Abstract
Cross-infection by Pseudomonas aeruginosa between unrelated patients with cystic fibrosis (CF) is believed to be uncommon. After detecting a genotypically identical strain of P. aeruginosa in five unrelated children with CF dying from severe lung disease, we determined its prevalence within a large CF clinic using pulsed-field gel electrophoresis and random amplified polymorphic DNA assays. The clinical status of P. aeruginosa–infected patients was also determined. Between September and December 1999, 152 patients, aged 3.9–20.7 years, provided sputum for culture. P. aeruginosa was detected in 118 children of mean (SD) age 13.5 (3.8) years. The genotyping techniques were concordant, showing that 65 (55%) infected patients carried an indistinguishable or closely related strain. No distinctive antibiogram or environmental reservoir was found. Patients with the clonal strain were more likely than those with unrelated isolates to have been hospitalized in the preceding 12 months for respiratory exacerbations. This study demonstrates extensive spread of a single, clonal strain of P. aeruginosa in a large pediatric CF clinic. Whether this strain is also more virulent than sporadic isolates remains to be determined. As transmissible strains could emerge elsewhere, other CF clinics may also need to consider molecular methods of surveillance for cross-infection.
Persistent airway infection by Pseudomonas aeruginosa among patients with cystic fibrosis (CF) is accompanied by deteriorating pulmonary function and reduced survival (1). The most likely source is believed to be from the environment where individual patients acquire their own unique strains (2, 3). Whereas siblings with CF are frequently found to have identical isolates (4, 5), unrelated patients with CF sharing common strains are reported to be unusual (6–9). Previous studies proposing person-to-person transmission of P. aeruginosa (10, 11) have been criticized for omitting molecular typing techniques (12). However, when these methods have been employed, cross-infection of P. aeruginosa between patients has been detected in some CF clinics, suggesting the emergence of transmissible strains (13–15). Nevertheless, these observations are regarded as unusual and insufficient to warrant additional infection-control measures (16).
During 1991–1995, while studying a cohort of 92 infants attending the CF clinic at the Royal Children's Hospital, Melbourne (17), we observed five unrelated cohort members die from severe lung disease before they were 5 years of age. A mucoid strain of P. aeruginosa had been newly identified in all five patients within 0.5–16 months of their deaths. Subsequently, pulsed-field gel electrophoresis (PFGE) testing of these and other P. aeruginosa isolates from this cohort revealed an identical macrorestriction pattern among 8 of 27 infected subjects, including all five who had died.
The cluster of deaths in these young patients sharing a genotypically identical strain of P. aeruginosa was unexpected. To investigate the possibility of an outbreak and to help guide infection-control policies, we performed a cross-sectional study to identify the distribution of the clonal strain within the CF clinic population, using two independent genomic fingerprinting methods. The clinical status of children with P. aeruginosa was also determined.
The clinic manages 326 (161 males) patients with CF. Affected infants are identified by newborn screening (18), followed by mutation analysis and sweat testing. Patients are seen at intervals of 3 months and hospitalized for severe exacerbations of lung disease (19). Standard hygienic precautions are employed, but at the time of this cross-sectional study, a policy segregating P. aeruginosa–infected from noninfected patients had not been adopted (11). While in hospital, patients shared rooms and equipment and often had physiotherapy together.
The study was a clinical audit, and written consent was not required by the Royal Children's Hospital Ethics Committee at the time the study was performed. However, pre- and posttest counseling for all families was provided by CF clinicians, and verbal consent was sought from parents, caregivers, and older patients before sputum samples were collected and tested. Specimen collection and chart review was undertaken between September and December 1999, when 312 (96%) patients were seen for their routine clinic appointment. Sputum was collected from all 152 (79 males) sputum producers (49% of the clinic), aged 3.9–20.7 years.
Patients with P. aeruginosa infection were assigned a National Institutes of Health score (20). A single blinded investigator (G.M.N.) reviewed the case records for age, sex, residential postcode, CF genotype, and, from the previous 12 months, for the number of hospitalizations for respiratory exacerbation, the best-recorded percent predicted FEV1, and whether there had been treatment with either inhaled antibiotics or recombinant human DNase.
Previously, in 1995, an environmental microbiologic survey of the CF clinic was conducted to detect Burkholderia cepacia complex isolates. Over 3 days, 28 individual samples were collected during normal working hours from sink drains and taps, shower recesses and taps, toilet door handles, rims, and flush buttons, patient equipment, including nebulizers, recreational facilities, and appliances from the inpatient wards, physiotherapy department, outpatient clinic rooms, and lung function laboratory. The environmental survey was repeated in late 1999 when 61 more samples were collected. The 12 P. aeruginosa isolates identified by the two surveys were stored at −70°C and were available for analysis.
