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Abstract

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Twenty-one strains of Burkholderia cepacia isolated from the environment, and 21 clinical strains isolated principally from sputum of cystic fibrosis (CF) patients, were characterized genotypically by macrorestriction analysis (genome fingerprinting) and PCR ribotyping, and phenotypically by susceptibility to antibiotics and the ability to macerate onion tissue. The plasmid content of the strains was also investigated. Environmental isolates showed a high degree of genetic variability, all strains differing from both one another and the CF isolates. The CF isolates were less variable, with common strains found in patients attending three geographically distinct CF centres. Phenotypic variation was found both within and between CF and environmental strains. Generally, CF isolates displayed higher levels of antibiotic resistance, while the ability to macerate onion tissue was more prevalent amongst environmental isolates. Plasmids were more frequently found in CF isolates, but were of similar size in both groups of strains. Such variability is not surprising in view of the existence of multiple genomovars within the B. cepacia complex.

The Gram-negative bacterium Burkholderia cepacia was originally described as a phytopathogen of onion ( Burkholder 1950) and subsequently as a saprophyte in soils and waters ( Morris & Roberts 1959). More recently, it has emerged as an opportunistic pathogen of man, particularly among cystic fibrosis (CF) and chronic granulomatous disease patients ( Govan et al. 1996 ), as a nosocomially acquired infection in a hospital intensive care unit ( Pegues et al. 1996 ), and amongst oncology patients ( Pegues et al. 1993 ). Burkholderia cepacia displays intrinsic resistance to a range of antibiotics ( Wilkinson & Pitt 1995), shows diverse degradative properties including the degradation of recalcitrant polychlorinated aromatic compounds, and produces a number of compounds antagonistic to phytopathogenic fungi. Such properties have led to interest in B. cepacia as a possible agent of bioremediation or biocontrol ( Govan et al. 1996 ).

Characterization of B. cepacia infecting CF patients in epidemiological studies in the UK ( Govan et al. 1996 ; Pitt et al. 1996 ) and the USA ( Johnson et al. 1994 ) has shown strong evidence of an epidemic strain of B. cepacia, and of person-to-person transmission amongst CF patients. The risk posed to CF patients from environmental reservoirs of B. cepacia strains and the relationship between clinical and environmental isolates is unclear, with contradictory evidence for phenotypic differences between isolates from CF patients and the environment ( Govan et al. 1996 ). Isolates from CF patients and nosocomially acquired infections have been shown to be incapable of causing the maceration of onion tissue in vitro whereas environmental isolates have been shown to macerate onion tissue and display high levels of pectinolytic activity ( Gonzalez 1979 ; Bevivino et al. 1994 ). However, a number of other CF isolates, including the epidemic strain, have been shown to cause maceration of onion by other workers ( Butler et al. 1995 ). The inability of environmental isolates to adhere to human uroepithelial cells has been suggested as evidence that environmental B. cepacia is incapable of causing infection in man ( Bevivino et al. 1994 ), though more recent evidence has suggested that the environment may be a potential source of infection to CF patients ( Cazzola et al. 1996 ). Such contradictory evidence has led to caution being advised in the use of B. cepacia as an agent of bioremediation or biocontrol until the relationship between clinical and environmental isolates of B. cepacia has been fully assessed ( Butler et al. 1995 ; Govan et al. 1996 ).

Phenotypic differences between B. cepacia strains such as those described above, along with genetic variability in both environmental and clinical isolates ( Pitt et al. 1996 ; Wise et al. 1996 ), have led to suggestions that the phylogeny of the species should be revised ( Govan et al. 1996 ; Vandamme et al. 1997 ). Yohalem & Lorbeer (1994 ) placed B. cepacia isolates into four groups or phenoms on the basis of a range of phenotypic properties. Genomic fingerprinting by pulsed field gel electrophoresis (PFGE) and arbitrarily primed PCR (AP-PCR) found four distinct genetic groups or genomovars amongst clinical and environmental isolates of B. cepacia ( Vandamme 1995; Revets et al. 1996 ). Genomovars have been defined as genetically different groups of bacteria that are indistinguishable by current phenotypically based typing methods ( Ursing et al. 1995 ). Recent evidence has suggested that B. cepacia is formed by a complex of five genomic species: B. cepacia genomovars I, II and IV, B. vietnamensis and the newly proposed species, B. multivorans ( Vandamme et al. 1997 ).

