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Environmental contamination with an epidemic strain of Pseudomonas aeruginosa in a Liverpool cystic fibrosis centre, and study of its survival on dry surfaces
Journal of Hospital Infection (2005) 59, 102–107
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Environmental contamination with an epidemic strain of Pseudomonas aeruginosa in a Liverpool cystic fibrosis centre, and study of its survival on dry surfaces S. Panageaa,*, C. Winstanleya, M.J. Walshawb, M.J. Ledsonb, C.A. Harta a
Department of Medical Microbiology and Genito-urinary Medicine, University of Liverpool, Liverpool L69 3GA, UK b Regional Adult Cystic Fibrosis Unit, Cardiothoracic Centre, Liverpool L14 3PE, UK Received 18 January 2004; accepted 10 September 2004
KEYWORDS Cystic fibrosis; Pseudomonas aeruginosa; Epidemic strain; Environmental contamination
Summary We conducted an environmental survey in the Liverpool adult cystic fibrosis (CF) centre in order to determine the extent of environmental contamination with an epidemic strain of Pseudomonas aeruginosa that colonizes most CF patients in Liverpool, and to identify possible reservoirs and routes of cross-infection. In addition, we studied the survival of this strain on dry surfaces, compared with that of other CF P. aeruginosa strains, to explore factors that might contribute to its high transmissibility. Samples were collected from staff, patients and the environment (drains, bath tubs, showers, dry surfaces, respiratory equipment and air) in the inpatient ward and outpatient clinic. P. aeruginosa strains were tested using a new polymerase chain reaction amplification assay specific for the Liverpool epidemic strain (LES). LES was isolated from patients’ hands, clothes and bed linen. Environmental contamination with LES was only detected in close proximity to colonized patients (external surfaces of their respiratory equipment, and spirometry machine tubing and chair) and was short-lived. No persistent environmental reservoirs were found. LES was detected in the majority of air samples from inside patients’ rooms, the ward corridor and the outpatient clinic. Survival of LES on dry surfaces was significantly longer than that for some other strains tested, but not compared with other strains shown not to be transmissible. Improved environmental survival on its own, therefore, cannot explain the high transmissibility of this epidemic strain. Our study suggests that airborne dissemination plays a significant role in patient-to-patient spread of LES, and confirms the need to segregate those
* Corresponding author. Tel.: C44 151 706 4410; fax: C44 151 706 5849. E-mail address: [email protected] 0195-6701/$ - see front matter Q 2004 The Hospital Infection Society. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.jhin.2004.09.018
Environmental contamination in cystic fibrosis
103
patients colonized by epidemic P. aeruginosa strains from all other CF patients. Q 2004 The Hospital Infection Society. Published by Elsevier Ltd. All rights reserved.
Introduction Pseudomonas aeruginosa is a major pathogen in cystic fibrosis (CF) patients and the leading cause of morbidity and mortality. Most CF patients are ultimately colonized with P. aeruginosa, and once chronic infection is established, it is virtually impossible to eradicate.1,2 However, the sources of acquisition and the means of transmission of the organism are not well understood. P. aeruginosa is ubiquitous in moist environments and it is found in many natural and domestic reservoirs including hospital sites. It has long been accepted that CF patients become colonized from diverse sources in the general environment.3 Indeed, most unrelated patients typically harbour their own unique strains of P. aeruginosa that persist for many years, indicating a low incidence of patient-to-patient spread or acquisition from a common source.4 On the other hand, cross-infection has been shown to occur in holiday camps5,6 and there are welldocumented reports of outbreaks involving highly transmissible, epidemic strains in a number of CF units including Liverpool.7–9 The mode of transmission of these epidemic strains is uncertain; however, direct patient-to-patient spread has been suggested given the failure to isolate the strains from the hospital environment. We conducted an environmental survey in the Liverpool adult CF centre in order to determine the extent of environmental contamination with the Liverpool epidemic strain (LES) to identify possible reservoirs and routes of cross-infection. In addition, we studied the survival of LES on dry surfaces compared with that of other CF P. aeruginosa strains to explore factors that might contribute to its high transmissibility.
Methods Accommodation The inpatient CF ward of the Cardiothoracic Centre, Liverpool, UK, consists of 12 single rooms including five with en-suite facilities. Communal areas include a lounge and kitchen, one bathroom, one shower room and two toilets. Hospitalized patients receive all treatment, including nebulization and
physiotherapy, in their own room with the door closed and are advised to avoid social contact with other patients on the ward. The outpatient clinic is located in another part of the hospital and consists of three consultation rooms, a treatment room and the lung function laboratory. While waiting for their appointment, patients are seated in the waiting room or in the corridor. At the time of the study, no segregation of patients with regard to their colonization with P. aeruginosa was in place.