Sputum and environmental samples were plated onto selective and nonselective media, to detect P. aeruginosa and other pathogens by standard techniques (17). P. aeruginosa colony morphotypes were identified by visual inspection (21) and tested for antibiotic susceptibility by disk diffusion (22). Multiple antibiotic resistance was defined as acquired resistance to all agents in two or more of the following classes: β-lactams, aminoglycosides, and fluoroquinolones (23).
An independent, blinded laboratory scientist randomly selected single colonies representing distinct P. aeruginosa morphotypes from each subject for molecular typing by PFGE following digestion with SpeI and DraI (24). Strains were judged to be indistinguishable if they had identical restriction patterns with both cutting enzymes. An isolate was considered to be closely related to the common strain if its PFGE pattern differed by a single genetic event such as a point mutation or an insertion or deletion of DNA, whereby fragments differed by two to three bands (25). The PFGE findings were confirmed by electrophoresis of random amplified polymorphic DNA, using primer 272 (5′-AGCGGGCCAA-3′) as previously described (3).
Patients were grouped according to the genotype and mucoid–nonmucoid phenotype of their P. aeruginosa isolate. The distribution of the National Institutes of Health scores was skewed, and for simplicity, the continuous clinical outcome measures were summarized using medians with statistical significance of between-group differences assessed by the Kruskal–Wallis test. Potential confounding effects of age, sex, and genotype were assessed using multiple regression after appropriate transformation of the outcome variable. Comparisons between proportions were made using Pearson's chi-square or Fisher's exact test and odds ratios. Stata software was used for all statistical analyses (26).
P. aeruginosa was detected in 118 of 152 (78%) sputum producers aged 4.6–20.7 (mean [SD], 13.5 [3.8]) years, including seven sibling pairs. Coinfection by one or more pathogens was identified in 51 sputum specimens (Staphylococcus aureus in 41, including 5 methicillin-resistant strains, Haemophilus influenzae in 13, Burkholderia cepacia complex in 2, and Stenotrophomonas maltophilia in 1).
Overall, 134 P. aeruginosa isolates (100 mucoid and 34 nonmucoid phenotype) from 118 sputum culture specimens (range, 1–2 isolates per patient) underwent molecular typing. PFGE and random amplified polymorphic DNA assays gave concordant results, showing that 65 of 118 (55%) children with P. aeruginosa in their sputum (including three sibling pairs) shared a strain genetically related to the birth cohort isolate (Figure 1)
Figure 1. Comparison of (A) PFGE* and (B) random amplified polymorphic DNA assays of clonal (Lanes 1–6) and unrelated (Lanes 7–12) Pseudomonas aeruginosa isolates. * SpeI macrorestriction profile.
[More] [Minimize]The clonal strain was exclusively mucoid in 37 children, produced mixed mucoid and nonmucoid colonies in 25 children, and was solely nonmucoid in only 3 cases. Unrelated second strains of P. aeruginosa (each with a distinct DNA fingerprint) were detected in seven patients infected by the clonal strain. In contrast, 40 of the remaining 53 children with CF having P. aeruginosa infection had a genotypically distinct isolate unrelated to the common clonal strain. The four remaining sibling pairs each shared a distinct isolate, whereas five unrelated children shared an indistinguishable mucoid isolate that was different from the common clonal strain.
Although significantly more resistant to ceftazidime, imipenem, gentamicin, and tobramycin than nonclonal isolates, the clonal strain could not be identified by a distinctive antibiogram (Table 1)
Resistance* (%) | |||||||
|---|---|---|---|---|---|---|---|
| Unrelated Strains | |||||||
| Antibiotic | Clonal Strain
(n = 65) | Mucoid
(n = 40) | Nonmucoid
(n = 13) | p Value† | |||
| Ticarcillin-clavulanate | 15 | 13 | 8 | 0.86 | |||
| Aztreonam | 55 | 31 | 38 | 0.06 | |||
| Ceftazidime | 56 | 23 | 15 | < 0.001 | |||
| Imipenem | 70 | 30 | 0 | < 0.001 | |||
| Gentamicin | 85 | 63 | 46 | 0.003 | |||
| Tobramycin | 54 | 30 | 31 | 0.04 | |||
| Amikacin | 61 | 63 | 62 | 1.0 | |||
| Ciprofloxacin | 21 | 5 | 23 | 0.05 | |||
| Multiresistant‡ | 14 | 13 | 23 | 0.59 | |||
The two independent environmental surveys yielded P. aeruginosa isolates in 10 sinks and 2 showers located in the inpatient wards, physiotherapy room, and pulmonary function laboratory. All were genotypically distinct and none were related to the clonal strain. No isolates were detected in other “wet areas” or patient nebulizers.
