APMIS 122: 85–91
© 2013 APMIS. Published by John Wiley & Sons Ltd. DOI 10.1111/apm.12243
Genotyping of Pseudomonas aeruginosa isolates from lung transplant recipients and aquatic environment– detected in-hospital transmission EWA JOHANSSON,1,2 CHRISTINA WELINDER-OLSSON1,2 and MARITA GILLJAM3,4 Clinical Microbiology, Sahlgrenska University Hospital, Gothenburg; 2Institute of Biomedicine, Sahlgrenska Academy, University of Gothenburg, Gothenburg; 3Department of Respiratory Medicine and Allergology, Sahlgrenska University Hospital, Gothenburg; 4Institute of Medicine, Sahlgrenska Academy, University of Gothenburg, Gothenburg, Sweden
1
Johansson E, Welinder-Olsson C, Gilljam M. Genotyping of Pseudomonas aeruginosa isolates from lung transplant recipients and aquatic environment–detected in-hospital transmission. APMIS 2014; 122: 85–91. Lung infection with Pseudomonas aeruginosa is common in lung transplant recipients and may lead to severe complications. Bacteriological surveillance aims to detect transmission of microbes between hospital environment and patients. We sought to determine whether genotyping of P. aeruginosa isolates could improve identifications of pathways of infection. From 2004 to 2009, we performed genotyping with multiple-locus variable number of tandem repeats analysis (MLVA) and pulsed-field gel electrophoresis (PFGE) of P. aeruginosa isolates cultured from lung transplant recipients at Sahlgrenska University Hospital, Gothenburg. During a small outbreak in 2008, cultivation and genotyping of isolates from sink and drains samples from the hospital ward were performed. Pseudomona aeruginosa from 11/18 patients were genotyped to unique strains. The remaining seven patients were carriers of a P. aeruginosa strain of cluster A genotype. Pseudomona aeruginosa was isolated in 4/8 water samples, typed by MLVA also as cluster A genotype and confirmed by PFGE to be similar or identical to the isolates from four transplanted patients. In conclusion, genotyping of isolates revealed a clonal relationship between patient and water isolates, indicating in-hospital transmission of P. aeruginosa. We suggest genotyping with MLVA for rapid routine surveillance, with the PFGE method used for extended, confirmatory analyses. Key words: Pseudomonas aeruginosa; lung transplant; transmission; surveillance; genotyping. Ewa Johansson, Clinical Microbiology, Sahlgrenska University hospital, PO Box 7193 S-402 34 Gothenburg, Sweden. e-mail: [email protected]
Pseudomonas aeruginosa are ubiquitous bacteria, able to persist in a large number of environments, and an important nosocomial pathogen (1). Pseudomonas aeruginosa, colonizing human airways, is difficult to eradicate and may cause increased morbidity and mortality in patients with cystic fibrosis (CF) as well as in lung transplant recipients and other immuno-compromised patients (2–7). Cross-infection with P. aeruginosa in the setting of lung transplantation is of concern, as early colonization may affect long-term outcome (8). Genomic finger-printing of P. aeruginosa in the CF clinics has shown to be essential for characterization of strains
Received 31 October 2012. Accepted 9 January 2013
(9–11). Pulsed-field gel electrophoresis (PFGE) offers a highly discriminatory method for genetic fingerprinting (12). However, this method is laborintensive, and the results are difficult to standardize and interpret. Consequently, other molecular methods have been used trying to discriminate strains with the same accuracy as PFGE. After PFGE, multilocus sequence typing (MLST) has become a popular genotyping technique. In a recent study, random amplified polymorphic DNA, MLST, and PFGE were compared and MLST demonstrated the highest level of agreement to PFGE to distinguish clonality of P. aeruginosa strains (13). However, MLST is an expensive method. Multiple-locus variable number of tandem repeats analysis (MLVA) is another genotyping method for P. aeruginosa strains 85
JOHANSSON et al.
(14, 15). Mansfeld et al. have compared MLVA with PFGE and MLST by typing 60 P. aeruginosa isolates from CF patients (16). They concluded that although MLVA and MLST had the same typeability, MLVA was selected as the preferred typing method based on slightly higher diversity, faster and cheaper compared with MLST (16). In the study, we used PFGE and MLVA to genotype isolates of P. aeruginosa to identify routes of in-hospital transmission at the transplant ward and to improve infection control. MATERIALS AND METHODS Patients and setting Airway samples from patients followed from 2004 to 2009 at the Gothenburg Program for Lung Transplantation were analyzed. Pre-transplant assessment, as well as periand post-transplant care, was undertaken at the Thoracic Intensive Care Unit (ICU), the Thoracic Transplant Ward and the Outpatient Clinic for Lung Transplantation, Sahlgrenska University Hospital.
analysis (Dice coefficient/UPGMA) and Tenover criteria (12). Designations used for PFGE genotype classifications include a capital letter (e.g., ‘A’); fingerprint patterns showing differences of one to six DNA fragments were indicated by a numerical suffix (e.g., ‘A-1’) to indicate clonal variation.
Multiple-locus variable number of tandem repeats analysis – Multiple-locus variable number of tandem repeats analysis typing was performed, adapted from the protocol of Onteniente et al. and Vu-Thien et al. (14, 15), with Q-buffer (Qiagen, Sollentuna, Sweden), using the primers for the following variable-number-of-tandemrepeats (VNTRs): ms211; ms212; ms214; ms213b; ms215b; ms216; ms217b; ms222b; ms223; and ms142 (MLVA10-Sweden). The size of each amplicon was determined and number of repeats was deduced, using the MLVA allele’s assignment table on the P. aeruginosa genotyping site (http://minisatellites.u-psud.fr/MLVAnet/). Strain PA01 was used as control. MLVA10-Sweden types were compared with the international database ‘pseudomonas2007’ created by Gilles (http://minisatellites.u-psud. fr/MLVAnet/).
RESULTS Clinical samples
Clinical outcome
Bacterial cultivations of sputum or bronchoalveolar lavage fluid samples were performed according to the reference methods for lower respiratory infections as recommended by Swedish Institute for Communicable Disease Control (Referensmetodik: Nedre luftv€ agsinfektioner, 2:a upplagan 2005).
Genotyping was performed on 24 isolates of P. aeruginosa obtained from airway samples from 18/ 72 (25%) culture-positive lung-transplanted patients. The isolates were collected from 2004 to 2009. Table 1 summarizes demographic, clinical, and microbiological data and Table 2 details genotyping data. All four patients with CF had pre-operative infections with P. aeruginosa, one patient with Burkholderia cenocepacia and another with Mycobacterium abscessus (18) as the main pathogens. For all CF patients, post-operative antibiotics were administered against P. aeruginosa and the other pathogens. For other patient disease groups, no patient had pre-operative P. aeruginosa infection and the standard post-operative antibiotic regimen of cefotaxim was administered. Infection with P. aeruginosa, contracted post-transplant, led to prolonged hospital stays and treatment with intravenous antibiotics within the first post-operative 6 months in 7/14 non-CF patients and after 14 months and 4 years, respectively, in two other patients. During 2 months in 2008, a cluster of five patients acquired P. aeruginosa airway infections and, as a consequence, sampling in the hospital environment was performed.
Environmental screening Specimens were collected as swabs from the surfaces of sink and shower drains at the Thoracic Transplant Ward of Sahlgrenska University Hospital, Gothenburg, Sweden, in June 2008 and additional samplings were performed at the adjacent Visceral Transplant ward in August 2008. The specimen swabs were cultivated at the Clinical Microbiology, Sahlgrenska University Hospital. Pseudomonas aeruginosa colonies were identified based on characteristic morphology, by Gram’s staining and oxidase test according to the Swedish reference methods for lower respiratory infections (see above).