Sample collection Samples in the inpatient ward were obtained during three visits one week apart from staffs’ hands and uniforms, patients’ hands, clothes and bed linen, and the inanimate environment including air. The patients sampled were known to be colonized with LES by recent testing of their respiratory secretions. One to three samples were collected from each environmental site including moist habitats (sink drains, bath tubs, showers) and dry surfaces (furniture, door handles, electronic equipment, respiratory equipment). Samples from staffs’ hands, dry environmental surfaces and the air were also collected in the outpatient clinic. Samples from moist sites were taken using sterile cotton wool swabs. Large surfaces such as furniture, doors and door handles, patient equipment, kitchen appliances, electronic equipment, toilet seats and flush handles were sampled using sterile cloths moistened with sterile water. Contact plates with Pseudomonas-selective agar were used to sample hands and uniforms of staff and hands, clothes and bed linen of patients colonized with LES. Air samples were obtained using the Casella slit sampler with a Pseudomonas-selective agar (SM559/SR102, Oxoid Ltd, Basingstoke, UK) plate working at a maximum volume of 700 L/min. The sampling time was 15 min.
Culture, identification and molecular typing Swabs were inoculated on Pseudomonas-selective agar. The cloths were placed in nutrient broth and, following overnight incubation at 37 8C, the broth was subcultured on Pseudomonas-selective agar.
104 All plates were incubated at 37 8C for 48 h. P. aeruginosa strains were identified by characteristic Gram-stain appearance, positive oxidase reaction and conventional biochemical tests using the API 20NE (biomerieux, Basingstoke, UK). All P. aeruginosa strains were tested by a polymerase chain reaction amplification assay using primers (PS21) specific for the identification of LES.10
Survival of P. aeruginosa strains on dry surfaces The strains tested included LES (mucoid and nonmucoid phenotype), reference strain PA01, two unique CF isolates from Liverpool patients, the Manchester UK epidemic strain, and three CF isolates implicated in epidemic spread from other UK units. Bacterial stock cultures were stored in 20% glycerol broth at K70 8C. Organisms were grown from frozen in nutrient broth at 37 8C for 24 h, washed twice, and resuspended in normal saline to a concentration of approximately 108 colony forming units (cfu)/mL. A 50-mL aliquot of each bacterial suspension was spread over an area of approximately 6 cm2 on multiple sterile Petri dishes and allowed to dry. Sequential quantitative cultures from these surfaces were obtained every hour for the first 6 h and then at 8, 9, 24 and 48 h after inoculation. Baseline viable bacterial counts were obtained immediately after inoculation for each strain. At each time point after inoculation, the test surface was rubbed vigorously for 10 s with a moistened sterile cotton swab. The swab was immersed in 1 mL of nutrient broth and mixed with a vortex mixer for 20 s. Viable counts were determined by serial 10-fold dilutions of this swab rinse solution and culturing 0.1-mL volumes on to blood agar plates. The swab/nutrient broth container was also incubated for 48 h before subculture on blood agar plates. Agar plates were incubated at 37 8C for 48 h before colonies were counted. The limit of our detection system was 10 cfu/mL. The experiment was repeated three times and the mean viable count at each time point was used to plot acturial survival curves for each strain. These were compared for statistically significant variation using the Log Rank test (Statview software).
Results Inpatient ward One hundred and fourteen samples from staff, patients and inanimate surfaces (Table I) and 23 air
S. Panagea et al. samples from 11 cubicles, the ward corridor and the lounge were examined (Table II). P. aeruginosa was isolated from the shared bathroom/toilet/shower handles and surfaces, as well as the toilet handles in two cubicles with en-suite facilities, but none of these isolates was LES. Only the shared toilet was persistently positive for P. aeruginosa on repeat sampling. The other sites were positive on only one occasion. The majority of the moist sites tested (wash basin, bath tub and shower drains) harboured P. aeruginosa (61%), but of these, only one sink drain in a colonized patient’s room was positive for LES (6%). This site also yielded non-epidemic P. aeruginosa. However, contamination with LES was short-lived; the sink drain tested negative for LES on repeat sampling one week later. LES was isolated from patients’ hands, bed linen and clothes. The external surface of their respiratory equipment was also found to be contaminated with LES, albeit transiently; repeat sampling after patients’ discharge and room cleaning was negative. LES was detected in 80% of the air samples taken inside the patients’ rooms and in 60% of the samples taken in the ward corridor. The number of organisms isolated ranged from 1 to 55 cfu/sample. Six air samples from inside the rooms were collected during patients’ physiotherapy and all were positive for LES. However, LES was detected in the room air even when the patients had been absent from their rooms for 1–3 h prior to testing. The air in two positive rooms tested negative 3 h after patient discharge and room cleaning. All the P. aeruginosa strains detected in the air were the epidemic strain.