Clinical measures all exhibited a pattern of poorest outcome in the clonal group, somewhat better in the nonclonal group, and best in the group with nonclonal, nonmucoid infection (Table 2)
Unrelated Strains | |||||
|---|---|---|---|---|---|
| Clonal Strain
(n = 61) | Mucoid
(n = 38) | Nonmucoid
(n = 12) | p Value | ||
| Age, yr | 14.4 (11.9, 17.5) | 13.6 (9.1, 15.9) | 14.0 (11.9, 15.4) | 0.13 | |
| Number of males, % | 29 (48) | 16 (42) | 8 (67) | 0.36 | |
| Metropolitan postcode, % | 39 (64) | 19 (50) | 7 (58) | 0.92 | |
| CF genotype, % | |||||
| ΔF508 homozygous | 36 (59) | 22 (58) | 10 (83) | 0.28 | |
| ΔF508 heterozygous | 14 (23) | 10 (26) | 1 (8) | 0.49 | |
| No copies of ΔF508 | 10 (16) | 5 (13) | 0 | 0.38 | |
| No genotype result | 1 (2) | 1 (3) | 1 (8) | 0.47 | |
| Inhaled antibiotic, %† | 37 (61) | 21 (55) | 7 (58) | 0.92 | |
| rhDNase, %† | 34 (56) | 15 (39) | 1 (8) | 0.002 | |
| Number, % hospitalized† | 35 (57) | 14 (37) | 2 (12) | 0.01 | |
| NIH score (reference 20) | 70.0 (58.8, 79.0) | 75.5 (66.8, 82.0) | 84.5 (76.8, 87.0) | 0.003 | |
| CXR score‡ | 9.0 (8.0, 11.0) | 8.0 (7.0, 9.0) | 6.5 (6.0, 8.8) | 0.02 | |
| Best % predicted FEV1† | 75.1 (65.0, 91.8) | 81.5 (67.3, 92.8) | 93.1 (82.5, 97.0) | 0.04 | |
By employing molecular typing techniques, we documented the extensive spread of a clonal P. aeruginosa strain in a large pediatric CF clinic. However, it is unknown whether this strain is also more virulent than other P. aeruginosa isolates detected in the clinic population. This is now the subject of a further study as although acquisition of the clonal strain was associated with the deaths of five preschool children with CF, the limitations of the cross-sectional design precluded firm conclusions being drawn over its impact in older patients.
Only half of the patients attending the clinic were sputum producers. Contemporaneous oropharyngeal cultures were not obtained from the remainder, as we have previously shown that these lack sensitivity and predictive accuracy for lower airway infection (24). However, during 1992–1999, 138 (90%) newly diagnosed children with CF were prospectively recruited into one of three cohorts (either at diagnosis, 1 year of age, or 2 years of age), and they participated in a longitudinal observational cohort study that included 3-monthly oropharyngeal and semiannual bronchial lavage cultures with storage of pathogenic isolates at −70°C (17, 24, 27). From these cohorts, lower respiratory P. aeruginosa infection was identified in 27 young children (lung biopsy and sputum in 1 case each, bronchial lavage only in 11 cases, and simultaneous bronchial lavage and oropharyngeal culture in 14 cases). The clonal strain was identified in eight of these infants, five of whom died after clonal strain acquisition. Just three children in the cohort had the clonal strain detected in both oropharyngeal and bronchial lavage cultures, only one of whom survived. An additional 16 infants had P. aeruginosa isolated exclusively from oropharyngeal culture; however, all these isolates were sporadic nonmucoid strains. This suggests that young children were an unlikely source for transmission of the clonal strain, and taking oropharyngeal cultures in the current study would have given little additional information. In contrast, the point prevalence for P. aeruginosa of 78% in sputum producers and 42% for all clinic patients was that expected for children with CF of this age (9, 28). The unusual finding was the detection of a clonal strain in 23% of clinic patients.