Genotyping
Pulsed-field gel electrophoresis – Genomic DNA from P. aeruginosa isolates were prepared according to method described by Gautom (17). The DNA were digested, using SpeI (Fermentas, Thermo Scientific, Helsingborg, Sweden) and the restriction fragments resolved by PFGE, following the manufacturer’s instructions (Bio-Rad Laboratories, Sundbyberg, Sweden). Pseudomonas aeruginosa strain PAO1 (ATCC BAA47, CCUG 49694) was included in each run as an internal reference. Computer analysis was achieved, using the BioNumerics software version 5.10 (Applied Maths, Kortrijk, Belgium). The relatedness of each PFGE fingerprint was determined, using cluster
86
Genotyping of P. aeruginosa
Genotyping of P. aeruginosa isolates had been performed pre-transplant for two CF patients (No. 13 © 2013 APMIS. Published by John Wiley & Sons Ltd
CROSS-INFECTION OF P. AERUGINOSA
Table 1. Genotyping of Pseudomonas aeruginosa in lung transplant recipients (2004–2009) No Age Gender Diagnosis Op Pseudomonas aeruginosa1 1st Positive2 Time post Genotyped op strains 1 60 M COPD SLT September 2002 5 months April 2006 2 28 F CF4,6 DLT December 2004 0 days January 2005 3
22
M
PPH
DLT
September 2005
1 year
4
62
F
IPF
SLT
June 2006
0.5 months
5 6
44 56
F F
CHD COPD
HLT SLT 928
June 2002 July 2007
7 8
55 59
M M
Sclerodermia EmphysemaA1
DLT DLT
November 2007 March 2008
1.5 months 2 years/ 3 months 5 months 3.5 months
9 10
57 60
M M
EmphysemaA1 IPF
DLT SLT 928
March 2008 March 2008
6 days 2 years/ 2 weeks 0.5 months
PFGE3 CR AI A-25
October 2005/ April 2006 June/November 2006 April 2008 July 2007
A-2-5/ A-2-5-25 EP 25
November 2007 March 2008
C-5-3-5-5 EC
April 2008 March 2008
CT-2 A-2-5-2c5
Antibiotic treatment p.o. i.v., p.o., inhaled p.o. p.o. i.v. i.v., p.o. p.o. i.v., p.o., inhaled i.v., p.o. i.v.
A-2-5-2b5
i.v., p.o., inhaled 12 63 M IPF SLT April 2008 1.5 months April 2008 A-2-5-35 i.v. 13 34 M CF4 DLT June 2008 0 days9 2004/2007/2008 A-35 i.v. 14 22 F CF4 DLT August 2008 0 days 2006/2008/2009 BF-2 i.v. 15 35 M Sarcoidosis DLT June 2009 14 months August 2009 GG i.v. 16 58 M EmphysemaA1 SLT July 2009 4 years July 2009 A-2-5-2b5 i.v. 17 14 F CF4,7 DLT Nov 2009 3 months November 2009 GS i.v. 18 67 F COPD SLT December 2009 1 month December 2009 GZ i.v. Age, at transplantation; COPD, chronic obstructive pulmonary disease; IPF, idiopathic pulmonary fibrosis; CF, cystic fibrosis; A1, alpha1antitrypsin-deficiency; CHD, congenital heart disease; PPH, primary pulmonary hypertension; SLT, single lung transplantation; DLT, double lung transplantation; HLT, heart and lung transplantation; i.v., intravenous; p.o., per oral; PFGE, pulsed-field gel electrophoresis. 1All strains were sensitive to at least one antibiotic in each group of quinolones, aminoglycosides, and betalactam antibiotics, respectively. 2First post-operative culture positive for P. aeruginosa. 3 See Table 2 for methods. 4Chronic genotyped P. aeruginosa pre-transplant. 5Cluster A strain; Pre-transplant co-infection with 6Mycobacterium abscessus and 7Burkholderia cenocepacia as the main pathogens. 8Re-transplanted other side due to chronic rejection, growth of P. aeruginosa after re-transplantation. 9Died in ICU and never transferred to the ward. 11
53
M
Sarcoidosis
SLT
March 2008
and 14, Table 1) and was repeated at transplantation (No. 2, 13, and 14) and on the first positive culture post-transplant (No. 2, 14, and 17). Genotyping was performed on the first post-operative P. aeruginosa isolates from 14 non-CF patients (Table 2). Only PFGE was performed for analyzing the isolates from patients 1 and 3, as MLVA was not used, as routine, at the beginning of the study period. The PFGE patterns obtained by SpeI digestion of genomic DNA were compared; 10 of the 17 patients had isolates with unique profiles, confirming that the strains were not genotypically related (Fig. 1). The isolate from patient number 6 could not be assigned a PFGE type, as the DNA was degraded during the preparation for analysis, but was later successfully classified by MLVA technique to a unique P. aeruginosa strain, with an MLVA code previously assigned to genotype 25 in France (C. Pourcel, personal communication). Isolates from six patients carrying assigned cluster A strains were genotyped using PFGE and MLVA10-Sweden during 2004–2008, while the cluster A strain from © 2013 APMIS. Published by John Wiley & Sons Ltd
March 2008
patient 16 was typed in June 2009, after relocation of the transplant program to a new building. Agreement between the two genotyping methods PFGE and MLVA10-Sweden results was good, with variations in MLVA at the 10 selected repeats providing discrimination to a similar extent as PFGE (Table 2). However, no amplified product was produced for 5/160 PCR analyses, probably explained by nucleotide variation in the primerannealing site. Isolates within PFGE clusters with variant PFGE profiles exhibited the same MLVA patterns. The specific P. aeruginosa cluster A seems to have a high mutation rate, resulting in several clonal variants. Cluster A strains isolated from the studied patients were differentiated according to seven different, but closely related, PFGE band patterns (Fig. 1). The method MLVA10-Sweden confirmed that all cluster A strains belonged to the same cluster (Table 2). Extensive growth of P. aeruginosa was recovered 2 months after the last patient analyzed in 2008 from four of eight samples collected from sink and
87
JOHANSSON et al.
Table 2. Genotype of Pseudomonas aeruginosa strains isolated from lung transplant recipients Patient VNTR code MLVA10- PFGE Sweden 211, 212, 214, 213, 215, 216, 217, 222, 223, 142 1 – – CR 2 6, 9, 5, 3, 2, 2, 3, 4, 2, 3 AI1 AI1 3 –/– –/– A-2/A-2 4 6, 6, 2, 2, 5, 2, 1, 3, 3, 42 A/A A-2-5/ A-2-5-2 5 3, 8, 2, 5, 2, 2, 3, 1, 5, 4 EP EP 3 6 8, 6, 2, 4.5, 1, 2, 1, 1, 25 1.5, 3 7 8, 9, 2, 2, 4, 2, 4, 3, 3, 4 C C-5-3-5-5 8 2, 4, 5, 1, 2, 1, 5, –, 4, 1 EC EC 9 3, 8, 5, 5, 5, 1, 3, 1, 2, 2 CT CT-2 10 6, 6, 2, 2, 5, 2, 1, 3, 3, 4 A A-2-5-2c 11 6, 6, 2, 2, 5, 2, 1, 3, 3, 4 A A-2-5-2b 12 6, 6, 2, 2, 5, 2, 1, 3, 3, 4 A A-2-5-3 13 –, 6, 2, 2, 5, 2, 1, 3, 3, 4 –/A/– A-3/A-3/ A-3 14 3, 8, 5, IS, 2.5, 1, –, 1, BF/–/– BF-2/ 2, 1 BF-2/ BF-2 15 3, 5, 4.5, 5, 6, 1, 2, 1, GG GG 4, 1 16 6, 6, 2, 2, 5, 2, 1, 3, 3, 4 A A-2-5-2b 17 3, 9, 4, 4, 2, 2, 5, 3, –, 4 GS GS 18 –, 9, 4, 2, 2, 1, 2, 1, 2, 2 GZ GZ Genotyping was performed once or twice (patient 2 and 4) with different methods post-transplant, except patient 13 for whom genotyping was performed three times pretransplant and patient 14 whom genotyping was performed once pre-transplant and twice post-transplant. VNTR, variable-number-of-tandem-repeats; MLVA, multiple-locus variable number of tandem repeat analysis; PFGE, pulsed-field gel electrophoresis; ‘–’, no amplified VNTR; IS, Insertion element. 1 Two letter strain AI is not related to cluster A strains. 2 Same VNTR code for both samples. 3 No PFGE pattern due to auto-degradation of DNA.