Outpatient clinic Sixteen samples were examined (Table III). LES was detected in the spirometry tubing and chair after use by colonized patients. Two subsequent samples from this site (2 and 3 h later) were negative. Overall, five of eight air samples (62.5%) were positive for LES. No other P. aeruginosa strains were detected in the air or on dry surfaces.
Survival of P. aeruginosa strains on dry surfaces Soon after drying (1 h after inoculation), the viable counts of the saline bacterial suspensions were reduced considerably (log10 reduction factors of 2.1–4.5). At 9 h, all strains were still recovered with log10 viable counts per mL ranging from 0.2 to 3.8. Viable organisms were still detectable 48 h after
Environmental contamination in cystic fibrosis Table I
105
Recovery of Pseudomonas aeruginosa from the inpatient ward inanimate environment, patients and staff
Site
Staffs’ hands and uniforms Patients’ hands Patients’ clothes Patients’ bed linen Wash basin, bath tub, shower drains Bathroom, toilet handles/surfaces Dry environmental surfacesa External surface of respiratory equipment
Number of sites/ people tested
Number of sites with non-epidemic P. aeruginosa
Number of sites with LES
Total number of samples
12 8 8 8 18
0 0 0 0 11 (61%)
0 2 (25%) 1 (12.5%) 2 (25%) 1 (6%)
12 8 8 8 40
4
4 (100%)
0
9
7b
0
0
14
15
0
3 (20%)
15
LES, Liverpool epidemic strain. a A single cloth was used to sample all dry surfaces in the same room including furniture, door handles, television, telephones etc. b Number of rooms tested.
drying for most strains, albeit at very low numbers (Figure 1). No significant difference was found in the survival of the mucoid and the non-mucoid phenotype of LES, or between LES and the reference strain PA01. LES survived significantly longer than the Manchester epidemic strain (PZ0.013), one unique Liverpool CF isolate b (PZ0.011) and one other epidemic UK strain B (PZ0.010). Comparison of LES survival with the other strains did not reveal any significant differences.
Discussion Recent reports of epidemic spread of transmissible P. aeruginosa strains among patients attending CF clinics worldwide have provided clear evidence to support the occurrence of cross-infection between CF patients, and have generated controversy on infection control practices and the management of these patients.3,11,12 LES not only colonizes most CF patients in Liverpool, but is also found in several Table II
other CF centres in the UK.13 We undertook the present study to investigate the means by which this strain is acquired and whether prolonged survival on dry surfaces contributes to its transmissibility. Extensive bacteriological screening of the inanimate hospital environment has identified P. aeruginosa in the majority of moist sites (wash basins, bath tubs and shower drains), and also on communal bathroom and toilet handles and surfaces. Only one of these sites (a sink drain inside a cubicle occupied by a known colonized patient) was transiently positive for LES, suggesting secondary contamination from the patient. P. aeruginosa is frequently isolated from moist sites in hospitals, but the importance of these reservoirs as a source of infection is uncertain.14 The role of contamination of the environment with P. aeruginosa in CF clinics has been the subject of several studies.15–19 Heavy environmental contamination was found in some studies,15,17,19 especially in moist sites, and transmission of P. aeruginosa from sinks to hands during handwashing has been shown.19 Survival for considerable periods of time in aerosols and on hands
Recovery of Pseudomonas aeruginosa from air samples in the inpatient ward
Site
Cubicle patient present Cubicle patient absent Ward corridor Lounge LES, Liverpool epidemic strain.
Number of samples with non-epidemic P. aeruginosa
Number of samples with LES
Total number of samples
0 0 0 0
10 2 3 0
11 4 5 1
106 Table III
S. Panagea et al. Recovery of Pseudomonas aeruginosa from air, staffs’ hands and dry surfaces in the outpatient clinic
Site
Air in consultation room Air in corridor Air in waiting room Air in spirometry room Staffs’ hands Dry surfacesa
Number of samples with non-epidemic P. aeruginosa
Number of samples with LES
Total number of samples
0 0 0 0 0 0
1 2 1 1 0 1
4 2 1 1 3 5
LES, Liverpool epidemic strain. a The dry surfaces tested included: door handles and chairs in the corridor and the waiting room (two samples), and spirometry machine tubing and chair (three samples).
has also been demonstrated.20 Improved hygienic precautions and segregation of patients prevented environmental contamination and was associated with reduced incidence of infection in Danish CF patients.18 However, even in situations of heavy environmental contamination, direct transmission from the environment to patients has only been demonstrated occasionally.19 Two recent studies investigating outbreaks with epidemic CF strains in Manchester and Melbourne failed to find contamination of the environment.7,21 No persistent environmental reservoirs for LES were detected in our study. We found, however, that colonized patients do indeed contaminate their hands, their clothes and their immediate surroundings (bed linen). LES was also detected on the external surface of their respiratory equipment and the spirometry machine tubing and chair following use by colonized patients, which raises the possibility of transmission to other patients either directly or via the hands of staff.
