- Email: [email protected]
Endemicity and inter-city spread of Burkholderia cepacia genomovar III in cystic fibrosis
Endemicity and inter-city spread of Burkholderia cepacia genomovar III in cystic fibrosis Janet S. Chen, MD, Kimberly A. Witzmann, MD, Theodore Spilker, BS, Robert J. Fink, MD, and John J. LiPuma, MD Objectives: We sought to determine whether the same Burkholderia cepacia complex strain has persisted as the dominant clonal lineage among patients in a large cystic fibrosis (CF) treatment center during the past 2 decades. Study design: The inter-city spread of B cepacia through transfer of a colonized patient and the impact of infection control measures in containing interpatient transmission were investigated. We analyzed all available B cepacia complex isolates recovered from 1981 to 1987 and from 1996 to 2000 at one large CF treatment center (Center A) and from 1997 to 2000 at another center (Center B). Incidence of B cepacia complex infection and infection control measures in both centers were assessed. Results: Seventeen (81%) of 21 Center A patients from whom B cepacia complex bacteria were recovered between 1981 and 1987 and 40 (97%) of 41 patients culture-positive between 1996 and 2000 were infected with the same genomovar III strain. Transfer of a colonized patient from Center A to Center B was associated with an increase in B cepacia complex infection in Center B, all of which was with the Center A dominant strain. This strain, designated PHDC, lacks both B cepacia epidemic strain and cblA markers. Conclusions: B cepacia complex strains may remain endemic in CF treatment centers for many years. Responsible bacterial and host factors and optimal infection control measures to prevent inter-patient spread remain to be identified. (J Pediatr 2001;139:643–9) Descriptions of rapid pulmonary deterioration culminating in death within weeks in a significant proportion of patients infected with Burkholderia cepacia underscored the impact of B cepacia in the population with cystic fibrosis (CF).1 The broad-spectrum an-
timicrobial resistance exhibited by most clinical isolates limits therapeutic options, and thus, prevention of B cepacia acquisition has become an important goal. However, much remains unknown about the epidemiology of B cepacia infection in CF, and optimal in-
From the Department of Pediatrics, St Christopher’s Hospital for Children, Philadelphia, Pennsylvania; Department of Pediatrics, Children’s National Medical Center, Washington, DC; and Department of Pediatrics and Communicable Diseases, University of Michigan, Ann Arbor.
Supported by a grant from the Cystic Fibrosis Foundation (to Dr LiPuma). Submitted for publication Feb 7, 2001; revision received June 1, 2001; accepted June 29, 2001. Reprint requests: John J. LiPuma, MD, 1150 W Medical Center Dr, 8328 MSRB III, Box 0646, Ann Arbor, MI 48109. Copyright © 2001 by Mosby, Inc. 0022-3476/2001/$35.00 + 0 9/21/118430 doi:10.1067/mpd.2001.118430
fection control policies have yet to be defined.2
See editorial, p 618. Early studies in which bacterial genotyping was used demonstrated person-to-person spread of some strains3,4 and the existence of dominant strain types that infected the majority of colonized persons at some CF treatment centers.5 Among these so-called epidemic strains, the ET12 lineage, responsible for epidemic spread in several centers, has been the most extensively studied.6 This strain is characterized by the presence of the B cepacia epidemic strain marker (BCESM)7 and expression of cblAencoded cable pili that mediate adherence to respiratory epithelial cells.8 Other epidemic strains have been less completely characterized, and additional factors that may contribute to BCESM
Burkholderia cepacia epidemic strain marker CF Cystic fibrosis PCR Polymerase chain reaction PFGE Pulsed field gel electrophoresis rep-PCR Repetitive extragenic palindromic PCR RAPD Randomly amplified polymorphic DNA
enhanced transmissibility have not been identified. Taxonomic studies have made it clear that bacteria previously identified merely as B cepacia by conventional tests actually belong to several distinct species or genomovars, referred to collectively as the B cepacia complex.9 Formal binomial designations have been 643
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provided for 4 of the 7 species described to date. These include B multivorans (genomovar II), B stabilis (genomovar IV), B vietnamiensis (genomovar V), and B ambifaria (genomovar VII).9-11 The remaining 3 species are still referred to as B cepacia genomovars I, III, and VI pending the identification of distinguishing phenotypic features.9,12 All 7 species have been recovered from sputum from persons with CF, but genomovar III accounts for the majority of isolates.13 Whether significant differences exist between species with respect to virulence, per se, has not been established. However, preliminary study has indicated that most epidemic strains are BCESM-positive genomovar III.7 In this study, we investigated B cepacia complex isolates recovered from a large CF treatment center in which a dominant strain type was identified several years ago.5 Genotyping analysis demonstrates that the same B cepacia complex strain continues to dominate in this center where it is now recovered from nearly all patients with B cepacia complex infections. This 20-year endemicity is punctuated by the inter-city spread of this strain to another large CF treatment center wherein its arrival was marked by a sharp increase in the incidence of B cepacia complex infection; this strain now predominates among colonized persons in this center.
METHODS Bacterial Isolates and Species Identification Putative B cepacia complex isolates, recovered by sputum culture from patients attending a large CF treatment center in Philadelphia (Center A) between 1981 and 1987 and genotyped during a previous investigation,5 were available from frozen stocks maintained in skim milk at –80°C. Additional putative B cepacia complex isolates recovered from patients in Center A between 1996 and 2000, as well as from patients receiving care in 644
THE JOURNAL OF PEDIATRICS NOVEMBER 2001 a CF treatment center in Washington, DC (Center B) between 1997 and 2000, were also available for study and similarly stored in skim milk at –80°C until they were analyzed. Isolates from frozen stock were recovered on Mueller-Hinton agar after incubation at 35°C for 24 to 48 hours. All isolates were confirmed as B cepacia complex based on growth on B cepacia– selective media,14 biochemical reactivity, and species-specific polymerase chain reaction (PCR) assays targeting ribosomal RNA and recA genes as previously described.15-17 The presence of B gladioli was confirmed by using a species-specific PCR assay.18 Any isolate not confirmed as a Burkholderia species was further analyzed by using the RapID NF Plus System (Remel, Norcross, Ga).