shower drains in rooms where patient 10, 11, and 12 had stayed. There were no strains of P. aeruginosa found in the samples from tap water in the ward corridor or from floor drain and sink in the anterooms. All four isolates revealed the MLVA10Sweden pattern, similar to the epidemic cluster A strain (Table 3). The more discriminating PFGE analysis confirmed these environmental isolates to be identical with the cluster A strains carried by four transplanted patients (Fig. 1 and Table 3). Due to overlapping periods of hospitalization and opportunities for close contact, there was possibility for direct transmission among patients 10, 11, and 12, but uncertain for the other four patients harboring the cluster A strain. The remaining 11 patients carried unique genotypes of P. aeruginosa. The environmental P. aeruginosa isolate from the adjacent ward displayed a unique PFGE pattern
88
and MLVA10-Sweden code, different from the patient isolates (Fig. 1 and Table 3).
DISCUSSION We describe a probable transmission of P. aeruginosa at a thoracic transplant ward, and the use of genotyping for identification and bacteriological surveillance. As P. aeruginosa is ubiquitous in the environment and also part of the endogenous micro-flora of hospitalized patients, the original source of the bacterium and the precise mode of transmission often remain unclear, although contaminated water and medical equipment, direct patient-to-patient transmission, as well as nurse-topatient transmission have been reported (1, 19, 20). Using genotyping methods, we identified unique P. aeruginosa strains in 11/18 (61%) patients, assumed to be cases of indigenous infection, while 7/18 (39%) shared the same genotype. For two CF patients with chronic P. aeruginosa infection, the identical strain was identified pre- and post-operatively, whereas for one patient with intermittent positive cultures, only the post-transplant strain had been genotyped. Over a 10-year study period, Cuttelod et al. (21) observed that endogenous sources of infection remained stable, although exogenous sources varied, concomitant to environmental contamination, infection control measures, and the genetics of P. aeruginosa itself. In the cases of three patients, there had been opportunity for close contact and we suspect crossinfection rather than an environmental origin of the infections. We are not able to give the exact room location for the remaining cluster A-infected patients at the ward; however, they all had access to the shower room with a contaminated floor drain. Acquisition of P. aeruginosa following exposure to contaminated tap water in the ICU has been described (22). However, the epidemiology of P. aeruginosa is complex, as shown for an outbreak in a German ICU, where the isolated environmental P. aeruginosa strains had no relation to the genotypes isolated from the patients (23). Sink and floor drain samplings from two patient rooms on the ward revealed that P. aeruginosa strains were identical or similar to the genotypes identified in cultures from patients hospitalized at the same time period, indicating prolonged contamination in the hospital ward environment. The PFGE and MLVA10-Sweden genotyping methods complemented each other. Using the discriminating but more labor-intensive PFGE, several of the cluster A strains exhibited different band patterns, complicating cluster analysis. However, using
© 2013 APMIS. Published by John Wiley & Sons Ltd
CROSS-INFECTION OF P. AERUGINOSA
Fig. 1. Cluster analysis of Pseudomonas aeruginosa chromosomal DNA digested with Spe1; restriction fragments were separated by PFGE. The dendrogram shows the relative similarities of the DNA band patterns from Swedish lung-transplanted patients (n = 17) and environmental strains from the transplant ward (n = 4) and adjacent ward (n = 1) at the Sahlgrenska hospital. The related cluster A strains are marked. The strain AI shares the first letter designation, ‘A’ with cluster A strains, although no genotypic relationship is indicated in the PFGE fingerprints.
Table 3. Genotype of environmental Pseudomonas aeruginosa strains isolated at two nearby medical care units Sampling site Time VNTR code MLVA PFGE 10-Sweden 211, 212, 214, 213, 215, 216, 217, 222, 223, 142 Transplant ward Sink, Room 7 June 2008 6, 6, 2, 2, 5, 2, 1, 3, 3, 4 A A-2-5-2c Floor drain, Room 7 June 2008 6, 6, 2, 2, 5, 2, 1, 3, 3, 4 A A-2-5-2d Sink, Room 8 June 2008 6, 6, 2, 2, 5, –, 1, 3, 3, 4 A A-2-5-3-4 Floor drain, Shower June 2008 6, 6, 2, 2, 5, 2, 1, 3, 3, 4 A A-2-5-2b-2 room Adjacent ward Sink August 2008 2, 9, 5, 8, 4, 1, 4, 4, 2, 4 EV EV VNTR, variable-number-of-tandem-repeats; MLVA, multiple-locus variable number of tandem repeat analysis; PFGE, pulsed-field gel electrophoresis. © 2013 APMIS. Published by John Wiley & Sons Ltd
89
JOHANSSON et al.
the less discriminating MLVA10-Sweden, we found all cluster A strains to belong to the same cluster, sharing the same VNTR code. Using the inter-laboratory reproducible MLVA technique, we compared the results to MLVA codes for P. aeruginosa isolates from other laboratories. We concluded that the PFGE profile of cluster A is identical to the previously described widespread clone C (24). We may also speculate that these P. aeruginosa cluster A/C strains, common to patients and environmental habitats (10), were the source of contamination at the lung transplant unit. Ruiz et al. detected a clear separation between clinical and environmental isolates and suggested that the origin of hospital infections is due to infection acquired by patients prior to hospital stay (25). As the tap water sample was not contaminated, and there was opportunity for close contact, patient-to-patient transmission is more likely than acquisition of P. aeruginosa from the environment. The P. aeruginosa cluster A genotype seems less prevalent at our hospital, as only one isolate with a unique pattern was detected at the adjacent ward. The phenotypes of resistant P. aeruginosa epidemic clones have been shown to undergo sporadic variation during the course of an outbreak (26). Using genotyping, rather than phenotyping techniques such as antibiotic susceptibility testing, has improved the characterization of P. aeruginosa isolates. The effects of implemented infection control measures can be effectively evaluated by molecular, epidemiological investigation (27). After relocation of the transplant unit to a new building, we are continuing the surveillance of P. aeruginosa and genotype new isolates; only one P. aeruginosa isolate has been typed to cluster A and there is no possibility of cross-infection in this patient. This study has limitations: genotyping was performed in only 25% of culture-positive patients and MLVA was missing for two patients, as the method had not yet been established for regular use at the time. In conclusion, this survey revealed cross-infection within a transplant unit, leading to infectious complications in lung transplant recipients. The study highlights the importance of microbiological surveillance in the environment of lung transplantation. We suggest genotyping, using MLVA10-Sweden for routine infection control and PFGE for extended analysis.