Figure 1 Survival of cystic fibrosis (CF) Pseudomonas aeruginosa strains on dry surfaces. %, Liverpool epidemic strain; &, Manchester; :, UK CF strain A; B UK CF strain B; $, UK CF strain C; C, unique CF strain a; 6, unique CF strain b.
Contamination was short-lived however, repeat sampling was negative following patient discharge and routine cleaning. Staffs’ hands and uniforms were also negative. Therefore, it appears that contamination of inanimate surfaces occurs only in close proximity to patients and is transient. Provided that standard hygienic and routine infection control measures are applied, inanimate surface contamination should play a minimal role in transmission. Nevertheless, the detection of LES in the majority of air samples in both the inpatient and outpatient sites is worrying. LES was isolated not only inside the cubicles, but also in the ward corridor, the clinic corridor and waiting room. It was found in the air not only in the presence of colonized patients, but also when patients were not in their rooms for up to 3 h prior to testing. Although the number of organisms was very small, the level of exposure required for acquisition is not known. The duration of airborne contamination was considerable and LES was the only P. aeruginosa strain isolated from the air, suggesting enhanced survival of this strain in aerosols. Air contamination with LES was also detected in the Manchester CF clinic.21 At present, it is unknown why some P. aeruginosa strains are capable of cross-infection in CF patients. Survival times of P. aeruginosa strains in aerosols have been shown to be dependent on strain characteristics,20 and increased survival time on dry surfaces has been suggested to contribute to increased transmissibility of some Burkholderia cepacia strains.22 We tested the hypothesis that prolonged survival of LES on dry surfaces contributes to its apparent higher transmissibility. We found that some P. aeruginosa strains, including LES, survive well in the environment under dry conditions. However, although LES survival was significantly longer compared with some strains
Environmental contamination in cystic fibrosis tested, it was not longer compared with other strains that have not been shown to be transmissible. Therefore, improved environmental survival on its own cannot explain the high transmissibility of this epidemic strain and we continue to look for other factors that can explain its prevalence in the UK CF community. In conclusion, we have shown ward and outpatient clinic environmental contamination with LES in this study. It appears to occur only in close proximity to colonized patients and to be shortlived. No persistent environmental reservoirs were detected. The presence of LES in the majority of air samples in both the inpatient and outpatient areas is particularly worrying, and it seems likely that airborne dissemination plays a significant role in patient-to-patient spread of this organism. We suggest that cohort segregation of patients who carry epidemic P. aeruginosa strains should be implemented.
References 1. Hart CA, Winstanley C. Persistent and aggressive bacteria in the lungs of cystic fibrosis children. Br Med Bull 2002;61: 81—96. 2. Govan JRW, Deretic V. Microbial pathogenesis in cystic fibrosis: mucoid Pseudomonas aeruginosa and Burkholderia cepacia. Microbiol Rev 1996;60:539—574. 3. Geddes DM. Of isolates and isolation: Pseudomonas aeruginosa in adults with cystic fibrosis. Lancet 2001;358: 522—523. 4. Govan JRW. Infection control in cystic fibrosis: methicillinresistant Staphylococcus aureus, Pseudomonas aeruginosa and the Burkholderia cepacia complex. J R Soc Med 2000; 93(Suppl. 38):40—45. 5. Ojeniyi B, Frederiksen B, Hoiby N. Pseudomonas aeruginosa cross-infection among patients with cystic fibrosis during a winter camp. Pediatr Pulmonol 2000;29:177—181. 6. Hunfeld KP, Schmidt C, Krackhardt B, et al. Risk of Pseudomonas aeruginosa cross-colonisation in patients with cystic fibrosis within a holiday camp: a molecularepidemiological study. Wien Klin Wochenschr 2000;112: 329—333. 7. Armstrong DS, Nixon GM, Carzino R, et al. Detection of a widespread clone of Pseudomonas aeruginosa in a pediatric cystic fibrosis clinic. Am J Respir Crit Care Med 2002;166: 983—987.