Isolate Genotyping Genomic DNA was prepared from all isolates by using the Easy-DNA Kit (Invitrogen, Carlsbad, Calif) with modifications previously reported.15 All isolates had an initial screening genotype analysis performed with randomly amplified polymorphic DNA (RAPD) typing as described19 with the following modifications. Five microliters of 5 mol/L betaine (SigmaAldrich, St Louis, Mo) and 0.7 µL of 10 mg/mL acetylated bovine serum albumin (Promega, Madison, Wis) were added to the final PCR mix. PCR was conducted by using a RapidCycler (Idaho Technologies, Idaho Falls, Idaho) thermal cycler in 0.2-mL thinwalled tubes with the following parameters: 4 cycles (with slope of 1.0°C per second) of 94°C for 60 seconds, 55°C for 60 seconds, and 72°C for 150 seconds; followed by 29 cycles (with slope of 0.7°C per second) of 94°C for 30 seconds, 55°C for 30 seconds, and 72°C for 75 seconds. Amplified DNA, along with a custom-synthesized DNA ladder of known fragment sizes, was electrophoresed in a 2% agarose gel (BioRad, Hercules, Calif ) at 2 V/cm for 1 hour and visualized by using
ethidium bromide staining and UV illumination. Isolates were further genotyped by using repetitive extragenic palindromic PCR (rep-PCR) with a BOX-A1R primer as previously described.20 PCR products were resolved on 20-cm-long 1.5% agarose gels in 0.5× tris-acetateEDTA buffer at 5 V/cm for 5 hours at 4°C. Gels were stained with ethidium bromide and visualized with UV illumination. Gel images were digitized by using a GelDoc 2000 gel analyzer (BioRad) and stored as tagged image file format files. Digitized images were converted, normalized with the DNA size marker, and analyzed with Molecular Analyst Fingerprinting Plus software (BioRad). The rolling-disk background subtraction method was applied, and similarity matrices of whole densitometric curves of the gel tracks were calculated by using pairwise Pearson’s product-moment correlation coefficient. Cluster analyses of similarity matrices were performed by the unweighted pair group method with arithmetic averages. A similarity coefficient of ≥0.8 was used to indicate isolates of the same genotype (ie, strain).21 A subset of isolates, including all those with unique profiles as determined by RAPD and rep-PCR analyses, were also analyzed by macrorestriction digest and pulsed field gel electrophoresis (PFGE). Single colonies of bacteria, recovered from growth on nonselective agar media, were suspended in 1 mL of SE buffer (0.07 mol/L NaCl, 2.5 mmol/L EDTA; pH 7.4), pelleted by centrifugation at 8000 rpm for 2 minutes, washed 3 additional times in 10 mL of SE buffer, and re-suspended in 0.5 mL of SE buffer. Optical density at 620 nm was adjusted to 1.0, and 200 µL of the cell suspension was homogenized with 200 µL of 2% low-meltingtemperature agarose (Sigma) in SE buffer. Agarose plugs were made in a plug mold refrigerated at 4°C for 30 minutes and then placed in 10 mL of PEN buffer (17 mmol/L N-lauryl sarco-
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VOLUME 139, NUMBER 5 sine, 1% sodium salt, 25 mmol/L EDTA; pH 9.6) containing 1 mg of protease. After incubation in a 37°C rocking incubator for 12 to 18 hours, plugs were washed for 30 minutes with 1× TE buffer (0.1 mmol/L TRIS-HCl, 0.01 mmol/L EDTA; pH 8.0) 5 times, and 2-mm wide sections were incubated at 37°C for 6 hours with SpeI (Promega). DNA fragments were separated in 1% agarose (BioRad) by using a CHEFDR III system (BioRad) at 5.0 V/cm with pulse times of 30 to 70 seconds for 25 hours. Gels were stained with ethidium bromide and visualized with UV illumination. Strain identity was determined by using published criteria.22
Putative Transmissibility Factors All isolates were analyzed by PCR for the presence of BCESM as described 13 with primers ESM-R1F (5´ –GACAACGAGCAACGCGTAA3´) and ESM-R2R (5´ -GGCTCATCTGCGTATCGA-3´), which are specific for esmR, the internal 834-bp open reading frame of the BCESM sequence.7 The presence of DNA sequences with homology to cblA was confirmed by dot-blot hybridization as described.13 For both PCR and dot-blot assays, isolate AU0355, a BCESM- and cblA-positive isolate of the ET12 clonal lineage originally isolated from a patient with CF in Toronto, was used as a positive control; B cepacia ATCC 25416 (American Type Culture Collection Manasas, Va; genomovar I, BCESMand cblA-negative)17 was used as a negative control.
Epidemiologic Data and Infection Control Measures The incidence and prevalence of B cepacia infection for Centers A and B were calculated for relevant years based on the total number of patients with CF being cared for in the respective center and the number reported by the respective clinical microbiology laboratory to have had at least one sputum culture positive for B cepacia. In
Center A, sputum cultures were obtained from most patients only once per year. Sputum cultures were obtained 4 to 6 times per year from patients at Center B. Infection control policies governing B cepacia infection in patients with CF were reviewed in Centers A and B for relevant years.
RESULTS Center A Isolates Bacterial isolates (n = 27), reported to be B cepacia by the referring laboratory, were recovered from 27 patients with CF at Center A between 1981 and 1987. These isolates had been genotyped by ribotype analysis during a previous investigation that demonstrated 14 of the isolates to be the same strain type.5 In the present study, analysis of these 27 isolates after recovery from frozen stocks confirmed that 21 were indeed B cepacia complex; however, among the remaining 6 isolates, 5 were actually Burkholderia gladioli, and one was identified as Ochrobacterium anthropi. (These 6 non– B cepacia isolates had unique ribotype profiles in the previous analysis.5) Among the 21 confirmed B cepacia complex isolates, 18 were identified as genomovar III, 2 were B multivorans, and 1 was B vietnamiensis. RAPD and rep-PCR analyses demonstrated that 17 of the 18 genomovar III isolates were the same strain; the remaining genomovar III isolate and, as expected, the B gladioli, B multivorans, and B vietnamiensis isolates had different DNA profiles (Fig 1). Isolates were available from 43 of the 44 patients with CF reported to have had at least one sputum culture positive for B cepacia while receiving care at Center A between 1996 and 2000. Species identification analysis confirmed that 41 of the 43 isolates were B cepacia genomovar III; the remaining 2 isolates were actually B gladioli and Achromobacter (Alcaligenes) xylosoxidans. RAPD and rep-PCR analyses demonstrated that 40 of the 41 genomovar III
Fig 1. Representative RAPD analysis. Each lane contains an isolate recovered from a different Center A patient between 1981 and 1987. Lane 1, Fragment size marker; lane 2, B gladioli; lane 3, B vietnamiensis; lane 4, the only B cepacia genomovar III recovered during this period with unique profile; lanes 5 through 8, isolates showing dominant B cepacia genomovar III strain profile.Thirteen additional patients were also colonized with the dominant strain. isolates were the same strain. This strain is the same as that which predominates among isolates recovered from Center A patients between 1981 and 1987 (Fig 2), indicating that this clonal lineage has been endemic in this center for 20 years. PFGE analysis was performed on a subset of 14 isolates from Center A, and the results were in agreement with those demonstrated by RAPD and rep-PCR typing (data not shown). Among the Center A patients were 4 from whom isolates were available during both the 1981-1987 and the 1996-2000 intervals. Genotype analyses indicated that 2 patients were apparently persistently colonized with the dominant strain type for at least 15 and 10 years, respectively. In the other 2 patients, isolation of B vietnamiensis and B gladioli, respectively, in 1986 was followed by recovery in 1997 and 2000, respectively, of the center-dominant genomovar III strain (Fig 3).
Center B Isolates Isolates were available from 20 of the 25 patients with CF reported to have had at least one sputum culture positive 645
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Fig 2. Representative rep-PCR analysis. Each lane contains an isolate recovered from a different Center A patient between 1996 and 2000. Lane 1, Fragment size marker; lane 2, the only B cepacia genomovar III recovered during this period with unique profile; lanes 3 through 5, isolates showing dominant B cepacia genomovar III strain profile.Thirty-seven additional patients were colonized with the dominant strain. Lane 6, Dominant B cepacia genomovar III strain recovered in Center A in 1981 (included for comparison). for B cepacia while receiving care in Center B between 1997 and 1999. No isolates cultured from CF sputum before 1997 were available. Species identification analysis confirmed that 19 isolates were B cepacia complex (18 genomovar III and 1 B multivorans), whereas the remaining was actually B gladioli. RAPD and rep-PCR analyses indicated that 17 of the 18 genomovar III isolates were the same strain. Furthermore, this strain was the same as that which predominated among colonized patients in Center A during the 1981-1987 and 1996-2000 intervals (Fig 4).