This study was funded by grant from Sahlgrenska University Hospital 95340. The authors thank Leif Larsson for the environmental sampling at the hospital and all members of the DNA laboratory at the Clinical Microbiology
90
for the skillful PFGE, G€ oran Dellgren and Gerdt Riise for reading of the manuscript. We further thank Ed Moore for constructive criticism of the manuscript. Finally, we are also grateful to Christine Pourcel from the Institute for Genetics and Microbiology in Paris, France for her excellent support with the MLVA method. The authors have no conflicts of interest.
REFERENCES 1. Kerr KG, Snelling AM. Pseudomonas aeruginosa: a formidable and ever-present adversary. J Hosp Infect 2009;73:338–44. 2. Cystic Fibrosis Foundation. Patient Registry 2005 Annual Report. Bethesda, MD: Cystic Fibrosis Foundation, 2006. 3. Emerson J, Rosenfeld M, McNamara S, Ramsey B, Gibson RL. Pseudomonas aeruginosa and other predictors of mortality and morbidity in young children with cystic fibrosis. Pediatr Pulmonol 2002;34:91–100. 4. Fagon JY, Chastre J, Vuagnat A, Trouillet JL, Novara A, Gibert C. Nosocomial pneumonia and mortality among patients in intensive care units. JAMA 1996;275:866–9. 5. Aaron SD, Vandemheen KL, Ramotar K, GiesbrechtLewis T, Tullis E, Freitag A, et al. Infection with transmissible strains of Pseudomonas aeruginosa and clinical outcomes in adults with cystic fibrosis. JAMA 2010;304:2145–53. 6. Zeglen S, Wojarski J, Wozniak-Grygiel E, Siola M, Jastrzebski D, Kucewicz-Czech E, et al. Frequency of Pseudomonas aeruginosa colonizations/infections in lung transplant recipients. Transplant Proc 2009;41:3222–4. 7. Sanders DB, Bittner RC, Rosenfeld M, Hoffman LR, Redding GJ, Goss CH. Failure to recover to baseline pulmonary function after cystic fibrosis pulmonary exacerbation. Am J Respir Crit Care Med 2010;182:627–32. 8. Kovats Z, S€ utto Z, Murak€ ozy G, Bohacs A, Czebe K, Lang G, et al. Airway pathogens during the first year after lung transplantation: a single-center experience. Transplant Proc 2011;43:1290–1. 9. Scott FW, Pitt TL. Identification and characterization of transmissible Pseudomonas aeruginosa strains in cystic fibrosis patients in England and Wales. J Med Microbiol 2004;53:609–15. 10. R€ omling U, Kader A, Sriramulu DD, Simm R, Kronvall G. Worldwide distribution of Pseudomonas aeruginosa clone C strains in the aquatic environment and cystic fibrosis patients. Environ Microbiol 2005;7:1029–38. 11. Johansson E, Welinder-Olsson C, Lindblad A, Gilljam M, Johannesson M, Meyer P, et al. Genomic fingerprinting of Pseudomonas aeruginosa from all Swedish CF Centres. A Scandinavian cystic fibrosis study consortium study. J Cyst Fibrosis 2007;6:114. 12. Tenover FC, Arbeit RD, Goering RV, Mickelsen PA, Murray BE, Persing DH, et al. Interpreting chromosomal DNA restriction patterns produced by pulsedfield gel electrophoresis: criteria for bacterial strain typing. J Clin Microbiol 1995;33:2233–9. © 2013 APMIS. Published by John Wiley & Sons Ltd
CROSS-INFECTION OF P. AERUGINOSA
13. Waters V, Zlosnik JEA, Yau YCW, Speert DP, Aaron SD, Guttman DS. Comparison of three typing methods for Pseudomonas aeruginosa isolates from patients with cystic fibrosis. Eur J Clin Microbiol Infect Dis 2012;31:3341–50. 14. Onteniente L, Brisse S, Tassios PT, Vergnaud G. Evaluation of the polymorphisms associated with tandem repeats for Pseudomonas aeruginosa strain typing. J Clin Microbiol 2003;41:4991–7. 15. Vu-Thien H, Corbineau G, Hormigos K, Fauroux B, Corvol H, Clement A, et al. Multiple-locus variablenumber tandem-repeat analysis for longitudinal survey of sources of Pseudomonas aeruginosa infection in cystic fibrosis patients. J Clin Microbiol 2007;45:3175–83. 16. Mansfeld R, Jongerden I, Bootsma M, Buiting A, Bonten M, Willems R. The population genetics of Pseudomonas aeruginosa isolates from different patient populations exhibits high-level host specificity. PLoS ONE 2010;5:e13482. 17. Gautom RK. Rapid pulsed-field gel electrophoresis protocol for typing of Escherichia coli 0157:H7 and other gram-negative organisms in 1 day. J Clin Microbiol 1997;35:2977–80. 18. Gilljam M, Schersten H, Silverborn M, J€ onsson B, Ericsson-Hollsing A. Lung transplantation in patients with cystic fibrosis and Mycobacterium abscessus infection. J Cystic Fibrosis 2010;9:272–6. 19. Schelenz S, French G. An outbreak of multidrug-resistant Pseudomonas aeruginosa infection associated with contamination of bronchoscopes and an endoscope washer-disinfector. J Hosp Infect 2000;46:23–30. 20. Moolenaar RL, Crutcher JM, San Joaquin VH, Sewell LV, Hutwagner LC, Carson LA, et al. A prolonged outbreak of Pseudomonas aeruginosa in a neonatal intensive care unit: did staff fingernails play a role in
© 2013 APMIS. Published by John Wiley & Sons Ltd
21.
22.
23.
24.
25.
26.
27.
disease transmission? Infect Control Hosp Epidemiol 2000;21:80–5. Cuttelod M, Senn L, Terletskiy V, Nahimana I, Petignat C, Eggimann P, et al. Molecular epidemiology of Pseudomonas aeruginosa in intensive care units over a 10-year period (1998-2007). Clin Microbiol Infect 2011;17:57–62. Rogues AM, Boulestreau H, Lasheras A, Boyer A, Gruson D, Merle C, et al. Contribution of tap water to patient colonisation with Pseudomonas aeruginosa in a medical intensive care unit. J Hosp Infect 2007;67:72–8. Kohlenberg A, Weitzel-Kage D, van der Linden P, Sohr D, V€ ogeler S, Kola A, et al. Outbreak of carbapenem-resistant Pseudomonas aeruginosa infection in a surgical intensive care unit. J Hosp Infect 2010;74:350–7. R€ omling U, Wingender J, M€ uller H, T€ ummler B. A major Pseudomonas aeruginosa clone common to patients and aquatic habitats. Appl Environ Microbiol 1994;60:1734–8. Ruiz L, Domınguez MA, Ruiz N, Vi~ nas M. Relationship between clinical and environmental isolates of Pseudomonas aeruginosa in a hospital setting. Arch Med Res 2004;35:251–7. Hocquet D, Bertrand X, K€ ohler T, Talon D, Plesiat P. Genetic and phenotypic variations of a resistant Pseudomonas aeruginosa epidemic clone. Antimicrob Agents Chemother 2003;47:1887–94. Petignat C, Francioli P, Nahimana I, Wenger A, Bille J, Schaller MD, et al. Exogenous sources of Pseudomonas aeruginosa in intensive care unit patients: implementation of infection control measures and follow-up with molecular typing. Infect Control Hosp Epidemiol 2006;27:953–7.
