3, 4, 6, 8, 9, 11, 15, 16, 17, 19, 20, and 21 did not show any rectal or nosocomial isolate. Clonal complexes 5 and 10, and clone 12, only harboured profiles from strains isolated from rectal swabs. PFGE-SpeI profiles of clone 7 were only recorded among nosocomial isolates (Figs 1 and 3c).

## Phenotypic characters

All the strains collected in this study were tested for their ability in forming biofilms and in growing with a bactericidal concentration of sodium hypochlorite. The ability in forming a biofilm was variable among these strains but strains with PFGE-SpeI profiles matching clones 6 and 22, and clonal complexes 16 and 17, showed very high capacities (indicated on Fig. 1 by a B+); mean OD<sub>600</sub> values (±SD), indicative of biofilm formation, were respectively 0.94 ± 0.18, 0.94 ± 0.20, 0.92 ± 0.21, and 1.54 ± 0.32, for these clones and clonal complexes. The biofilm formation

<footnote>*Involved an Irgasan enrichment step.</footnote>
<figure><img src="image_2.png" /></figure>
<figure><img src="image_3.png" /><figcaption>**Figure 2** Pulsed field gel electrophoresis profiles of *Spel* restricted genomic DNA of a selection of clones and clonal complexes of *Pseudomonas aeruginosa* detected in this study. These profiles (indicated by CC) were numbered, and these numbers match those indicated on Figs 1 and 3. Scale inferred from a DNA size marker is shown. poe lab codes match those of Fig. 1.</figcaption></figure>

OD~600~ value for *P. aeruginosa* strain PA01 was 0.60 ± 0.13. Some nosocomial and rectal strains not belonging to these groups, i.e. poe50, poe116, poe117, poe119, poe120, and poe127, also showed high production of biofilms with mean OD~600~ values (±SD), respectively, of 0.84 ± 0.10, 0.80 ± 0.12, 1.12 ± 0.23, 0.86 ± 0.16, 0.82 ± 0.15, and 0.92 ± 0.25. Strains belonging to the PFGE-SpeI groups 3, 4, 6, 15, 16, and 22, and the nosocomial strains poe50, poe82, and poe119 were found to be able to grow with a bactericidal concentration of sodium hypochlorite (indicated on Fig. 1 by CH+).

## Detection of *Pseudomonas aeruginosa* virulence genes

Fourteen genes involved in *P. aeruginosa* virulence properties were searched among the waterborne, rectal, and nosocomial isolates of this study: *exoS*, *exoT*, *exoU*, and *exoY* that encode cytotoxins secreted by the type three secretion system; *lasA*, *lasB*, and *aprA* that encode, respectively, elastase A, elastase B, and an alkaline protease; *algD*, a gene involved in alginate production; *toxA* that encodes a cytotoxin with an ADP-ribosyl transferase activity; *rhlAB*, involved in the synthesis of biosurfactants named rhamnolipids; and *lasI*, *lasR*, *rhlI*, and *rhlR* that

are involved in quorum-sensing and genetic regulation of virulence factors. All these genes, except *exoU* and *exoS*, were detected among all the strains tested. *exoU* was detected among 53% (71/133) of the strains, and *exoS* among 47% (62/133). Strains harbouring *exoU* are indicated by an asterisk in Fig. 1.

## Discussion

*Pseudomonas aeruginosa* is one of the most important aetiological agents of nosocomial infections. Several studies have investigated the sources of contamination of ICU patients by *P. aeruginosa*, and showed that cross-contamination between patients and water networks could explain several nosocomial outbreaks. However, strains involved in these outbreaks were considered transient colonizers of the hospital units, with multi-resistant strains emerging from the successive uses of antibiotic therapies. Rarely, a *P. aeruginosa* population of a unit was analysed outside the scope of an epidemic outbreak. Here, we have initiated a long-term study addressing issues regarding *P. aeruginosa* colonization of a long-stay hospital care unit (looking after medullary, cranial traumatized, polytraumatized wounded patients, or patients with

**Figure 1** Dendrogram showing the number of DNA band changes between pair of pulsed field gel electrophoresis profiles of *Spel* restricted genomic DNA of the hospital *Pseudomonas aeruginosa* isolates; 168 strains were analysed. Branch length is proportional to the number of changes between a pair of profiles. Strains were given poe lab numbers and an additional code indicating their origin and their sampling date. W indicates a strain isolated from a water sample; A indicates a strain isolated from a tap nozzle aerator (grid); R, from a rectal swab; and P, from an infected patient. Room numbers, going from 1 to 14, are indicated after these one-letter codes, and the maintenance cabinet is indicated by 'mc'. After the room number or code, the sampling date is indicated by a three-letter code for the month, followed by the last number of the year (from 2004 to 2006). In parentheses, the number of strains isolated from the same sample and showing identical profiles is indicated. Ledges numbered from 1 to 22, in circles, indicate clones and clonal complexes. Strains, clones, or clonal complexes showing a B+ on their respective tree branch had a high capacity to form biofilms, and those with a CH+ grew in the presence of a bactericidal concentration of sodium hypochlorite. The terms 'water, grids, and patients' indicated in italic on the figure refer to the source of the clones or clonal complexes; grids being used to indicate tap nozzle aerators. The dendrogram was computed using a maximum parsimony method. * Indicates the strains harboring *exoU* but not *exoS*.
<table><caption>(a)</caption><thead><tr><th>Date</th><th colspan="7">2004</th><th colspan="3">2005</th><th colspan="1">2006</th></tr><tr><th>CC</th><th>Sept</th><th>Oct</th><th>Nov</th><th>Dec</th><th>Jan</th><th>Feb</th><th>March</th><th>May</th><th>Jan</th><th>Feb</th><th>March</th><th>May</th></tr></thead><tbody><tr><td>1</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>2</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>3</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>4</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>6</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>8</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>9</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>11</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>15</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>16</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>17</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>19</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>20</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>21</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>22</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr></tbody></table>