Epidemiologic Data and Infection Control Measures During the late 1980s, the prevalence of B cepacia complex infection among patients with CF in Center A was ~20%. The annual incidence peaked in 1988 with a rate of 7.8%, followed by a rate of 5.8% in 1989. In early 1990, cohorting of hospitalized 646
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Fig 3. PFGE analysis of isolates from Center A. Lane 1, Fragment size marker; lane 2, dominant B cepacia genomovar III strain recovered in 1981; lanes 3 and 4, B cepacia genomovar III recovered from patient 1 in 1985 and 2000, respectively; lanes 5 and 6, B cepacia genomovar III recovered from patient 2 in 1986 and 1996, respectively; lanes 7 and 8, B gladioli and B cepacia genomovar III recovered from patient 3 in 1986 and 1997, respectively; lanes 9 and 10, B vietnamiensis and B cepacia genomovar III recovered from patient 4 in 1986 and 2000, respectively. patients with CF on the basis of B cepacia colonization status was initiated and was associated with a drop in incidence rates to 2.1% and 3.7% in 1990 and 1991, respectively. In mid 1996, hospitalized non-colonized patients were prohibited from sharing rooms, and in early 1998, this policy was expanded so that all patients with CF, irrespective of B cepacia colonization status, were in separate rooms when hospitalized. In 1997, separate waiting rooms were established for outpatients. Between 1992 and 1999, the prevalence of B cepacia complex infection in Center A dropped from 15% to 7%; incidence rates remained <2% during this time. Since 1995, 2 to 4 patients per year have been reported to have had B cepacia complex first recovered from sputum culture. Between 1990 and 1995, the prevalence of B cepacia complex infection in Center B ranged from 2.6% to 5.9%, with an annual incidence of <2% (Fig 4). In September 1993, a patient with
CF from Center A, infected with that center’s dominant genomovar III strain since 1989, transferred care to Center B. This patient was not hospitalized at Center B during the remainder of 1993 but was an inpatient twice in 1994 and 3 times during each year from 1995 to 1997. The patient had 6 outpatient visits per year during this period and was described by Center B caregivers as very social and an active participant in CF-related social activities. The incidence of B cepacia complex infection in Center B increased to 8.8% in 1996 and 5.7% in 1997; 23 patients became infected during these 2 years. Among the patients from whom B cepacia complex isolates were available for study, 18 of 19 were colonized with genomovar III, and 17 of these were colonized with the dominant strain. The only patient infected with a nondominant strain had the first positive culture in 1994, whereas 15 of the 17 with the dominant strain had first positive cultures after 1995. The 2 remaining patients with the dominant strain included the patient who transferred from Center A in 1993 and another patient reported to be B cepacia culture–positive since 1988 (unfortunately, isolates recovered from this patient before 1997 were not available for analysis). Thus, nearly all of the patients colonized with B cepacia complex in Center B after 1993 were infected with the Center A dominant strain. In early 1997, cohorting of hospitalized patients on the basis of B cepacia colonization status was initiated in Center B. Furthermore, colonized inpatients were placed in contact isolation and were required to wear mask and gloves when out of their rooms. A hospital-wide educational program regarding infection control was introduced at that time. In the outpatient setting, infected patients were restricted to separate clinic days during which no non-colonized patients were seen. Patients were required to wear masks while in the waiting room, and particular attention was given to disinfection
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VOLUME 139, NUMBER 5 of clinic rooms and equipment. The annual incidence of B cepacia complex infection has been <1% since that time.
DISCUSSION Epidemiologic studies of B cepacia infection in patients with CF have been hampered by difficulties with accurate identification of Burkholderia and related species and by the lack of consensus methods for strain typing. The recent recognition that several distinct species of bacteria actually comprise the B cepacia complex and the development of reliable PCR-based assays for their identification now provide a critical framework for further study. By using bacterial genotyping and a polyphasic identification scheme incorporating phenotypic and species-specific PCR assays, we have identified a B cepacia genomovar III strain, designated PHDC, which infects nearly all B cepacia– infected patients receiving care in large CF treatment centers in 2 US cities. We further demonstrated that this clone has not only been endemic but has indeed represented the dominant strain among infected patients in one center for at least the past 20 years. The transfer of an infected patient between centers was associated with a marked increase in incidence of B cepacia complex infection caused by this strain. In both centers, reinforcement of more rigid infection control measures significantly reduced the rate of infection. These findings highlight a number of important aspects of B cepacia complex infection in patients with CF. The finding in the late 1980s of specific B cepacia complex strains that were recovered from multiple patients5 suggested that some strains have an enhanced ability to colonize or be transmitted among patients. The identification and characterization of such so-called epidemic strains is important in that it may provide valuable insights into virulence mechanisms and host factors involved
Fig 4. Cluster analysis of rep-PCR profiles. Each track contains an isolate recovered from a different patient from either Center A or Center B during the year indicated. On the scale, similarity coefficients are expressed as percentages. Not shown are an additional 14 genomovar III isolates from Center A during 1981 to 1987, 37 from Center A during 1996 to 2000, and 14 from Center B during 1997 to 1999, all of which also clustered with the dominant strain (ie, with a similarity coefficient of >0.8) as described in the text.The bottom tracks show the only 3 genomovar III isolates available for analysis that are not of the dominant strain type as described in the text.
Fig 5. Annual incidence and prevalence of B cepacia complex infection in Center B. Strict infection control initiated in early 1997 as described in text. *Percent of patients with CF receiving care in Center B.
in infection in CF. Our finding that PHDC has stably persisted within a group of patients with CF for 2 decades further suggests that this strain possesses features contributing to its apparent adaptation to the CF host. Nevertheless, the identification of B cepacia complex transmissibility or colonization factors has been problematic. Few epidemic strains have been de-
scribed in detail. Several strains recovered from multiple patients contained a conserved 1.4-kb genomic fragment not found in strains recovered from single patients.7 This fragment, termed the B cepacia epidemic strain marker (BCESM), encodes an approximately 834-bp open reading frame, esmR, with homology to negative transcriptional regulators; however, the role of this 647
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putative gene in virulence remains unknown. ET12, a genomovar III strain that dominates among patients with CF in Ontario, Canada, and is associated with inter-patient spread in the United Kingdom, has been the most completely studied epidemic lineage. In addition to esmR, this strain elaborates large peritrichous pili, termed cable pili. The gene encoding cable pili, cblA, has been characterized, as has the cognate epithelial cell receptor for the associated adhesin.8,23 A recent study of B cepacia complex isolates recovered from over 600 patients with CF in the United States demonstrated that only one (an ET12 strain) contained the complete cblA sequence.13 Thus, although adherence is an important virulence mechanism for numerous pathogenic bacterial species, the role of cable pili and the role of adherence in general in B cepacia complex infection in CF remain unclear. Our results indicate that PHDC contains neither esmR nor cblA sequences. Therefore, although these markers have potential roles in the virulence of some epidemic strains, their presence is clearly not essential in all epidemic lineages. Recent recommendations to stratify infection control measures on the basis of the presence or absence of these markers would therefore seem ill-advised.24 Our study also illustrates the utility and limitations of cohorting patients with CF on the basis of B cepacia colonization status as an infection control measure. This approach has long been reported as a successful intervention in reducing spread of B cepacia in patients with CF.25 We similarly show that the initiation of cohorting was associated with a decrease in incidence of B cepacia complex infection in both Centers A and B. However, it is clear that B cepacia complex bacteria are frequently misidentified by routine analyses.26 In our study several sputum culture isolates identified as “B cepacia” by the referring laboratories had indeed been misidentified. In fact, 6 (22%) of the 27 648
THE JOURNAL OF PEDIATRICS NOVEMBER 2001 Center A isolates described in a previous report5 are now identified as species other than B cepacia complex. Isolate misidentification that results in a patient being inappropriately labeled as “B cepacia–positive,” and thus exposed to a cohort of genuinely infected individuals, presents an unfortunate and avoidable risk. Furthermore, exposure of an infected patient to persons harboring different (perhaps more virulent) B cepacia complex strains also represents an unnecessary risk. In this study we identified 2 Center A patients in whom isolation of B vietnamiensis and B gladioli, respectively, were followed some time later by recovery of the dominant genomovar III strain. It is not clear whether the apparent “replacement” of one Burkholderia species with another in these patients resulted from patient cohorting; however, such a scenario is certainly feasible. We also identified 2 patients who were apparently infected with the dominant genomovar III strain for periods of at least 10 to 15 years. Although a comprehensive analysis of clinical outcomes relative to infecting B cepacia complex strain was beyond the scope of this study, we are aware that both patients have remained relatively well for the past several years, despite apparent persistent infection. Several other patients infected with the dominant strain have died; some of these deaths have been associated with B cepacia sepsis (“cepacia syndrome”). The reasons for the variable outcomes associated with infection with the same B cepacia complex strain are not known. Nevertheless, it is clear that although some B cepacia complex strains, such as PHDC and ET12, may have an enhanced capacity for spread or pulmonary infection in patients with CF, this does not necessarily indicate greater virulence per se. Until a more complete understanding of B cepacia complex pathogenic mechanisms and CF host factors is available, predictions of clinical outcome based solely on infecting strain may not be possible.