Sharing of a single P. aeruginosa strain by a large group of unrelated children attending the same clinic suggests person-to-person transmission. In contrast, simple breakdown of infection-control measures should have meant the spread of several different strains. Although acquisition from a common hospital source is possible, two environmental surveys 4 years apart failed to detect the clonal strain. A role for flexible bronchoscopy as a means of transmission was examined. However, this was believed to be unlikely as of the eight patients from the infant cohort study who became infected by the clonal strain, two had never undergone bronchial lavage. Diagnosis of infection in these children was confirmed by either lung biopsy or sputum culture. Moreover, routine surveillance cultures of bronchoscope suction channels conducted before each bronchoscopy were consistently sterile for bacteria and fungi (27). Nevertheless, the clonal strain was first detected in a 4-month-old infant with CF dying from a fulminant pneumonia in 1991, whereas the original environmental survey was not undertaken until 4 years later. It is possible that a common source of infection may have only existed before these surveys were conducted. Alternatively, exposure to a common source outside the hospital is unlikely as patients were unrelated (except for three sibling pairs) and lived in geographically diverse areas.
Past studies have also suggested that unrelated patients with CF can be infected by distinct strains of P. aeruginosa, implying either a low incidence of patient-to-patient spread or acquisition from a common source (3, 28, 29). Indeed, transmission within CF centers has been suspected for several years (10, 11, 30, 31). Recently, two studies from the United Kingdom (13, 14) have presented compelling evidence for spread of a single P. aeruginosa clone among unrelated patients attending a CF clinic. The present study also suggests that cross-infection is not confined to British centers. Moreover, the Melbourne clonal strain is genotypically distinct from the epidemic strains in Liverpool (13) and Manchester (14) (JRW Govan, personal communication).
Whereas the clonal strain's origins in the Melbourne clinic are unknown, the emergence and spread of common strains in other centers has been assumed to be from the combined induction and selection of antibiotic resistance, followed by patient-to-patient transmission within the hospital setting (13). This is consistent with our earlier observation of hospitalization during infancy being an important risk factor for acquiring P. aeruginosa (32). Although our strain was not multiresistant, it was more likely to be resistant to antibiotics commonly used within the clinic than were other unrelated isolates.
Infection with the clonal strain was not associated with an adverse clinical outcome in this cross-sectional study. Nevertheless, in the 12 months preceding the survey, patients with this strain were more likely to be hospitalized for severe respiratory exacerbations. Whether these resulted from persistent infection or provided an opportunity for clonal strain acquisition is unknown. Detection of the clonal strain in five children who died before the age of 5 years in our clinic is worrying and suggests increased virulence. Furthermore, in the 18-month period since completing the study, children with the clonal strain were more likely to die from lung disease than those infected by other P. aeruginosa isolates (12/75 versus 3/61; odds ratio 3.7; 95% confidence interval 1.0, 17). It is not clear, however, whether this strain is more virulent (33) or whether the increase in mortality and morbidity represents acquisition of a mucoid strain that has already adapted to the CF lung (1–3). To help address mechanisms of acquisition, including superinfection and virulence, further analysis of clinical data and sequential isolates from the infant cohort collected during the 1990s is being undertaken to identify risk factors for clonal acquisition and to evaluate the effects on lung disease (27).
Following these findings, adherence to standard precautions for infection control within the Royal Children's Hospital CF clinic has been reinforced. In an attempt to further reduce clonal strain acquisition, cohort segregation according to P. aeruginosa culture status and genotype during hospitalization and clinic attendance has been introduced (11, 30). Those uninfected or with unrelated sporadic P. aeruginosa isolates are being followed prospectively to determine whether cohort segregation reduces further transmission of the clonal strain.
This study is the first to report evidence of extensive P. aeruginosa cross-infection in an Australian CF clinic. The outbreak probably would have remained undetected had not a study of CF lung microbiology been undertaken. Presently, most CF centers in Australia do not use molecular typing of P. aeruginosa isolates or recommend segregation of patients on the basis of P. aeruginosa culture status. As there are only a few reports describing PFGE analysis of P. aeruginosa strains within individual clinics, the sharing of clonal strains may be more common than currently recognized. Unfortunately, simple phenotypic markers, including antibiotic susceptibility profiles, cannot reliably identify epidemic strains (14, 34).
Whether molecular surveillance of P. aeruginosa should be adopted in CF clinics elsewhere is controversial (35). There is uncertainty over the relationship between strain transmissibility, virulence, and effects on morbidity and mortality. Testing is expensive, and there is a substantial human, as well as financial, cost to segregation. For all CF clinics, basic hygiene measures, particularly hand washing, continue to be the cornerstone of cross-infection–control strategies. Additional policies should be determined by local conditions (4–6); however, in the future, regular genotypic screening of P. aeruginosa isolates may become necessary to identify episodes of cross-infection. Should these be identified, improved infection-control, including patient segregation, may become necessary to prevent further spread of clonal strains.
The authors thank Professor David Speert, Department of Pediatrics, University of British Columbia for kindly performing blinded random amplified polymorphic DNA analysis on a subset of P. aeruginosa isolates as a means of independently validating our findings.
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