91
© 2013 APMIS. Published by John Wiley & Sons Ltd. DOI 10.1111/apm.12243
Genotyping of Pseudomonas aeruginosa isolates from lung transplant recipients and aquatic environment– detected in-hospital transmission EWA JOHANSSON,1,2 CHRISTINA WELINDER-OLSSON1,2 and MARITA GILLJAM3,4 Clinical Microbiology, Sahlgrenska University Hospital, Gothenburg; 2Institute of Biomedicine, Sahlgrenska Academy, University of Gothenburg, Gothenburg; 3Department of Respiratory Medicine and Allergology, Sahlgrenska University Hospital, Gothenburg; 4Institute of Medicine, Sahlgrenska Academy, University of Gothenburg, Gothenburg, Sweden
1
Johansson E, Welinder-Olsson C, Gilljam M. Genotyping of Pseudomonas aeruginosa isolates from lung transplant recipients and aquatic environment–detected in-hospital transmission. APMIS 2014; 122: 85–91. Lung infection with Pseudomonas aeruginosa is common in lung transplant recipients and may lead to severe complications. Bacteriological surveillance aims to detect transmission of microbes between hospital environment and patients. We sought to determine whether genotyping of P. aeruginosa isolates could improve identifications of pathways of infection. From 2004 to 2009, we performed genotyping with multiple-locus variable number of tandem repeats analysis (MLVA) and pulsed-field gel electrophoresis (PFGE) of P. aeruginosa isolates cultured from lung transplant recipients at Sahlgrenska University Hospital, Gothenburg. During a small outbreak in 2008, cultivation and genotyping of isolates from sink and drains samples from the hospital ward were performed. Pseudomona aeruginosa from 11/18 patients were genotyped to unique strains. The remaining seven patients were carriers of a P. aeruginosa strain of cluster A genotype. Pseudomona aeruginosa was isolated in 4/8 water samples, typed by MLVA also as cluster A genotype and confirmed by PFGE to be similar or identical to the isolates from four transplanted patients. In conclusion, genotyping of isolates revealed a clonal relationship between patient and water isolates, indicating in-hospital transmission of P. aeruginosa. We suggest genotyping with MLVA for rapid routine surveillance, with the PFGE method used for extended, confirmatory analyses. Key words: Pseudomonas aeruginosa; lung transplant; transmission; surveillance; genotyping. Ewa Johansson, Clinical Microbiology, Sahlgrenska University hospital, PO Box 7193 S-402 34 Gothenburg, Sweden. e-mail: [email protected]
Pseudomonas aeruginosa are ubiquitous bacteria, able to persist in a large number of environments, and an important nosocomial pathogen (1). Pseudomonas aeruginosa, colonizing human airways, is difficult to eradicate and may cause increased morbidity and mortality in patients with cystic fibrosis (CF) as well as in lung transplant recipients and other immuno-compromised patients (2–7). Cross-infection with P. aeruginosa in the setting of lung transplantation is of concern, as early colonization may affect long-term outcome (8). Genomic finger-printing of P. aeruginosa in the CF clinics has shown to be essential for characterization of strains
Received 31 October 2012. Accepted 9 January 2013
(9–11). Pulsed-field gel electrophoresis (PFGE) offers a highly discriminatory method for genetic fingerprinting (12). However, this method is laborintensive, and the results are difficult to standardize and interpret. Consequently, other molecular methods have been used trying to discriminate strains with the same accuracy as PFGE. After PFGE, multilocus sequence typing (MLST) has become a popular genotyping technique. In a recent study, random amplified polymorphic DNA, MLST, and PFGE were compared and MLST demonstrated the highest level of agreement to PFGE to distinguish clonality of P. aeruginosa strains (13). However, MLST is an expensive method. Multiple-locus variable number of tandem repeats analysis (MLVA) is another genotyping method for P. aeruginosa strains 85
JOHANSSON et al.
(14, 15). Mansfeld et al. have compared MLVA with PFGE and MLST by typing 60 P. aeruginosa isolates from CF patients (16). They concluded that although MLVA and MLST had the same typeability, MLVA was selected as the preferred typing method based on slightly higher diversity, faster and cheaper compared with MLST (16). In the study, we used PFGE and MLVA to genotype isolates of P. aeruginosa to identify routes of in-hospital transmission at the transplant ward and to improve infection control. MATERIALS AND METHODS Patients and setting Airway samples from patients followed from 2004 to 2009 at the Gothenburg Program for Lung Transplantation were analyzed. Pre-transplant assessment, as well as periand post-transplant care, was undertaken at the Thoracic Intensive Care Unit (ICU), the Thoracic Transplant Ward and the Outpatient Clinic for Lung Transplantation, Sahlgrenska University Hospital.
analysis (Dice coefficient/UPGMA) and Tenover criteria (12). Designations used for PFGE genotype classifications include a capital letter (e.g., ‘A’); fingerprint patterns showing differences of one to six DNA fragments were indicated by a numerical suffix (e.g., ‘A-1’) to indicate clonal variation.
Multiple-locus variable number of tandem repeats analysis – Multiple-locus variable number of tandem repeats analysis typing was performed, adapted from the protocol of Onteniente et al. and Vu-Thien et al. (14, 15), with Q-buffer (Qiagen, Sollentuna, Sweden), using the primers for the following variable-number-of-tandemrepeats (VNTRs): ms211; ms212; ms214; ms213b; ms215b; ms216; ms217b; ms222b; ms223; and ms142 (MLVA10-Sweden). The size of each amplicon was determined and number of repeats was deduced, using the MLVA allele’s assignment table on the P. aeruginosa genotyping site (http://minisatellites.u-psud.fr/MLVAnet/). Strain PA01 was used as control. MLVA10-Sweden types were compared with the international database ‘pseudomonas2007’ created by Gilles (http://minisatellites.u-psud. fr/MLVAnet/).
RESULTS Clinical samples
Clinical outcome
Bacterial cultivations of sputum or bronchoalveolar lavage fluid samples were performed according to the reference methods for lower respiratory infections as recommended by Swedish Institute for Communicable Disease Control (Referensmetodik: Nedre luftv€ agsinfektioner, 2:a upplagan 2005).
Genotyping was performed on 24 isolates of P. aeruginosa obtained from airway samples from 18/ 72 (25%) culture-positive lung-transplanted patients. The isolates were collected from 2004 to 2009. Table 1 summarizes demographic, clinical, and microbiological data and Table 2 details genotyping data. All four patients with CF had pre-operative infections with P. aeruginosa, one patient with Burkholderia cenocepacia and another with Mycobacterium abscessus (18) as the main pathogens. For all CF patients, post-operative antibiotics were administered against P. aeruginosa and the other pathogens. For other patient disease groups, no patient had pre-operative P. aeruginosa infection and the standard post-operative antibiotic regimen of cefotaxim was administered. Infection with P. aeruginosa, contracted post-transplant, led to prolonged hospital stays and treatment with intravenous antibiotics within the first post-operative 6 months in 7/14 non-CF patients and after 14 months and 4 years, respectively, in two other patients. During 2 months in 2008, a cluster of five patients acquired P. aeruginosa airway infections and, as a consequence, sampling in the hospital environment was performed.
Environmental screening Specimens were collected as swabs from the surfaces of sink and shower drains at the Thoracic Transplant Ward of Sahlgrenska University Hospital, Gothenburg, Sweden, in June 2008 and additional samplings were performed at the adjacent Visceral Transplant ward in August 2008. The specimen swabs were cultivated at the Clinical Microbiology, Sahlgrenska University Hospital. Pseudomonas aeruginosa colonies were identified based on characteristic morphology, by Gram’s staining and oxidase test according to the Swedish reference methods for lower respiratory infections (see above).