Finally, our study also demonstrates the utility of different genotyping methods for B cepacia complex bacteria. In the absence of a standardized genotyping scheme for these species, we used complementary methods for strain identification. RAPD analysis is routinely used for isolate genotyping in this laboratory. This method is simple, rapid, inexpensive, and well suited to ascertaining strain identity among a small number of isolates within a short time. However, inter-assay variation in DNA profiles makes it difficult to analyze large numbers of isolates or to compare new profiles with those from previous assays in ongoing studies. In contrast, rep-PCR, although more timeconsuming, yields highly reproducible profiles that can be stored in a database for comparison with profiles generated in several different experiments. This method has been used in similar studies of large numbers of Pseudomonas isolates to determine clonal relatedness.21 PFGE is both time-consuming and expensive. Nevertheless, it is currently the most widely used bacterial genotyping method, and consensus interpretive criteria have been developed.22 The results obtained with all 3 genotyping methods were in agreement and allowed us to unequivocally identify PHDC as the dominant strain type in Centers A and B.
REFERENCES 1. Isles A, Maclusky I, Corey M, Gold R, Prober C, Fleming P, et al. Pseudomonas cepacia infection in cystic fibrosis: an emerging problem. J Pediatr 1984; 104:206-10. 2. LiPuma JJ. Burkholderia cepacia epidemiology and pathogenesis: implications for infection control. Curr Opin Pulm Med 1998;4:337-341. 3. LiPuma JJ, Dasen SE, Nielson DW, Stem RC, Stull TL. Person-to-person transmission of Pseudomonas cepacia between patients with cystic fibrosis. Lancet 1990;336:1094-6. 4. Govan JR, Brown PH, Maddison J, Doherty CJ, Nelson JW, Dodd M, et al. Evidence for transmission of Pseudomonas cepacia by social contact in cystic fibrosis. Lancet 1993;342:15-9.
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VOLUME 139, NUMBER 5 5. LiPuma JJ, Mortensen JE, Dasen SE, Edlind TD, Schidlow DV, Bums JL, et al. Ribotype analysis of Pseudomonas cepacia from cystic fibrosis treatment centers. J Pediatr 1988;113:859-62. 6. Sun L, Jiang RZ, Steinbach S, Holmes A, Campanelli C, Forstner J, et al. The emergence of a highly transmissible lineage of cbl+ Pseudomonas (Burkholderia) cepacia causing CF centre epidemics in North America and Britain. Nat Med 1995;1:661-6. 7. Mahenthiralingam E, Simpson DA, Speert DP. Identification and characterization of a novel DNA marker associated with epidemic Burkholderia cepacia strains recovered from patients with cystic fibrosis. J Clin Microbiol 1997;35:808-16. 8. Sajjan US, Sun L, Goldstein R, Forstner JF. Cable (cbl) type II pili of cystic fibrosis-associated Burkholderia (Pseudomonas) cepacia: nucleotide sequence of the cblA major subunit pilin gene and novel morphology of the assembled appendage fibers. J Bacteriol 1995;177:1030-8. 9. Vandamme P, Holmes B, Vancanneyt M, Coenye T, Hoste B, Coopman R, et al. Occurrence of multiple genomovars of Burkholderia cepacia in cystic fibrosis patients and proposal of Burkholderia multivorans sp. nov. Int J Syst Bacteriol 1997;47:1188-200. 10. Vandamme P, Mahenthiralingam E, Holmes B, Coenye T, Hoste B, DeVos P, et al. Identification and population structure of Burkholderia stabilis sp. nov. (formerly Burkholderia cepacia genomovar IV). J Clin Microbiol 2000; 38:1042-7. 11. Coenye T, Mahenthiralingam E, Henry D, LiPuma JJ, Laevens S, Gillis M, et al. Burkholderia ambifaria sp. nov., a novel member of the Burkholderia cepacia complex comprising biocontrol and cystic fibrosis-related isolates. Int J Syst Evol Microbiol. 2001;51:1481-90.
12. Coenye T, LiPuma J, Henry D, Hoste B, Mahenthiralingam E, Vandemeulebroecke K, et al. Burkholderia cepacia genomovar VI, a new member of the Burkholderia cepacia complex isolated from cystic fibrosis patients. Int J Syst Bacteriol. 2001;51:271-9. 13. LiPuma JJ, Spilker T, Gill LH, Campbell PW III, Liu L, Mahenthiralingam E. Disproportionate distribution of Burkholderia cepacia complex species and transmissibility factors in cystic fibrosis. Am J Respir Crit Care Med 2001;164:92-6. 14. Henry DA, Campbell ME, LiPuma JJ, Speert DP. Identification of Burkholderia cepacia isolates from patients with cystic fibrosis and use of a simple new selective medium. J Clin Microbiol 1997; 35:614-9. 15. LiPuma JJ, Dulaney BJ, McMenamin JD, Whitby PW, Stull TL, Coenye T, et al. Development of rRNA-based PCR assays for identification of Burkholderia cepacia complex isolates recovered from cystic fibrosis patients. J Clin Microbiol 1999;37:3167-70. 16. McMenamin JD, Zaccone TM, Coenye T, Vandamme P, LiPuma JJ. Misidentification of Burkholderia cepacia in US cystic fibrosis treatment centers. An analysis of 1,051 recent sputum isolates. Chest 2000;117:1611-65. 17. Mahenthiralingam E, Bischof J, Byrne SK, Radomski C, Davies JE, Av-Gay Y, et al. DNA-based diagnostic approaches for identification of Burkholderia cepacia complex, Burkholderia vietnarniensis, Burkholderia multivorans, Burkholderia stabilis, and Burkholderia cepacia genomovars I and II. J Clin Microbiol 2000;38:316573. 18. Whitby PW, Carter KB, Hatter KL, LiPuma JJ, Stull TL. Identification of members of the Burkholderia cepacia complex by species-specific PCR. J Clin Microbiol 2000;38:2962-5.
19. Mahenthiralingam E, Campbell ME, Henry DA, Speert DP. Epidemiology of Burkholderia cepacia infection in patients with cystic fibrosis: analysis by randomly amplified polymorphic DNA fingerprinting. J Clin Microbiol 1996;34:2914-20. 20. Rademaker JLW, Louws FJ, de Bruijn FJ. Characterization of the diversity of ecologically important microbes by rep-PCR fingerprinting. In: Akkermans ADL, van Elsas JD, de Bruijn FJ, editors. Molecular microbial ecology manual. Suppl 3. Dordrecht, The Netherlands: Kluwer Academic Publishers; 1998. p. 1-26. 21. Cho J-C, Tiedje JM. Biogeography and degree of endemicity of fluorescent Pseudomonas strains in soil. Appl Environ Microbiol 2000;66:5448-56. 22. Tenover FC, Arbeit RD, Goering RV, Mickelsen PA, Murray BE, Persing DH, et al. Interpreting chromosomal DNA restriction patterns produced by pulsed-field gel electrophoresis: criteria for bacterial strain typing. J Clin Microbiol 1995;33:2233-9. 23. Sajjan US, Sylvester FA, Forstner JF. Cable-piliated Burkholderia cepacia binds to cytokeratin 13 of epithelial cells. Infect Immun 2000;68:1787-95. 24. Clode FE, Kaufmann ME, Malnick H, Pitt TL. Distribution of genes encoding putative transmissibility factors among epidemic and non-epidemic strains of Burkholderia cepacia from cystic fibrosis patients in the United Kingdom. J Clin Microbiol 2000;38:1763-6. 25. Thomassen MJ, Demko CA, Doershuk CF, Stem RC, Klinger JD. Pseudomonas cepacia: decrease in colonization in patients with cystic fibrosis. Am Rev Respir Dis 1986;134:669-71. 26. Blecker-Shelly D, Spilker T, Gracely EJ, Coenye T, Vandamme P, LiPuma JJ. Utility of commercial systems for identification of Burkholderia cepacia complex from cysticfibrosis sputum culture. J Clin Microbiol 2000;38:3112-5.