<table><caption>(b)</caption><thead><tr><th>Room</th><th>1</th><th>2</th><th>3</th><th>4</th><th>5</th><th>6</th><th>7</th><th>8</th><th>9</th><th>10</th><th>11</th><th>12</th><th>13</th><th>14</th><th>mc</th></tr></thead><tbody><tr><td>CC</td><td>1</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>1</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>2</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>3</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>4</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>6</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>8</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>9</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>11</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>15</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>16</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>17</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>19</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>20</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>21</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>22</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr></tbody></table>

<table><caption>(c)</caption><thead><tr><th>Sample</th><th>Water</th><th>Aerator</th><th>Rectal</th><th>Patient</th></tr></thead><tbody><tr><td>CC</td><td></td><td></td><td></td><td></td></tr><tr><td>1</td><td></td><td></td><td></td><td></td></tr><tr><td>2</td><td></td><td></td><td></td><td></td></tr><tr><td>3</td><td></td><td></td><td></td><td></td></tr><tr><td>4</td><td></td><td></td><td></td><td></td></tr><tr><td>5</td><td></td><td></td><td></td><td></td></tr><tr><td>6</td><td></td><td></td><td></td><td></td></tr><tr><td>7</td><td></td><td></td><td></td><td></td></tr><tr><td>8</td><td></td><td></td><td></td><td></td></tr><tr><td>9</td><td></td><td></td><td></td><td></td></tr><tr><td>10</td><td></td><td></td><td></td><td></td></tr><tr><td>11</td><td></td><td></td><td></td><td></td></tr><tr><td>12</td><td></td><td></td><td></td><td></td></tr><tr><td>13</td><td></td><td></td><td></td><td></td></tr><tr><td>14</td><td></td><td></td><td></td><td></td></tr><tr><td>15</td><td></td><td></td><td></td><td></td></tr><tr><td>16</td><td></td><td></td><td></td><td></td></tr><tr><td>17</td><td></td><td></td><td></td><td></td></tr><tr><td>18</td><td></td><td></td><td></td><td></td></tr><tr><td>19</td><td></td><td></td><td></td><td></td></tr><tr><td>20</td><td></td><td></td><td></td><td></td></tr><tr><td>21</td><td></td><td></td><td></td><td></td></tr><tr><td>22</td><td></td><td></td><td></td><td></td></tr></tbody></table>

**Figure 3** Illustration of the spatio-temporal infra-specific dynamics among the hospital *Pseudomonas aeruginosa* population. CC indicates clones or clonal complexes. CC and room numbers match those of Figs 1 and 2. A black box indicates the presence of a clone or clonal complex. (a) Persistence of *P. aeruginosa* clones and clonal complexes in the hospital unit water network; (b) Spread of *P. aeruginosa* clones and clonal complexes among the unit. Only samples from the water network were considered. (c) Distribution of the *P. aeruginosa* clones and clonal complexes among all samples [water, tap nozzle aerators, rectal swabs, and nosocomial infection (patient)] analysed in this study.

important after-effects of cerebral vascular accidents). This first report of the data collected over a 2-year period suggests an important turnover among the hospital water network of *P. aeruginosa* population but permanent resident clones appeared to have been selected, and been able to resist to the hospital disinfection and cleansing measures. One of these *P. aeruginosa* clones was found involved in a nosocomial infection but no outbreak was recorded during this study.

The water network of the investigated unit was found to contain a perennial population of *P. aeruginosa*, with cells being detected in 68% of the water samples and 74% of the tap nozzle aerators. This is the highest occurrence of *P. aeruginosa* recorded so far in a water network. Previous results suggested colonization between 16% and 60% of hospital water taps. These differences could be because of the procedures used. In this study, not only typical blue-green *P. aeruginosa* colonies growing on PAB-CN medium were considered; in fact, all bacterial colony morphotypes growing on the PAB-CN medium were submitted to a *P. aeruginosa* identification scheme involving phenotypic analyses and a PCR test using a species-specific gene maker. Using this approach, 6 out of 14

room water networks were found permanently colonized by *P. aeruginosa*. The other water taps, including the one in the maintenance cabinet, were only colonized occasionally. Water from the outdoor tap of the building never showed any culturable *P. aeruginosa* cells.