Genotyping
Pulsed-field gel electrophoresis – Genomic DNA from P. aeruginosa isolates were prepared according to method described by Gautom (17). The DNA were digested, using SpeI (Fermentas, Thermo Scientific, Helsingborg, Sweden) and the restriction fragments resolved by PFGE, following the manufacturer’s instructions (Bio-Rad Laboratories, Sundbyberg, Sweden). Pseudomonas aeruginosa strain PAO1 (ATCC BAA47, CCUG 49694) was included in each run as an internal reference. Computer analysis was achieved, using the BioNumerics software version 5.10 (Applied Maths, Kortrijk, Belgium). The relatedness of each PFGE fingerprint was determined, using cluster
86
Genotyping of P. aeruginosa
Genotyping of P. aeruginosa isolates had been performed pre-transplant for two CF patients (No. 13 © 2013 APMIS. Published by John Wiley & Sons Ltd
CROSS-INFECTION OF P. AERUGINOSA
Table 1. Genotyping of Pseudomonas aeruginosa in lung transplant recipients (2004–2009) No Age Gender Diagnosis Op Pseudomonas aeruginosa1 1st Positive2 Time post Genotyped op strains 1 60 M COPD SLT September 2002 5 months April 2006 2 28 F CF4,6 DLT December 2004 0 days January 2005 3
22
M
PPH
DLT
September 2005
1 year
4
62
F
IPF
SLT
June 2006
0.5 months
5 6
44 56
F F
CHD COPD
HLT SLT 928
June 2002 July 2007
7 8
55 59
M M
Sclerodermia EmphysemaA1
DLT DLT
November 2007 March 2008
1.5 months 2 years/ 3 months 5 months 3.5 months
9 10
57 60
M M
EmphysemaA1 IPF
DLT SLT 928
March 2008 March 2008
6 days 2 years/ 2 weeks 0.5 months
PFGE3 CR AI A-25
October 2005/ April 2006 June/November 2006 April 2008 July 2007
A-2-5/ A-2-5-25 EP 25
November 2007 March 2008
C-5-3-5-5 EC
April 2008 March 2008
CT-2 A-2-5-2c5
Antibiotic treatment p.o. i.v., p.o., inhaled p.o. p.o. i.v. i.v., p.o. p.o. i.v., p.o., inhaled i.v., p.o. i.v.
A-2-5-2b5
i.v., p.o., inhaled 12 63 M IPF SLT April 2008 1.5 months April 2008 A-2-5-35 i.v. 13 34 M CF4 DLT June 2008 0 days9 2004/2007/2008 A-35 i.v. 14 22 F CF4 DLT August 2008 0 days 2006/2008/2009 BF-2 i.v. 15 35 M Sarcoidosis DLT June 2009 14 months August 2009 GG i.v. 16 58 M EmphysemaA1 SLT July 2009 4 years July 2009 A-2-5-2b5 i.v. 17 14 F CF4,7 DLT Nov 2009 3 months November 2009 GS i.v. 18 67 F COPD SLT December 2009 1 month December 2009 GZ i.v. Age, at transplantation; COPD, chronic obstructive pulmonary disease; IPF, idiopathic pulmonary fibrosis; CF, cystic fibrosis; A1, alpha1antitrypsin-deficiency; CHD, congenital heart disease; PPH, primary pulmonary hypertension; SLT, single lung transplantation; DLT, double lung transplantation; HLT, heart and lung transplantation; i.v., intravenous; p.o., per oral; PFGE, pulsed-field gel electrophoresis. 1All strains were sensitive to at least one antibiotic in each group of quinolones, aminoglycosides, and betalactam antibiotics, respectively. 2First post-operative culture positive for P. aeruginosa. 3 See Table 2 for methods. 4Chronic genotyped P. aeruginosa pre-transplant. 5Cluster A strain; Pre-transplant co-infection with 6Mycobacterium abscessus and 7Burkholderia cenocepacia as the main pathogens. 8Re-transplanted other side due to chronic rejection, growth of P. aeruginosa after re-transplantation. 9Died in ICU and never transferred to the ward. 11
53
M
Sarcoidosis
SLT
March 2008
and 14, Table 1) and was repeated at transplantation (No. 2, 13, and 14) and on the first positive culture post-transplant (No. 2, 14, and 17). Genotyping was performed on the first post-operative P. aeruginosa isolates from 14 non-CF patients (Table 2). Only PFGE was performed for analyzing the isolates from patients 1 and 3, as MLVA was not used, as routine, at the beginning of the study period. The PFGE patterns obtained by SpeI digestion of genomic DNA were compared; 10 of the 17 patients had isolates with unique profiles, confirming that the strains were not genotypically related (Fig. 1). The isolate from patient number 6 could not be assigned a PFGE type, as the DNA was degraded during the preparation for analysis, but was later successfully classified by MLVA technique to a unique P. aeruginosa strain, with an MLVA code previously assigned to genotype 25 in France (C. Pourcel, personal communication). Isolates from six patients carrying assigned cluster A strains were genotyped using PFGE and MLVA10-Sweden during 2004–2008, while the cluster A strain from © 2013 APMIS. Published by John Wiley & Sons Ltd
March 2008
patient 16 was typed in June 2009, after relocation of the transplant program to a new building. Agreement between the two genotyping methods PFGE and MLVA10-Sweden results was good, with variations in MLVA at the 10 selected repeats providing discrimination to a similar extent as PFGE (Table 2). However, no amplified product was produced for 5/160 PCR analyses, probably explained by nucleotide variation in the primerannealing site. Isolates within PFGE clusters with variant PFGE profiles exhibited the same MLVA patterns. The specific P. aeruginosa cluster A seems to have a high mutation rate, resulting in several clonal variants. Cluster A strains isolated from the studied patients were differentiated according to seven different, but closely related, PFGE band patterns (Fig. 1). The method MLVA10-Sweden confirmed that all cluster A strains belonged to the same cluster (Table 2). Extensive growth of P. aeruginosa was recovered 2 months after the last patient analyzed in 2008 from four of eight samples collected from sink and
87
JOHANSSON et al.
Table 2. Genotype of Pseudomonas aeruginosa strains isolated from lung transplant recipients Patient VNTR code MLVA10- PFGE Sweden 211, 212, 214, 213, 215, 216, 217, 222, 223, 142 1 – – CR 2 6, 9, 5, 3, 2, 2, 3, 4, 2, 3 AI1 AI1 3 –/– –/– A-2/A-2 4 6, 6, 2, 2, 5, 2, 1, 3, 3, 42 A/A A-2-5/ A-2-5-2 5 3, 8, 2, 5, 2, 2, 3, 1, 5, 4 EP EP 3 6 8, 6, 2, 4.5, 1, 2, 1, 1, 25 1.5, 3 7 8, 9, 2, 2, 4, 2, 4, 3, 3, 4 C C-5-3-5-5 8 2, 4, 5, 1, 2, 1, 5, –, 4, 1 EC EC 9 3, 8, 5, 5, 5, 1, 3, 1, 2, 2 CT CT-2 10 6, 6, 2, 2, 5, 2, 1, 3, 3, 4 A A-2-5-2c 11 6, 6, 2, 2, 5, 2, 1, 3, 3, 4 A A-2-5-2b 12 6, 6, 2, 2, 5, 2, 1, 3, 3, 4 A A-2-5-3 13 –, 6, 2, 2, 5, 2, 1, 3, 3, 4 –/A/– A-3/A-3/ A-3 14 3, 8, 5, IS, 2.5, 1, –, 1, BF/–/– BF-2/ 2, 1 BF-2/ BF-2 15 3, 5, 4.5, 5, 6, 1, 2, 1, GG GG 4, 1 16 6, 6, 2, 2, 5, 2, 1, 3, 3, 4 A A-2-5-2b 17 3, 9, 4, 4, 2, 2, 5, 3, –, 4 GS GS 18 –, 9, 4, 2, 2, 1, 2, 1, 2, 2 GZ GZ Genotyping was performed once or twice (patient 2 and 4) with different methods post-transplant, except patient 13 for whom genotyping was performed three times pretransplant and patient 14 whom genotyping was performed once pre-transplant and twice post-transplant. VNTR, variable-number-of-tandem-repeats; MLVA, multiple-locus variable number of tandem repeat analysis; PFGE, pulsed-field gel electrophoresis; ‘–’, no amplified VNTR; IS, Insertion element. 1 Two letter strain AI is not related to cluster A strains. 2 Same VNTR code for both samples. 3 No PFGE pattern due to auto-degradation of DNA.