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timicrobial resistance exhibited by most clinical isolates limits therapeutic options, and thus, prevention of B cepacia acquisition has become an important goal. However, much remains unknown about the epidemiology of B cepacia infection in CF, and optimal in-
From the Department of Pediatrics, St Christopher’s Hospital for Children, Philadelphia, Pennsylvania; Department of Pediatrics, Children’s National Medical Center, Washington, DC; and Department of Pediatrics and Communicable Diseases, University of Michigan, Ann Arbor.
Supported by a grant from the Cystic Fibrosis Foundation (to Dr LiPuma). Submitted for publication Feb 7, 2001; revision received June 1, 2001; accepted June 29, 2001. Reprint requests: John J. LiPuma, MD, 1150 W Medical Center Dr, 8328 MSRB III, Box 0646, Ann Arbor, MI 48109. Copyright © 2001 by Mosby, Inc. 0022-3476/2001/$35.00 + 0 9/21/118430 doi:10.1067/mpd.2001.118430
fection control policies have yet to be defined.2
See editorial, p 618. Early studies in which bacterial genotyping was used demonstrated person-to-person spread of some strains3,4 and the existence of dominant strain types that infected the majority of colonized persons at some CF treatment centers.5 Among these so-called epidemic strains, the ET12 lineage, responsible for epidemic spread in several centers, has been the most extensively studied.6 This strain is characterized by the presence of the B cepacia epidemic strain marker (BCESM)7 and expression of cblAencoded cable pili that mediate adherence to respiratory epithelial cells.8 Other epidemic strains have been less completely characterized, and additional factors that may contribute to BCESM
Burkholderia cepacia epidemic strain marker CF Cystic fibrosis PCR Polymerase chain reaction PFGE Pulsed field gel electrophoresis rep-PCR Repetitive extragenic palindromic PCR RAPD Randomly amplified polymorphic DNA
enhanced transmissibility have not been identified. Taxonomic studies have made it clear that bacteria previously identified merely as B cepacia by conventional tests actually belong to several distinct species or genomovars, referred to collectively as the B cepacia complex.9 Formal binomial designations have been 643
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provided for 4 of the 7 species described to date. These include B multivorans (genomovar II), B stabilis (genomovar IV), B vietnamiensis (genomovar V), and B ambifaria (genomovar VII).9-11 The remaining 3 species are still referred to as B cepacia genomovars I, III, and VI pending the identification of distinguishing phenotypic features.9,12 All 7 species have been recovered from sputum from persons with CF, but genomovar III accounts for the majority of isolates.13 Whether significant differences exist between species with respect to virulence, per se, has not been established. However, preliminary study has indicated that most epidemic strains are BCESM-positive genomovar III.7 In this study, we investigated B cepacia complex isolates recovered from a large CF treatment center in which a dominant strain type was identified several years ago.5 Genotyping analysis demonstrates that the same B cepacia complex strain continues to dominate in this center where it is now recovered from nearly all patients with B cepacia complex infections. This 20-year endemicity is punctuated by the inter-city spread of this strain to another large CF treatment center wherein its arrival was marked by a sharp increase in the incidence of B cepacia complex infection; this strain now predominates among colonized persons in this center.
METHODS Bacterial Isolates and Species Identification Putative B cepacia complex isolates, recovered by sputum culture from patients attending a large CF treatment center in Philadelphia (Center A) between 1981 and 1987 and genotyped during a previous investigation,5 were available from frozen stocks maintained in skim milk at –80°C. Additional putative B cepacia complex isolates recovered from patients in Center A between 1996 and 2000, as well as from patients receiving care in 644
THE JOURNAL OF PEDIATRICS NOVEMBER 2001 a CF treatment center in Washington, DC (Center B) between 1997 and 2000, were also available for study and similarly stored in skim milk at –80°C until they were analyzed. Isolates from frozen stock were recovered on Mueller-Hinton agar after incubation at 35°C for 24 to 48 hours. All isolates were confirmed as B cepacia complex based on growth on B cepacia– selective media,14 biochemical reactivity, and species-specific polymerase chain reaction (PCR) assays targeting ribosomal RNA and recA genes as previously described.15-17 The presence of B gladioli was confirmed by using a species-specific PCR assay.18 Any isolate not confirmed as a Burkholderia species was further analyzed by using the RapID NF Plus System (Remel, Norcross, Ga).
Isolate Genotyping Genomic DNA was prepared from all isolates by using the Easy-DNA Kit (Invitrogen, Carlsbad, Calif) with modifications previously reported.15 All isolates had an initial screening genotype analysis performed with randomly amplified polymorphic DNA (RAPD) typing as described19 with the following modifications. Five microliters of 5 mol/L betaine (SigmaAldrich, St Louis, Mo) and 0.7 µL of 10 mg/mL acetylated bovine serum albumin (Promega, Madison, Wis) were added to the final PCR mix. PCR was conducted by using a RapidCycler (Idaho Technologies, Idaho Falls, Idaho) thermal cycler in 0.2-mL thinwalled tubes with the following parameters: 4 cycles (with slope of 1.0°C per second) of 94°C for 60 seconds, 55°C for 60 seconds, and 72°C for 150 seconds; followed by 29 cycles (with slope of 0.7°C per second) of 94°C for 30 seconds, 55°C for 30 seconds, and 72°C for 75 seconds. Amplified DNA, along with a custom-synthesized DNA ladder of known fragment sizes, was electrophoresed in a 2% agarose gel (BioRad, Hercules, Calif ) at 2 V/cm for 1 hour and visualized by using
ethidium bromide staining and UV illumination. Isolates were further genotyped by using repetitive extragenic palindromic PCR (rep-PCR) with a BOX-A1R primer as previously described.20 PCR products were resolved on 20-cm-long 1.5% agarose gels in 0.5× tris-acetateEDTA buffer at 5 V/cm for 5 hours at 4°C. Gels were stained with ethidium bromide and visualized with UV illumination. Gel images were digitized by using a GelDoc 2000 gel analyzer (BioRad) and stored as tagged image file format files. Digitized images were converted, normalized with the DNA size marker, and analyzed with Molecular Analyst Fingerprinting Plus software (BioRad). The rolling-disk background subtraction method was applied, and similarity matrices of whole densitometric curves of the gel tracks were calculated by using pairwise Pearson’s product-moment correlation coefficient. Cluster analyses of similarity matrices were performed by the unweighted pair group method with arithmetic averages. A similarity coefficient of ≥0.8 was used to indicate isolates of the same genotype (ie, strain).21 A subset of isolates, including all those with unique profiles as determined by RAPD and rep-PCR analyses, were also analyzed by macrorestriction digest and pulsed field gel electrophoresis (PFGE). Single colonies of bacteria, recovered from growth on nonselective agar media, were suspended in 1 mL of SE buffer (0.07 mol/L NaCl, 2.5 mmol/L EDTA; pH 7.4), pelleted by centrifugation at 8000 rpm for 2 minutes, washed 3 additional times in 10 mL of SE buffer, and re-suspended in 0.5 mL of SE buffer. Optical density at 620 nm was adjusted to 1.0, and 200 µL of the cell suspension was homogenized with 200 µL of 2% low-meltingtemperature agarose (Sigma) in SE buffer. Agarose plugs were made in a plug mold refrigerated at 4°C for 30 minutes and then placed in 10 mL of PEN buffer (17 mmol/L N-lauryl sarco-
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VOLUME 139, NUMBER 5 sine, 1% sodium salt, 25 mmol/L EDTA; pH 9.6) containing 1 mg of protease. After incubation in a 37°C rocking incubator for 12 to 18 hours, plugs were washed for 30 minutes with 1× TE buffer (0.1 mmol/L TRIS-HCl, 0.01 mmol/L EDTA; pH 8.0) 5 times, and 2-mm wide sections were incubated at 37°C for 6 hours with SpeI (Promega). DNA fragments were separated in 1% agarose (BioRad) by using a CHEFDR III system (BioRad) at 5.0 V/cm with pulse times of 30 to 70 seconds for 25 hours. Gels were stained with ethidium bromide and visualized with UV illumination. Strain identity was determined by using published criteria.22
Putative Transmissibility Factors All isolates were analyzed by PCR for the presence of BCESM as described 13 with primers ESM-R1F (5´ –GACAACGAGCAACGCGTAA3´) and ESM-R2R (5´ -GGCTCATCTGCGTATCGA-3´), which are specific for esmR, the internal 834-bp open reading frame of the BCESM sequence.7 The presence of DNA sequences with homology to cblA was confirmed by dot-blot hybridization as described.13 For both PCR and dot-blot assays, isolate AU0355, a BCESM- and cblA-positive isolate of the ET12 clonal lineage originally isolated from a patient with CF in Toronto, was used as a positive control; B cepacia ATCC 25416 (American Type Culture Collection Manasas, Va; genomovar I, BCESMand cblA-negative)17 was used as a negative control.