107 8. Jones MA, Govan JRW, Doherty CJ, et al. Spread of a multiresistant strain of Pseudomonas aeruginosa in an adult cystic fibrosis clinic. Lancet 2001;358:557—558. 9. Cheng K, Smyth RL, Govan JRW, et al. Spread of a b-lactamresistant Pseudomonas aeruginosa in a cystic fibrosis clinic. Lancet 1996;348:639—642. 10. Parsons YN, Panagea S, Smart CHM, Walshaw MJ, Hart CA, Winstanley C. Use of subtractive hybridisation to identify a diagnostic probe for a cystic fibrosis epidemic strain of Pseudomonas aeruginosa. J Clin Microbiol 2002;40: 4607—4611. 11. Ramsey BW. To cohort or not to cohort. How transmissible is Pseudomonas aeruginosa? Am J Respir Crit Care Med 2002; 166:906—907. 12. Pitt TL. Cross infection of cystic fibrosis patients with Pseudomonas aeruginosa. Thorax 2002;57:921. 13. Scott F, Pitt TL. Identification and characterisation of transmissible Pseudomonas aeruginosa strains in UK cystic fibrosis centres. [Abstract 146]. J Cystic Fibrosis 2003; 2(Suppl. 2):39. 14. Ayliffe GAJ, Babb JR, Collins BJ, Lowbury EJL, Newsom SWB. Pseudomonas aeruginosa in hospital sinks. Lancet 1974;2: 578—581. 15. Zimakoff J, Hoiby N, Rosendal K, Guilbert JP. Epidemiology of Pseudomonas aeruginosa and the role of contamination of the environment in a cystic fibrosis clinic. J Hosp Infect 1983; 4:31—40. 16. Speert DP, Campbell ME. Hospital epidemiology of Pseudomonas aeruginosa from patients with cystic fibrosis. J Hosp Infect 1987;9:11—21. 17. Bobhammer J, Fiedler B, Gudowius P, von der Hardt H, Romling U, Tummler B. Comparative hygienic surveillance of contamination with pseudomonads in a cystic fibrosis ward over a 4-year period. J Hosp Infect 1995;31:261—274. 18. Zembrzuska-Sadkowska E, Sneum M, Ojeniyi B, Heiden L, Hoiby N. Epidemiology of Pseudomonas aeruginosa infection and the role of contamination of the environment in the Danish cystic fibrosis centre. J Hosp Infect 1995;29:1—7. 19. Doring G, Jansen S, Noll H, et al. Distribution and transmission of Pseudomonas aeruginosa and Burkholderia cepacia in a hospital ward. Pediatr Pulmonol 1996;21: 90—100. 20. Doring G, Ulrich M, Muller W, et al. Generation of Pseudomonas aeruginosa aerosols during hand-washing from contaminated sink drains, transmission to hands of hospital personnel, and its prevention by use of a new heating device. Zentralbl Hyg 1991;191:494—505. 21. Jones AM, Govan JRW, Doherty CJ, et al. Identification of airborne dissemination of epidemic multiresistant strains of Pseudomonas aeruginosa at a CF centre during a cross infection outbreak. Thorax 2003;58:525—527. 22. Drabick JA, Gracely EJ, Heidecker GJ, LiPuma JJ. Survival of Burkholderia cepacia on environmental surfaces. J Hosp Infect 1996;32:267—276.
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Environmental contamination with an epidemic strain of Pseudomonas aeruginosa in a Liverpool cystic fibrosis centre, and study of its survival on dry surfaces S. Panageaa,*, C. Winstanleya, M.J. Walshawb, M.J. Ledsonb, C.A. Harta a
Department of Medical Microbiology and Genito-urinary Medicine, University of Liverpool, Liverpool L69 3GA, UK b Regional Adult Cystic Fibrosis Unit, Cardiothoracic Centre, Liverpool L14 3PE, UK Received 18 January 2004; accepted 10 September 2004
KEYWORDS Cystic fibrosis; Pseudomonas aeruginosa; Epidemic strain; Environmental contamination
Summary We conducted an environmental survey in the Liverpool adult cystic fibrosis (CF) centre in order to determine the extent of environmental contamination with an epidemic strain of Pseudomonas aeruginosa that colonizes most CF patients in Liverpool, and to identify possible reservoirs and routes of cross-infection. In addition, we studied the survival of this strain on dry surfaces, compared with that of other CF P. aeruginosa strains, to explore factors that might contribute to its high transmissibility. Samples were collected from staff, patients and the environment (drains, bath tubs, showers, dry surfaces, respiratory equipment and air) in the inpatient ward and outpatient clinic. P. aeruginosa strains were tested using a new polymerase chain reaction amplification assay specific for the Liverpool epidemic strain (LES). LES was isolated from patients’ hands, clothes and bed linen. Environmental contamination with LES was only detected in close proximity to colonized patients (external surfaces of their respiratory equipment, and spirometry machine tubing and chair) and was short-lived. No persistent environmental reservoirs were found. LES was detected in the majority of air samples from inside patients’ rooms, the ward corridor and the outpatient clinic. Survival of LES on dry surfaces was significantly longer than that for some other strains tested, but not compared with other strains shown not to be transmissible. Improved environmental survival on its own, therefore, cannot explain the high transmissibility of this epidemic strain. Our study suggests that airborne dissemination plays a significant role in patient-to-patient spread of LES, and confirms the need to segregate those
* Corresponding author. Tel.: C44 151 706 4410; fax: C44 151 706 5849. E-mail address: [email protected] 0195-6701/$ - see front matter Q 2004 The Hospital Infection Society. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.jhin.2004.09.018
Environmental contamination in cystic fibrosis
103
patients colonized by epidemic P. aeruginosa strains from all other CF patients. Q 2004 The Hospital Infection Society. Published by Elsevier Ltd. All rights reserved.