Rectal carriage of *P. aeruginosa* was observed among 60% of the patients of the unit; a number in line with other reports (Bonten et al. 1999; Bertrand et al. 2001). Rectal carriage could have been one of the main sources in the origin of the presence of *P. aeruginosa* in the water network. The PFGE-SpeI data of this study support this conclusion. In three instances, the PFGE-SpeI profiles of strains from tap nozzle aerators were found to form a clonal lineage with rectal isolates. In one case, strains having identical PFGE-SpeI profiles were found to come from a patient's rectal swab and the tap nozzle aerator of a same room, suggesting a recent cross-contamination. In the other two cases, clones of the rectal isolates of two patients were recovered from neighbouring rooms, suggesting contamination by the medical staff involved in the hygienic care of the patients. Interestingly, none of these strains was isolated from the water samples, and none was detected on the other sampling dates. This could be
because of the descaling and cleansing procedures that
were used by the hospital staff. The limited distribution
of these strains suggested an unlikely diffusion through
water pipes. The PFGE-SpeI data set also suggested cross-
contamination of patients by rectal strains. For instance,
clone 12 grouped rectal strains collected at the same time
but from patients of three different rooms. Interestingly,
a rectal strain of this same complex was still recovered
4 months later from one of these patients, suggesting per-
sistence through time (without any sign of infection).

However, one clonal complex (no. 16) and two clones (nos 19 and 22), recorded in this study, could have had undergone a diffusion through the water pipes. Complex 16 and clone 22 were recovered, respectively, from five and eleven rooms of the unit, while clone 19 was only recovered from water samples. Clone 22 was detected in the tap water but not the tap nozzle aerator of the maintenance cabinet of the medical unit. These three groups of strains were not detected among the rectal swabs. All these observations are thus pointing towards a diffusion of these strains through the water pipes. However, it is *P. aeruginosa* rectal carriage that appeared most likely to be at the origin of their presence among the water network; *P. aeruginosa* strains being undetectable from the water tap located outside the medical building.

The PFGE-SpeI dataset showed a great diversity associ-
ated with an important turnover in the composition of
the investigated water network of Ps. aeruginosa popula-
tion. Such a great genetic diversity among a hospital
water network was previously observed (Ferroni et al.
1998; Blanc et al. 2004) but not always (Reuter et al.
2002). High strain turnovers were not notified. Over the
32 distinct PFGE-SpeI profiles recorded among the water
isolates, only 30% were observed at more than one sam-
pling time. Furthermore, only two clonal lineages, 16 and
22, were recorded throughout the 2-year period of this
study. Despite the possibility of having missed some
strains in the course of this study because of the sampling
procedure used, we consider these data as being indicative
of a globally poor ability of most P. aeruginosa strains to
survive over a long period to the living conditions pre-
vailing in a water network. However, some clones or clon-
nal complexes appeared to have properties favouring their
persistence in such a biotope. In fact, the most persistent
clones were found to be very good biofilm producers and
to grow under a bactericidal concentration of sodium
hypochlorite. Furthermore, some of the observed genetic
diversifications of water clones into clonal complexes
might be indicative of genetic adaptations for the water
network. These adaptations might have involved processes
similar to those observed among bacterial colonizers of
CF patients (e.g. Smith et al. 2006; Menard et al. 2007),
and could have led to better adapted genetic variants.

However, analysis of a set of *P. aeruginosa* genes involved
in virulence among the collected strains did not reveal
any particular events that could have led to the genetic
diversification observed. All the strains analysed were
found to have the set of genes that can lead to acute
infections. Only *exoS* and *exoU* showed a peculiar distri-
bution, with these genes never being found together in a
same strain. Such an observation was previously made by
other authors (Feltman *et al.* 2001; Wolfgang *et al.* 2003;
Finnan *et al.* 2004).

Overall, this 2-year study demonstrated that a highly
diverse population of *P. aeruginosa* can be found in a
hospital water network. This population appeared to
have undergone important changes but some clones and
clonal complexes were found to be highly successful.
Some of these were found to have disseminated
throughout the unit, to have persisted for over at least
2 years, and in some cases to show genetic changes that
could be signs of adaptations towards the living condi-
tions prevailing in the water network. All the water net-
work isolates appeared to possess the genetic
background for the colonization of weakened indivi-
duals. Overall, avoiding contamination of the water
network by rectal *P. aeruginosa* seems one of the key
issues in the prevention of *P. aeruginosa* dissemination
throughout a hospital unit. However, once strains have
got adapted to a water network, their eradication can be
very hard to achieve.

Acknowledgements

Raphaël Lavenir was supported by a PhD fellowship from
the ‘Prospective’ Rhône-Alpes Region (France) program.
The authors thank the CNRS, Université Lyon 1, and
Rhône-Alpes Region (France) for having financially sup-
ported parts of this work. This work was also partly funded
by the Agence Nationale de la Recherche (ANR) 05 SEST
project #009-01, and the AFSSET #ES-2005-008 project.
The authors thank Claire Monnez (UMR 5557; CNRS, Uni-
versité Lyon 1 and ENVL) for her technical assistance.

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