Epidemiologic Data and Infection Control Measures The incidence and prevalence of B cepacia infection for Centers A and B were calculated for relevant years based on the total number of patients with CF being cared for in the respective center and the number reported by the respective clinical microbiology laboratory to have had at least one sputum culture positive for B cepacia. In
Center A, sputum cultures were obtained from most patients only once per year. Sputum cultures were obtained 4 to 6 times per year from patients at Center B. Infection control policies governing B cepacia infection in patients with CF were reviewed in Centers A and B for relevant years.
RESULTS Center A Isolates Bacterial isolates (n = 27), reported to be B cepacia by the referring laboratory, were recovered from 27 patients with CF at Center A between 1981 and 1987. These isolates had been genotyped by ribotype analysis during a previous investigation that demonstrated 14 of the isolates to be the same strain type.5 In the present study, analysis of these 27 isolates after recovery from frozen stocks confirmed that 21 were indeed B cepacia complex; however, among the remaining 6 isolates, 5 were actually Burkholderia gladioli, and one was identified as Ochrobacterium anthropi. (These 6 non– B cepacia isolates had unique ribotype profiles in the previous analysis.5) Among the 21 confirmed B cepacia complex isolates, 18 were identified as genomovar III, 2 were B multivorans, and 1 was B vietnamiensis. RAPD and rep-PCR analyses demonstrated that 17 of the 18 genomovar III isolates were the same strain; the remaining genomovar III isolate and, as expected, the B gladioli, B multivorans, and B vietnamiensis isolates had different DNA profiles (Fig 1). Isolates were available from 43 of the 44 patients with CF reported to have had at least one sputum culture positive for B cepacia while receiving care at Center A between 1996 and 2000. Species identification analysis confirmed that 41 of the 43 isolates were B cepacia genomovar III; the remaining 2 isolates were actually B gladioli and Achromobacter (Alcaligenes) xylosoxidans. RAPD and rep-PCR analyses demonstrated that 40 of the 41 genomovar III
Fig 1. Representative RAPD analysis. Each lane contains an isolate recovered from a different Center A patient between 1981 and 1987. Lane 1, Fragment size marker; lane 2, B gladioli; lane 3, B vietnamiensis; lane 4, the only B cepacia genomovar III recovered during this period with unique profile; lanes 5 through 8, isolates showing dominant B cepacia genomovar III strain profile.Thirteen additional patients were also colonized with the dominant strain. isolates were the same strain. This strain is the same as that which predominates among isolates recovered from Center A patients between 1981 and 1987 (Fig 2), indicating that this clonal lineage has been endemic in this center for 20 years. PFGE analysis was performed on a subset of 14 isolates from Center A, and the results were in agreement with those demonstrated by RAPD and rep-PCR typing (data not shown). Among the Center A patients were 4 from whom isolates were available during both the 1981-1987 and the 1996-2000 intervals. Genotype analyses indicated that 2 patients were apparently persistently colonized with the dominant strain type for at least 15 and 10 years, respectively. In the other 2 patients, isolation of B vietnamiensis and B gladioli, respectively, in 1986 was followed by recovery in 1997 and 2000, respectively, of the center-dominant genomovar III strain (Fig 3).
Center B Isolates Isolates were available from 20 of the 25 patients with CF reported to have had at least one sputum culture positive 645
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Fig 2. Representative rep-PCR analysis. Each lane contains an isolate recovered from a different Center A patient between 1996 and 2000. Lane 1, Fragment size marker; lane 2, the only B cepacia genomovar III recovered during this period with unique profile; lanes 3 through 5, isolates showing dominant B cepacia genomovar III strain profile.Thirty-seven additional patients were colonized with the dominant strain. Lane 6, Dominant B cepacia genomovar III strain recovered in Center A in 1981 (included for comparison). for B cepacia while receiving care in Center B between 1997 and 1999. No isolates cultured from CF sputum before 1997 were available. Species identification analysis confirmed that 19 isolates were B cepacia complex (18 genomovar III and 1 B multivorans), whereas the remaining was actually B gladioli. RAPD and rep-PCR analyses indicated that 17 of the 18 genomovar III isolates were the same strain. Furthermore, this strain was the same as that which predominated among colonized patients in Center A during the 1981-1987 and 1996-2000 intervals (Fig 4).