Introduction Pseudomonas aeruginosa is a major pathogen in cystic fibrosis (CF) patients and the leading cause of morbidity and mortality. Most CF patients are ultimately colonized with P. aeruginosa, and once chronic infection is established, it is virtually impossible to eradicate.1,2 However, the sources of acquisition and the means of transmission of the organism are not well understood. P. aeruginosa is ubiquitous in moist environments and it is found in many natural and domestic reservoirs including hospital sites. It has long been accepted that CF patients become colonized from diverse sources in the general environment.3 Indeed, most unrelated patients typically harbour their own unique strains of P. aeruginosa that persist for many years, indicating a low incidence of patient-to-patient spread or acquisition from a common source.4 On the other hand, cross-infection has been shown to occur in holiday camps5,6 and there are welldocumented reports of outbreaks involving highly transmissible, epidemic strains in a number of CF units including Liverpool.7–9 The mode of transmission of these epidemic strains is uncertain; however, direct patient-to-patient spread has been suggested given the failure to isolate the strains from the hospital environment. We conducted an environmental survey in the Liverpool adult CF centre in order to determine the extent of environmental contamination with the Liverpool epidemic strain (LES) to identify possible reservoirs and routes of cross-infection. In addition, we studied the survival of LES on dry surfaces compared with that of other CF P. aeruginosa strains to explore factors that might contribute to its high transmissibility.
Methods Accommodation The inpatient CF ward of the Cardiothoracic Centre, Liverpool, UK, consists of 12 single rooms including five with en-suite facilities. Communal areas include a lounge and kitchen, one bathroom, one shower room and two toilets. Hospitalized patients receive all treatment, including nebulization and
physiotherapy, in their own room with the door closed and are advised to avoid social contact with other patients on the ward. The outpatient clinic is located in another part of the hospital and consists of three consultation rooms, a treatment room and the lung function laboratory. While waiting for their appointment, patients are seated in the waiting room or in the corridor. At the time of the study, no segregation of patients with regard to their colonization with P. aeruginosa was in place.
Sample collection Samples in the inpatient ward were obtained during three visits one week apart from staffs’ hands and uniforms, patients’ hands, clothes and bed linen, and the inanimate environment including air. The patients sampled were known to be colonized with LES by recent testing of their respiratory secretions. One to three samples were collected from each environmental site including moist habitats (sink drains, bath tubs, showers) and dry surfaces (furniture, door handles, electronic equipment, respiratory equipment). Samples from staffs’ hands, dry environmental surfaces and the air were also collected in the outpatient clinic. Samples from moist sites were taken using sterile cotton wool swabs. Large surfaces such as furniture, doors and door handles, patient equipment, kitchen appliances, electronic equipment, toilet seats and flush handles were sampled using sterile cloths moistened with sterile water. Contact plates with Pseudomonas-selective agar were used to sample hands and uniforms of staff and hands, clothes and bed linen of patients colonized with LES. Air samples were obtained using the Casella slit sampler with a Pseudomonas-selective agar (SM559/SR102, Oxoid Ltd, Basingstoke, UK) plate working at a maximum volume of 700 L/min. The sampling time was 15 min.
Culture, identification and molecular typing Swabs were inoculated on Pseudomonas-selective agar. The cloths were placed in nutrient broth and, following overnight incubation at 37 8C, the broth was subcultured on Pseudomonas-selective agar.
104 All plates were incubated at 37 8C for 48 h. P. aeruginosa strains were identified by characteristic Gram-stain appearance, positive oxidase reaction and conventional biochemical tests using the API 20NE (biomerieux, Basingstoke, UK). All P. aeruginosa strains were tested by a polymerase chain reaction amplification assay using primers (PS21) specific for the identification of LES.10
Survival of P. aeruginosa strains on dry surfaces The strains tested included LES (mucoid and nonmucoid phenotype), reference strain PA01, two unique CF isolates from Liverpool patients, the Manchester UK epidemic strain, and three CF isolates implicated in epidemic spread from other UK units. Bacterial stock cultures were stored in 20% glycerol broth at K70 8C. Organisms were grown from frozen in nutrient broth at 37 8C for 24 h, washed twice, and resuspended in normal saline to a concentration of approximately 108 colony forming units (cfu)/mL. A 50-mL aliquot of each bacterial suspension was spread over an area of approximately 6 cm2 on multiple sterile Petri dishes and allowed to dry. Sequential quantitative cultures from these surfaces were obtained every hour for the first 6 h and then at 8, 9, 24 and 48 h after inoculation. Baseline viable bacterial counts were obtained immediately after inoculation for each strain. At each time point after inoculation, the test surface was rubbed vigorously for 10 s with a moistened sterile cotton swab. The swab was immersed in 1 mL of nutrient broth and mixed with a vortex mixer for 20 s. Viable counts were determined by serial 10-fold dilutions of this swab rinse solution and culturing 0.1-mL volumes on to blood agar plates. The swab/nutrient broth container was also incubated for 48 h before subculture on blood agar plates. Agar plates were incubated at 37 8C for 48 h before colonies were counted. The limit of our detection system was 10 cfu/mL. The experiment was repeated three times and the mean viable count at each time point was used to plot acturial survival curves for each strain. These were compared for statistically significant variation using the Log Rank test (Statview software).