shower drains in rooms where patient 10, 11, and 12 had stayed. There were no strains of P. aeruginosa found in the samples from tap water in the ward corridor or from floor drain and sink in the anterooms. All four isolates revealed the MLVA10Sweden pattern, similar to the epidemic cluster A strain (Table 3). The more discriminating PFGE analysis confirmed these environmental isolates to be identical with the cluster A strains carried by four transplanted patients (Fig. 1 and Table 3). Due to overlapping periods of hospitalization and opportunities for close contact, there was possibility for direct transmission among patients 10, 11, and 12, but uncertain for the other four patients harboring the cluster A strain. The remaining 11 patients carried unique genotypes of P. aeruginosa. The environmental P. aeruginosa isolate from the adjacent ward displayed a unique PFGE pattern
88
and MLVA10-Sweden code, different from the patient isolates (Fig. 1 and Table 3).
DISCUSSION We describe a probable transmission of P. aeruginosa at a thoracic transplant ward, and the use of genotyping for identification and bacteriological surveillance. As P. aeruginosa is ubiquitous in the environment and also part of the endogenous micro-flora of hospitalized patients, the original source of the bacterium and the precise mode of transmission often remain unclear, although contaminated water and medical equipment, direct patient-to-patient transmission, as well as nurse-topatient transmission have been reported (1, 19, 20). Using genotyping methods, we identified unique P. aeruginosa strains in 11/18 (61%) patients, assumed to be cases of indigenous infection, while 7/18 (39%) shared the same genotype. For two CF patients with chronic P. aeruginosa infection, the identical strain was identified pre- and post-operatively, whereas for one patient with intermittent positive cultures, only the post-transplant strain had been genotyped. Over a 10-year study period, Cuttelod et al. (21) observed that endogenous sources of infection remained stable, although exogenous sources varied, concomitant to environmental contamination, infection control measures, and the genetics of P. aeruginosa itself. In the cases of three patients, there had been opportunity for close contact and we suspect crossinfection rather than an environmental origin of the infections. We are not able to give the exact room location for the remaining cluster A-infected patients at the ward; however, they all had access to the shower room with a contaminated floor drain. Acquisition of P. aeruginosa following exposure to contaminated tap water in the ICU has been described (22). However, the epidemiology of P. aeruginosa is complex, as shown for an outbreak in a German ICU, where the isolated environmental P. aeruginosa strains had no relation to the genotypes isolated from the patients (23). Sink and floor drain samplings from two patient rooms on the ward revealed that P. aeruginosa strains were identical or similar to the genotypes identified in cultures from patients hospitalized at the same time period, indicating prolonged contamination in the hospital ward environment. The PFGE and MLVA10-Sweden genotyping methods complemented each other. Using the discriminating but more labor-intensive PFGE, several of the cluster A strains exhibited different band patterns, complicating cluster analysis. However, using
© 2013 APMIS. Published by John Wiley & Sons Ltd
CROSS-INFECTION OF P. AERUGINOSA
Fig. 1. Cluster analysis of Pseudomonas aeruginosa chromosomal DNA digested with Spe1; restriction fragments were separated by PFGE. The dendrogram shows the relative similarities of the DNA band patterns from Swedish lung-transplanted patients (n = 17) and environmental strains from the transplant ward (n = 4) and adjacent ward (n = 1) at the Sahlgrenska hospital. The related cluster A strains are marked. The strain AI shares the first letter designation, ‘A’ with cluster A strains, although no genotypic relationship is indicated in the PFGE fingerprints.
Table 3. Genotype of environmental Pseudomonas aeruginosa strains isolated at two nearby medical care units Sampling site Time VNTR code MLVA PFGE 10-Sweden 211, 212, 214, 213, 215, 216, 217, 222, 223, 142 Transplant ward Sink, Room 7 June 2008 6, 6, 2, 2, 5, 2, 1, 3, 3, 4 A A-2-5-2c Floor drain, Room 7 June 2008 6, 6, 2, 2, 5, 2, 1, 3, 3, 4 A A-2-5-2d Sink, Room 8 June 2008 6, 6, 2, 2, 5, –, 1, 3, 3, 4 A A-2-5-3-4 Floor drain, Shower June 2008 6, 6, 2, 2, 5, 2, 1, 3, 3, 4 A A-2-5-2b-2 room Adjacent ward Sink August 2008 2, 9, 5, 8, 4, 1, 4, 4, 2, 4 EV EV VNTR, variable-number-of-tandem-repeats; MLVA, multiple-locus variable number of tandem repeat analysis; PFGE, pulsed-field gel electrophoresis. © 2013 APMIS. Published by John Wiley & Sons Ltd
89
JOHANSSON et al.
the less discriminating MLVA10-Sweden, we found all cluster A strains to belong to the same cluster, sharing the same VNTR code. Using the inter-laboratory reproducible MLVA technique, we compared the results to MLVA codes for P. aeruginosa isolates from other laboratories. We concluded that the PFGE profile of cluster A is identical to the previously described widespread clone C (24). We may also speculate that these P. aeruginosa cluster A/C strains, common to patients and environmental habitats (10), were the source of contamination at the lung transplant unit. Ruiz et al. detected a clear separation between clinical and environmental isolates and suggested that the origin of hospital infections is due to infection acquired by patients prior to hospital stay (25). As the tap water sample was not contaminated, and there was opportunity for close contact, patient-to-patient transmission is more likely than acquisition of P. aeruginosa from the environment. The P. aeruginosa cluster A genotype seems less prevalent at our hospital, as only one isolate with a unique pattern was detected at the adjacent ward. The phenotypes of resistant P. aeruginosa epidemic clones have been shown to undergo sporadic variation during the course of an outbreak (26). Using genotyping, rather than phenotyping techniques such as antibiotic susceptibility testing, has improved the characterization of P. aeruginosa isolates. The effects of implemented infection control measures can be effectively evaluated by molecular, epidemiological investigation (27). After relocation of the transplant unit to a new building, we are continuing the surveillance of P. aeruginosa and genotype new isolates; only one P. aeruginosa isolate has been typed to cluster A and there is no possibility of cross-infection in this patient. This study has limitations: genotyping was performed in only 25% of culture-positive patients and MLVA was missing for two patients, as the method had not yet been established for regular use at the time. In conclusion, this survey revealed cross-infection within a transplant unit, leading to infectious complications in lung transplant recipients. The study highlights the importance of microbiological surveillance in the environment of lung transplantation. We suggest genotyping, using MLVA10-Sweden for routine infection control and PFGE for extended analysis.