Epidemiologic Data and Infection Control Measures During the late 1980s, the prevalence of B cepacia complex infection among patients with CF in Center A was ~20%. The annual incidence peaked in 1988 with a rate of 7.8%, followed by a rate of 5.8% in 1989. In early 1990, cohorting of hospitalized 646
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Fig 3. PFGE analysis of isolates from Center A. Lane 1, Fragment size marker; lane 2, dominant B cepacia genomovar III strain recovered in 1981; lanes 3 and 4, B cepacia genomovar III recovered from patient 1 in 1985 and 2000, respectively; lanes 5 and 6, B cepacia genomovar III recovered from patient 2 in 1986 and 1996, respectively; lanes 7 and 8, B gladioli and B cepacia genomovar III recovered from patient 3 in 1986 and 1997, respectively; lanes 9 and 10, B vietnamiensis and B cepacia genomovar III recovered from patient 4 in 1986 and 2000, respectively. patients with CF on the basis of B cepacia colonization status was initiated and was associated with a drop in incidence rates to 2.1% and 3.7% in 1990 and 1991, respectively. In mid 1996, hospitalized non-colonized patients were prohibited from sharing rooms, and in early 1998, this policy was expanded so that all patients with CF, irrespective of B cepacia colonization status, were in separate rooms when hospitalized. In 1997, separate waiting rooms were established for outpatients. Between 1992 and 1999, the prevalence of B cepacia complex infection in Center A dropped from 15% to 7%; incidence rates remained <2% during this time. Since 1995, 2 to 4 patients per year have been reported to have had B cepacia complex first recovered from sputum culture. Between 1990 and 1995, the prevalence of B cepacia complex infection in Center B ranged from 2.6% to 5.9%, with an annual incidence of <2% (Fig 4). In September 1993, a patient with
CF from Center A, infected with that center’s dominant genomovar III strain since 1989, transferred care to Center B. This patient was not hospitalized at Center B during the remainder of 1993 but was an inpatient twice in 1994 and 3 times during each year from 1995 to 1997. The patient had 6 outpatient visits per year during this period and was described by Center B caregivers as very social and an active participant in CF-related social activities. The incidence of B cepacia complex infection in Center B increased to 8.8% in 1996 and 5.7% in 1997; 23 patients became infected during these 2 years. Among the patients from whom B cepacia complex isolates were available for study, 18 of 19 were colonized with genomovar III, and 17 of these were colonized with the dominant strain. The only patient infected with a nondominant strain had the first positive culture in 1994, whereas 15 of the 17 with the dominant strain had first positive cultures after 1995. The 2 remaining patients with the dominant strain included the patient who transferred from Center A in 1993 and another patient reported to be B cepacia culture–positive since 1988 (unfortunately, isolates recovered from this patient before 1997 were not available for analysis). Thus, nearly all of the patients colonized with B cepacia complex in Center B after 1993 were infected with the Center A dominant strain. In early 1997, cohorting of hospitalized patients on the basis of B cepacia colonization status was initiated in Center B. Furthermore, colonized inpatients were placed in contact isolation and were required to wear mask and gloves when out of their rooms. A hospital-wide educational program regarding infection control was introduced at that time. In the outpatient setting, infected patients were restricted to separate clinic days during which no non-colonized patients were seen. Patients were required to wear masks while in the waiting room, and particular attention was given to disinfection
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VOLUME 139, NUMBER 5 of clinic rooms and equipment. The annual incidence of B cepacia complex infection has been <1% since that time.
DISCUSSION Epidemiologic studies of B cepacia infection in patients with CF have been hampered by difficulties with accurate identification of Burkholderia and related species and by the lack of consensus methods for strain typing. The recent recognition that several distinct species of bacteria actually comprise the B cepacia complex and the development of reliable PCR-based assays for their identification now provide a critical framework for further study. By using bacterial genotyping and a polyphasic identification scheme incorporating phenotypic and species-specific PCR assays, we have identified a B cepacia genomovar III strain, designated PHDC, which infects nearly all B cepacia– infected patients receiving care in large CF treatment centers in 2 US cities. We further demonstrated that this clone has not only been endemic but has indeed represented the dominant strain among infected patients in one center for at least the past 20 years. The transfer of an infected patient between centers was associated with a marked increase in incidence of B cepacia complex infection caused by this strain. In both centers, reinforcement of more rigid infection control measures significantly reduced the rate of infection. These findings highlight a number of important aspects of B cepacia complex infection in patients with CF. The finding in the late 1980s of specific B cepacia complex strains that were recovered from multiple patients5 suggested that some strains have an enhanced ability to colonize or be transmitted among patients. The identification and characterization of such so-called epidemic strains is important in that it may provide valuable insights into virulence mechanisms and host factors involved
Fig 4. Cluster analysis of rep-PCR profiles. Each track contains an isolate recovered from a different patient from either Center A or Center B during the year indicated. On the scale, similarity coefficients are expressed as percentages. Not shown are an additional 14 genomovar III isolates from Center A during 1981 to 1987, 37 from Center A during 1996 to 2000, and 14 from Center B during 1997 to 1999, all of which also clustered with the dominant strain (ie, with a similarity coefficient of >0.8) as described in the text.The bottom tracks show the only 3 genomovar III isolates available for analysis that are not of the dominant strain type as described in the text.
Fig 5. Annual incidence and prevalence of B cepacia complex infection in Center B. Strict infection control initiated in early 1997 as described in text. *Percent of patients with CF receiving care in Center B.
in infection in CF. Our finding that PHDC has stably persisted within a group of patients with CF for 2 decades further suggests that this strain possesses features contributing to its apparent adaptation to the CF host. Nevertheless, the identification of B cepacia complex transmissibility or colonization factors has been problematic. Few epidemic strains have been de-
scribed in detail. Several strains recovered from multiple patients contained a conserved 1.4-kb genomic fragment not found in strains recovered from single patients.7 This fragment, termed the B cepacia epidemic strain marker (BCESM), encodes an approximately 834-bp open reading frame, esmR, with homology to negative transcriptional regulators; however, the role of this 647
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putative gene in virulence remains unknown. ET12, a genomovar III strain that dominates among patients with CF in Ontario, Canada, and is associated with inter-patient spread in the United Kingdom, has been the most completely studied epidemic lineage. In addition to esmR, this strain elaborates large peritrichous pili, termed cable pili. The gene encoding cable pili, cblA, has been characterized, as has the cognate epithelial cell receptor for the associated adhesin.8,23 A recent study of B cepacia complex isolates recovered from over 600 patients with CF in the United States demonstrated that only one (an ET12 strain) contained the complete cblA sequence.13 Thus, although adherence is an important virulence mechanism for numerous pathogenic bacterial species, the role of cable pili and the role of adherence in general in B cepacia complex infection in CF remain unclear. Our results indicate that PHDC contains neither esmR nor cblA sequences. Therefore, although these markers have potential roles in the virulence of some epidemic strains, their presence is clearly not essential in all epidemic lineages. Recent recommendations to stratify infection control measures on the basis of the presence or absence of these markers would therefore seem ill-advised.24 Our study also illustrates the utility and limitations of cohorting patients with CF on the basis of B cepacia colonization status as an infection control measure. This approach has long been reported as a successful intervention in reducing spread of B cepacia in patients with CF.25 We similarly show that the initiation of cohorting was associated with a decrease in incidence of B cepacia complex infection in both Centers A and B. However, it is clear that B cepacia complex bacteria are frequently misidentified by routine analyses.26 In our study several sputum culture isolates identified as “B cepacia” by the referring laboratories had indeed been misidentified. In fact, 6 (22%) of the 27 648
THE JOURNAL OF PEDIATRICS NOVEMBER 2001 Center A isolates described in a previous report5 are now identified as species other than B cepacia complex. Isolate misidentification that results in a patient being inappropriately labeled as “B cepacia–positive,” and thus exposed to a cohort of genuinely infected individuals, presents an unfortunate and avoidable risk. Furthermore, exposure of an infected patient to persons harboring different (perhaps more virulent) B cepacia complex strains also represents an unnecessary risk. In this study we identified 2 Center A patients in whom isolation of B vietnamiensis and B gladioli, respectively, were followed some time later by recovery of the dominant genomovar III strain. It is not clear whether the apparent “replacement” of one Burkholderia species with another in these patients resulted from patient cohorting; however, such a scenario is certainly feasible. We also identified 2 patients who were apparently infected with the dominant genomovar III strain for periods of at least 10 to 15 years. Although a comprehensive analysis of clinical outcomes relative to infecting B cepacia complex strain was beyond the scope of this study, we are aware that both patients have remained relatively well for the past several years, despite apparent persistent infection. Several other patients infected with the dominant strain have died; some of these deaths have been associated with B cepacia sepsis (“cepacia syndrome”). The reasons for the variable outcomes associated with infection with the same B cepacia complex strain are not known. Nevertheless, it is clear that although some B cepacia complex strains, such as PHDC and ET12, may have an enhanced capacity for spread or pulmonary infection in patients with CF, this does not necessarily indicate greater virulence per se. Until a more complete understanding of B cepacia complex pathogenic mechanisms and CF host factors is available, predictions of clinical outcome based solely on infecting strain may not be possible.