Results Inpatient ward One hundred and fourteen samples from staff, patients and inanimate surfaces (Table I) and 23 air
S. Panagea et al. samples from 11 cubicles, the ward corridor and the lounge were examined (Table II). P. aeruginosa was isolated from the shared bathroom/toilet/shower handles and surfaces, as well as the toilet handles in two cubicles with en-suite facilities, but none of these isolates was LES. Only the shared toilet was persistently positive for P. aeruginosa on repeat sampling. The other sites were positive on only one occasion. The majority of the moist sites tested (wash basin, bath tub and shower drains) harboured P. aeruginosa (61%), but of these, only one sink drain in a colonized patient’s room was positive for LES (6%). This site also yielded non-epidemic P. aeruginosa. However, contamination with LES was short-lived; the sink drain tested negative for LES on repeat sampling one week later. LES was isolated from patients’ hands, bed linen and clothes. The external surface of their respiratory equipment was also found to be contaminated with LES, albeit transiently; repeat sampling after patients’ discharge and room cleaning was negative. LES was detected in 80% of the air samples taken inside the patients’ rooms and in 60% of the samples taken in the ward corridor. The number of organisms isolated ranged from 1 to 55 cfu/sample. Six air samples from inside the rooms were collected during patients’ physiotherapy and all were positive for LES. However, LES was detected in the room air even when the patients had been absent from their rooms for 1–3 h prior to testing. The air in two positive rooms tested negative 3 h after patient discharge and room cleaning. All the P. aeruginosa strains detected in the air were the epidemic strain.
Outpatient clinic Sixteen samples were examined (Table III). LES was detected in the spirometry tubing and chair after use by colonized patients. Two subsequent samples from this site (2 and 3 h later) were negative. Overall, five of eight air samples (62.5%) were positive for LES. No other P. aeruginosa strains were detected in the air or on dry surfaces.
Survival of P. aeruginosa strains on dry surfaces Soon after drying (1 h after inoculation), the viable counts of the saline bacterial suspensions were reduced considerably (log10 reduction factors of 2.1–4.5). At 9 h, all strains were still recovered with log10 viable counts per mL ranging from 0.2 to 3.8. Viable organisms were still detectable 48 h after
Environmental contamination in cystic fibrosis Table I
105
Recovery of Pseudomonas aeruginosa from the inpatient ward inanimate environment, patients and staff
Site
Staffs’ hands and uniforms Patients’ hands Patients’ clothes Patients’ bed linen Wash basin, bath tub, shower drains Bathroom, toilet handles/surfaces Dry environmental surfacesa External surface of respiratory equipment
Number of sites/ people tested
Number of sites with non-epidemic P. aeruginosa
Number of sites with LES
Total number of samples
12 8 8 8 18
0 0 0 0 11 (61%)
0 2 (25%) 1 (12.5%) 2 (25%) 1 (6%)
12 8 8 8 40
4
4 (100%)
0
9
7b
0
0
14
15
0
3 (20%)
15
LES, Liverpool epidemic strain. a A single cloth was used to sample all dry surfaces in the same room including furniture, door handles, television, telephones etc. b Number of rooms tested.
drying for most strains, albeit at very low numbers (Figure 1). No significant difference was found in the survival of the mucoid and the non-mucoid phenotype of LES, or between LES and the reference strain PA01. LES survived significantly longer than the Manchester epidemic strain (PZ0.013), one unique Liverpool CF isolate b (PZ0.011) and one other epidemic UK strain B (PZ0.010). Comparison of LES survival with the other strains did not reveal any significant differences.