This study was funded by grant from Sahlgrenska University Hospital 95340. The authors thank Leif Larsson for the environmental sampling at the hospital and all members of the DNA laboratory at the Clinical Microbiology
90
for the skillful PFGE, G€ oran Dellgren and Gerdt Riise for reading of the manuscript. We further thank Ed Moore for constructive criticism of the manuscript. Finally, we are also grateful to Christine Pourcel from the Institute for Genetics and Microbiology in Paris, France for her excellent support with the MLVA method. The authors have no conflicts of interest.
REFERENCES 1. Kerr KG, Snelling AM. Pseudomonas aeruginosa: a formidable and ever-present adversary. J Hosp Infect 2009;73:338–44. 2. Cystic Fibrosis Foundation. Patient Registry 2005 Annual Report. Bethesda, MD: Cystic Fibrosis Foundation, 2006. 3. Emerson J, Rosenfeld M, McNamara S, Ramsey B, Gibson RL. Pseudomonas aeruginosa and other predictors of mortality and morbidity in young children with cystic fibrosis. Pediatr Pulmonol 2002;34:91–100. 4. Fagon JY, Chastre J, Vuagnat A, Trouillet JL, Novara A, Gibert C. Nosocomial pneumonia and mortality among patients in intensive care units. JAMA 1996;275:866–9. 5. Aaron SD, Vandemheen KL, Ramotar K, GiesbrechtLewis T, Tullis E, Freitag A, et al. Infection with transmissible strains of Pseudomonas aeruginosa and clinical outcomes in adults with cystic fibrosis. JAMA 2010;304:2145–53. 6. Zeglen S, Wojarski J, Wozniak-Grygiel E, Siola M, Jastrzebski D, Kucewicz-Czech E, et al. Frequency of Pseudomonas aeruginosa colonizations/infections in lung transplant recipients. Transplant Proc 2009;41:3222–4. 7. Sanders DB, Bittner RC, Rosenfeld M, Hoffman LR, Redding GJ, Goss CH. Failure to recover to baseline pulmonary function after cystic fibrosis pulmonary exacerbation. Am J Respir Crit Care Med 2010;182:627–32. 8. Kovats Z, S€ utto Z, Murak€ ozy G, Bohacs A, Czebe K, Lang G, et al. Airway pathogens during the first year after lung transplantation: a single-center experience. Transplant Proc 2011;43:1290–1. 9. Scott FW, Pitt TL. Identification and characterization of transmissible Pseudomonas aeruginosa strains in cystic fibrosis patients in England and Wales. J Med Microbiol 2004;53:609–15. 10. R€ omling U, Kader A, Sriramulu DD, Simm R, Kronvall G. Worldwide distribution of Pseudomonas aeruginosa clone C strains in the aquatic environment and cystic fibrosis patients. Environ Microbiol 2005;7:1029–38. 11. Johansson E, Welinder-Olsson C, Lindblad A, Gilljam M, Johannesson M, Meyer P, et al. Genomic fingerprinting of Pseudomonas aeruginosa from all Swedish CF Centres. A Scandinavian cystic fibrosis study consortium study. J Cyst Fibrosis 2007;6:114. 12. Tenover FC, Arbeit RD, Goering RV, Mickelsen PA, Murray BE, Persing DH, et al. Interpreting chromosomal DNA restriction patterns produced by pulsedfield gel electrophoresis: criteria for bacterial strain typing. J Clin Microbiol 1995;33:2233–9. © 2013 APMIS. Published by John Wiley & Sons Ltd
CROSS-INFECTION OF P. AERUGINOSA
13. Waters V, Zlosnik JEA, Yau YCW, Speert DP, Aaron SD, Guttman DS. Comparison of three typing methods for Pseudomonas aeruginosa isolates from patients with cystic fibrosis. Eur J Clin Microbiol Infect Dis 2012;31:3341–50. 14. Onteniente L, Brisse S, Tassios PT, Vergnaud G. Evaluation of the polymorphisms associated with tandem repeats for Pseudomonas aeruginosa strain typing. J Clin Microbiol 2003;41:4991–7. 15. Vu-Thien H, Corbineau G, Hormigos K, Fauroux B, Corvol H, Clement A, et al. Multiple-locus variablenumber tandem-repeat analysis for longitudinal survey of sources of Pseudomonas aeruginosa infection in cystic fibrosis patients. J Clin Microbiol 2007;45:3175–83. 16. Mansfeld R, Jongerden I, Bootsma M, Buiting A, Bonten M, Willems R. The population genetics of Pseudomonas aeruginosa isolates from different patient populations exhibits high-level host specificity. PLoS ONE 2010;5:e13482. 17. Gautom RK. Rapid pulsed-field gel electrophoresis protocol for typing of Escherichia coli 0157:H7 and other gram-negative organisms in 1 day. J Clin Microbiol 1997;35:2977–80. 18. Gilljam M, Schersten H, Silverborn M, J€ onsson B, Ericsson-Hollsing A. Lung transplantation in patients with cystic fibrosis and Mycobacterium abscessus infection. J Cystic Fibrosis 2010;9:272–6. 19. Schelenz S, French G. An outbreak of multidrug-resistant Pseudomonas aeruginosa infection associated with contamination of bronchoscopes and an endoscope washer-disinfector. J Hosp Infect 2000;46:23–30. 20. Moolenaar RL, Crutcher JM, San Joaquin VH, Sewell LV, Hutwagner LC, Carson LA, et al. A prolonged outbreak of Pseudomonas aeruginosa in a neonatal intensive care unit: did staff fingernails play a role in
© 2013 APMIS. Published by John Wiley & Sons Ltd
21.
22.
23.
24.
25.
26.
27.
disease transmission? Infect Control Hosp Epidemiol 2000;21:80–5. Cuttelod M, Senn L, Terletskiy V, Nahimana I, Petignat C, Eggimann P, et al. Molecular epidemiology of Pseudomonas aeruginosa in intensive care units over a 10-year period (1998-2007). Clin Microbiol Infect 2011;17:57–62. Rogues AM, Boulestreau H, Lasheras A, Boyer A, Gruson D, Merle C, et al. Contribution of tap water to patient colonisation with Pseudomonas aeruginosa in a medical intensive care unit. J Hosp Infect 2007;67:72–8. Kohlenberg A, Weitzel-Kage D, van der Linden P, Sohr D, V€ ogeler S, Kola A, et al. Outbreak of carbapenem-resistant Pseudomonas aeruginosa infection in a surgical intensive care unit. J Hosp Infect 2010;74:350–7. R€ omling U, Wingender J, M€ uller H, T€ ummler B. A major Pseudomonas aeruginosa clone common to patients and aquatic habitats. Appl Environ Microbiol 1994;60:1734–8. Ruiz L, Domınguez MA, Ruiz N, Vi~ nas M. Relationship between clinical and environmental isolates of Pseudomonas aeruginosa in a hospital setting. Arch Med Res 2004;35:251–7. Hocquet D, Bertrand X, K€ ohler T, Talon D, Plesiat P. Genetic and phenotypic variations of a resistant Pseudomonas aeruginosa epidemic clone. Antimicrob Agents Chemother 2003;47:1887–94. Petignat C, Francioli P, Nahimana I, Wenger A, Bille J, Schaller MD, et al. Exogenous sources of Pseudomonas aeruginosa in intensive care unit patients: implementation of infection control measures and follow-up with molecular typing. Infect Control Hosp Epidemiol 2006;27:953–7.
91