Finally, our study also demonstrates the utility of different genotyping methods for B cepacia complex bacteria. In the absence of a standardized genotyping scheme for these species, we used complementary methods for strain identification. RAPD analysis is routinely used for isolate genotyping in this laboratory. This method is simple, rapid, inexpensive, and well suited to ascertaining strain identity among a small number of isolates within a short time. However, inter-assay variation in DNA profiles makes it difficult to analyze large numbers of isolates or to compare new profiles with those from previous assays in ongoing studies. In contrast, rep-PCR, although more timeconsuming, yields highly reproducible profiles that can be stored in a database for comparison with profiles generated in several different experiments. This method has been used in similar studies of large numbers of Pseudomonas isolates to determine clonal relatedness.21 PFGE is both time-consuming and expensive. Nevertheless, it is currently the most widely used bacterial genotyping method, and consensus interpretive criteria have been developed.22 The results obtained with all 3 genotyping methods were in agreement and allowed us to unequivocally identify PHDC as the dominant strain type in Centers A and B.
REFERENCES 1. Isles A, Maclusky I, Corey M, Gold R, Prober C, Fleming P, et al. Pseudomonas cepacia infection in cystic fibrosis: an emerging problem. J Pediatr 1984; 104:206-10. 2. LiPuma JJ. Burkholderia cepacia epidemiology and pathogenesis: implications for infection control. Curr Opin Pulm Med 1998;4:337-341. 3. LiPuma JJ, Dasen SE, Nielson DW, Stem RC, Stull TL. Person-to-person transmission of Pseudomonas cepacia between patients with cystic fibrosis. Lancet 1990;336:1094-6. 4. Govan JR, Brown PH, Maddison J, Doherty CJ, Nelson JW, Dodd M, et al. Evidence for transmission of Pseudomonas cepacia by social contact in cystic fibrosis. Lancet 1993;342:15-9.
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VOLUME 139, NUMBER 5 5. LiPuma JJ, Mortensen JE, Dasen SE, Edlind TD, Schidlow DV, Bums JL, et al. Ribotype analysis of Pseudomonas cepacia from cystic fibrosis treatment centers. J Pediatr 1988;113:859-62. 6. Sun L, Jiang RZ, Steinbach S, Holmes A, Campanelli C, Forstner J, et al. The emergence of a highly transmissible lineage of cbl+ Pseudomonas (Burkholderia) cepacia causing CF centre epidemics in North America and Britain. Nat Med 1995;1:661-6. 7. Mahenthiralingam E, Simpson DA, Speert DP. Identification and characterization of a novel DNA marker associated with epidemic Burkholderia cepacia strains recovered from patients with cystic fibrosis. J Clin Microbiol 1997;35:808-16. 8. Sajjan US, Sun L, Goldstein R, Forstner JF. Cable (cbl) type II pili of cystic fibrosis-associated Burkholderia (Pseudomonas) cepacia: nucleotide sequence of the cblA major subunit pilin gene and novel morphology of the assembled appendage fibers. J Bacteriol 1995;177:1030-8. 9. Vandamme P, Holmes B, Vancanneyt M, Coenye T, Hoste B, Coopman R, et al. Occurrence of multiple genomovars of Burkholderia cepacia in cystic fibrosis patients and proposal of Burkholderia multivorans sp. nov. Int J Syst Bacteriol 1997;47:1188-200. 10. Vandamme P, Mahenthiralingam E, Holmes B, Coenye T, Hoste B, DeVos P, et al. Identification and population structure of Burkholderia stabilis sp. nov. (formerly Burkholderia cepacia genomovar IV). J Clin Microbiol 2000; 38:1042-7. 11. Coenye T, Mahenthiralingam E, Henry D, LiPuma JJ, Laevens S, Gillis M, et al. Burkholderia ambifaria sp. nov., a novel member of the Burkholderia cepacia complex comprising biocontrol and cystic fibrosis-related isolates. Int J Syst Evol Microbiol. 2001;51:1481-90.
12. Coenye T, LiPuma J, Henry D, Hoste B, Mahenthiralingam E, Vandemeulebroecke K, et al. Burkholderia cepacia genomovar VI, a new member of the Burkholderia cepacia complex isolated from cystic fibrosis patients. Int J Syst Bacteriol. 2001;51:271-9. 13. LiPuma JJ, Spilker T, Gill LH, Campbell PW III, Liu L, Mahenthiralingam E. Disproportionate distribution of Burkholderia cepacia complex species and transmissibility factors in cystic fibrosis. Am J Respir Crit Care Med 2001;164:92-6. 14. Henry DA, Campbell ME, LiPuma JJ, Speert DP. Identification of Burkholderia cepacia isolates from patients with cystic fibrosis and use of a simple new selective medium. J Clin Microbiol 1997; 35:614-9. 15. LiPuma JJ, Dulaney BJ, McMenamin JD, Whitby PW, Stull TL, Coenye T, et al. Development of rRNA-based PCR assays for identification of Burkholderia cepacia complex isolates recovered from cystic fibrosis patients. J Clin Microbiol 1999;37:3167-70. 16. McMenamin JD, Zaccone TM, Coenye T, Vandamme P, LiPuma JJ. Misidentification of Burkholderia cepacia in US cystic fibrosis treatment centers. An analysis of 1,051 recent sputum isolates. Chest 2000;117:1611-65. 17. Mahenthiralingam E, Bischof J, Byrne SK, Radomski C, Davies JE, Av-Gay Y, et al. DNA-based diagnostic approaches for identification of Burkholderia cepacia complex, Burkholderia vietnarniensis, Burkholderia multivorans, Burkholderia stabilis, and Burkholderia cepacia genomovars I and II. J Clin Microbiol 2000;38:316573. 18. Whitby PW, Carter KB, Hatter KL, LiPuma JJ, Stull TL. Identification of members of the Burkholderia cepacia complex by species-specific PCR. J Clin Microbiol 2000;38:2962-5.
19. Mahenthiralingam E, Campbell ME, Henry DA, Speert DP. Epidemiology of Burkholderia cepacia infection in patients with cystic fibrosis: analysis by randomly amplified polymorphic DNA fingerprinting. J Clin Microbiol 1996;34:2914-20. 20. Rademaker JLW, Louws FJ, de Bruijn FJ. Characterization of the diversity of ecologically important microbes by rep-PCR fingerprinting. In: Akkermans ADL, van Elsas JD, de Bruijn FJ, editors. Molecular microbial ecology manual. Suppl 3. Dordrecht, The Netherlands: Kluwer Academic Publishers; 1998. p. 1-26. 21. Cho J-C, Tiedje JM. Biogeography and degree of endemicity of fluorescent Pseudomonas strains in soil. Appl Environ Microbiol 2000;66:5448-56. 22. Tenover FC, Arbeit RD, Goering RV, Mickelsen PA, Murray BE, Persing DH, et al. Interpreting chromosomal DNA restriction patterns produced by pulsed-field gel electrophoresis: criteria for bacterial strain typing. J Clin Microbiol 1995;33:2233-9. 23. Sajjan US, Sylvester FA, Forstner JF. Cable-piliated Burkholderia cepacia binds to cytokeratin 13 of epithelial cells. Infect Immun 2000;68:1787-95. 24. Clode FE, Kaufmann ME, Malnick H, Pitt TL. Distribution of genes encoding putative transmissibility factors among epidemic and non-epidemic strains of Burkholderia cepacia from cystic fibrosis patients in the United Kingdom. J Clin Microbiol 2000;38:1763-6. 25. Thomassen MJ, Demko CA, Doershuk CF, Stem RC, Klinger JD. Pseudomonas cepacia: decrease in colonization in patients with cystic fibrosis. Am Rev Respir Dis 1986;134:669-71. 26. Blecker-Shelly D, Spilker T, Gracely EJ, Coenye T, Vandamme P, LiPuma JJ. Utility of commercial systems for identification of Burkholderia cepacia complex from cysticfibrosis sputum culture. J Clin Microbiol 2000;38:3112-5.
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