Discussion Recent reports of epidemic spread of transmissible P. aeruginosa strains among patients attending CF clinics worldwide have provided clear evidence to support the occurrence of cross-infection between CF patients, and have generated controversy on infection control practices and the management of these patients.3,11,12 LES not only colonizes most CF patients in Liverpool, but is also found in several Table II
other CF centres in the UK.13 We undertook the present study to investigate the means by which this strain is acquired and whether prolonged survival on dry surfaces contributes to its transmissibility. Extensive bacteriological screening of the inanimate hospital environment has identified P. aeruginosa in the majority of moist sites (wash basins, bath tubs and shower drains), and also on communal bathroom and toilet handles and surfaces. Only one of these sites (a sink drain inside a cubicle occupied by a known colonized patient) was transiently positive for LES, suggesting secondary contamination from the patient. P. aeruginosa is frequently isolated from moist sites in hospitals, but the importance of these reservoirs as a source of infection is uncertain.14 The role of contamination of the environment with P. aeruginosa in CF clinics has been the subject of several studies.15–19 Heavy environmental contamination was found in some studies,15,17,19 especially in moist sites, and transmission of P. aeruginosa from sinks to hands during handwashing has been shown.19 Survival for considerable periods of time in aerosols and on hands
Recovery of Pseudomonas aeruginosa from air samples in the inpatient ward
Site
Cubicle patient present Cubicle patient absent Ward corridor Lounge LES, Liverpool epidemic strain.
Number of samples with non-epidemic P. aeruginosa
Number of samples with LES
Total number of samples
0 0 0 0
10 2 3 0
11 4 5 1
106 Table III
S. Panagea et al. Recovery of Pseudomonas aeruginosa from air, staffs’ hands and dry surfaces in the outpatient clinic
Site
Air in consultation room Air in corridor Air in waiting room Air in spirometry room Staffs’ hands Dry surfacesa
Number of samples with non-epidemic P. aeruginosa
Number of samples with LES
Total number of samples
0 0 0 0 0 0
1 2 1 1 0 1
4 2 1 1 3 5
LES, Liverpool epidemic strain. a The dry surfaces tested included: door handles and chairs in the corridor and the waiting room (two samples), and spirometry machine tubing and chair (three samples).
has also been demonstrated.20 Improved hygienic precautions and segregation of patients prevented environmental contamination and was associated with reduced incidence of infection in Danish CF patients.18 However, even in situations of heavy environmental contamination, direct transmission from the environment to patients has only been demonstrated occasionally.19 Two recent studies investigating outbreaks with epidemic CF strains in Manchester and Melbourne failed to find contamination of the environment.7,21 No persistent environmental reservoirs for LES were detected in our study. We found, however, that colonized patients do indeed contaminate their hands, their clothes and their immediate surroundings (bed linen). LES was also detected on the external surface of their respiratory equipment and the spirometry machine tubing and chair following use by colonized patients, which raises the possibility of transmission to other patients either directly or via the hands of staff.
Figure 1 Survival of cystic fibrosis (CF) Pseudomonas aeruginosa strains on dry surfaces. %, Liverpool epidemic strain; &, Manchester; :, UK CF strain A; B UK CF strain B; $, UK CF strain C; C, unique CF strain a; 6, unique CF strain b.
Contamination was short-lived however, repeat sampling was negative following patient discharge and routine cleaning. Staffs’ hands and uniforms were also negative. Therefore, it appears that contamination of inanimate surfaces occurs only in close proximity to patients and is transient. Provided that standard hygienic and routine infection control measures are applied, inanimate surface contamination should play a minimal role in transmission. Nevertheless, the detection of LES in the majority of air samples in both the inpatient and outpatient sites is worrying. LES was isolated not only inside the cubicles, but also in the ward corridor, the clinic corridor and waiting room. It was found in the air not only in the presence of colonized patients, but also when patients were not in their rooms for up to 3 h prior to testing. Although the number of organisms was very small, the level of exposure required for acquisition is not known. The duration of airborne contamination was considerable and LES was the only P. aeruginosa strain isolated from the air, suggesting enhanced survival of this strain in aerosols. Air contamination with LES was also detected in the Manchester CF clinic.21 At present, it is unknown why some P. aeruginosa strains are capable of cross-infection in CF patients. Survival times of P. aeruginosa strains in aerosols have been shown to be dependent on strain characteristics,20 and increased survival time on dry surfaces has been suggested to contribute to increased transmissibility of some Burkholderia cepacia strains.22 We tested the hypothesis that prolonged survival of LES on dry surfaces contributes to its apparent higher transmissibility. We found that some P. aeruginosa strains, including LES, survive well in the environment under dry conditions. However, although LES survival was significantly longer compared with some strains
Environmental contamination in cystic fibrosis tested, it was not longer compared with other strains that have not been shown to be transmissible. Therefore, improved environmental survival on its own cannot explain the high transmissibility of this epidemic strain and we continue to look for other factors that can explain its prevalence in the UK CF community. In conclusion, we have shown ward and outpatient clinic environmental contamination with LES in this study. It appears to occur only in close proximity to colonized patients and to be shortlived. No persistent environmental reservoirs were detected. The presence of LES in the majority of air samples in both the inpatient and outpatient areas is particularly worrying, and it seems likely that airborne dissemination plays a significant role in patient-to-patient spread of this organism. We suggest that cohort segregation of patients who carry epidemic P. aeruginosa strains should be implemented.
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