Although strains of B. cepacia infecting CF patients in the UK and North America have been well characterized phenotypically and by molecular typing methods ( Pitt et al. 1996 ; Henry et al. 1997 ), there has been little charaterization of environmental isolates found in the UK. Butler et al. (1995) investigated the phenotypic properties of 12 environmental isolates of B. cepacia and undertook genomic analysis by macrorestriction digests followed by PFGE. These isolates were found to differ both genetically and phenotypically from each other and from isolates infecting CF patients. However, most of these isolates were obtained from tropical houses in botanical gardens. These isolates could be described as coming from an unusual habitat in terms of the environmental population of B. cepacia that occurs in the UK, and CF patients are much less likely to come into contact with such isolates than with those occurring in the natural rural and urban environment of the UK. In this study, isolates obtained from the environment (soils, rhizosphere and waters) from both urban and rural areas were characterized phenotypically by their ability to macerate onion tissue in vitro ( Lelliott & Stead 1987) and their susceptibility to a range of antibiotics, genetically through molecular typing by PCR ribotyping, PFGE macrorestriction analysis, and through their plasmid content and size. These isolates were compared with B. cepacia strains isolated from CF patients living in the South Wales area, and with isolates from other European CF centres, to investigate relationships and variations between B. cepacia isolates from CF patients and those in the environment to which the patients may be exposed.

Materials and methods

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Bacterial strains

Twenty-one environmental strains were isolated from soils, waters and rhizosphere at 106 sites in South Wales. Sites were chosen from urban, rural and industrial areas to give a full representation of environmental B. cepacia in South Wales. For soil samples, 2–3 g soil were collected into a sterile universal container, 10 ml sterile water were added and the soil suspended by vortex mixing for 5 min. The sample was allowed to settle before 100 μl of the aqueous layer was removed and plated directly onto each of the selective media used. For the rhizosphere samples, plant roots were gently removed from soil and treated as for the soil samples. For water samples, around 10 ml water and sediment were collected from streams and rivers in an upstream direction, or from the edges of lakes and reservoirs. Samples were vortex mixed for 5 min. After mixing, 100 μl of the sample were plated directly onto each of the selective media.

Each sample was plated onto a range of selective media: B. cepacia selective agar (Mast Diagnostics, Bootle, UK); TB-T agar ( Hagedoorn et al. 1987 ); TB-T agar supplemented with 5 g ml−1 gentamicin; and a minimal agar medium containing 20% onion homogenate and tetracycline at 20 g ml−1 with and without the addition of gentamicin at 5 g ml−1 ( Wigley 1998). The samples were incubated at 30 °C for 48 h. Colonies found growing were Gram stained and all Gram-negative rods were sub-cultured onto fresh media prior to identification, principally by the API 20 NE system (BioMeriuex, Marcy L’Etoile, France). Twenty-one isolates were identified as B. cepacia, a recovery rate of 20% from samples tested. The use of a range of selective media was found to increase the recovery rate of B. cepacia by around 70% compared with the use of the commercial B. cepacia medium alone, suggesting an underestimation of B. cepacia in the environment in some previous studies ( Fisher et al. 1993 ; Mortensen et al. 1995 ).

Twenty-one isolates from the sputum of CF patients attending the Cardiff CF centre were kindly provided by Mr Alan Paull of the Public Health Laboratory Service, Cardiff. A number of isolates from CF centres in Strasbourg and Scotland were kindly provided from the collection of Prof. J.R.W. Govan, University of Edinburgh. The species type strain NCPPB 2993 (ACTC 25416) was obtained from the National Collection of Plant Pathogenic Bacteria (Harpenden, Hertfordshire, UK). Stock isolates were maintained at −70 °C in nutrient broth with 20% glycerol as a cryoprotectant.

In vitro maceration of onion tissue

In vitro maceration of onion tissue was investigated by a modified version of the method of Lelliott & Stead (1987). Clean, sound, disease-free onions (Allium cepa) were wiped with 90% v/v alcohol before cutting aseptically into slices of approximately 5 mm in thickness. Onion slices were placed into sterile Petri dishes and nicks of approximately 2 mm made in the tissue surface. Overnight nutrient broth cultures (100 μl) of each B. cepacia isolate to be tested (approximately 107 cfu ml−1) were inoculated onto the surface of the onion tissue, with sterile saline and Pseudomonas aeruginosa isolates as controls. Onion slices were incubated at 30 °C for 48 h, after which onion maceration and discoloration were assessed with the aid of a sterile needle as a probe.

Antibiotic susceptibility

Antibiotic resistance profiles were determined by the modified Kirby Bauer Disc Diffusion test using the WHO recommended method ( Vandepitte et al. 1991 ). The following discs used were selected on the basis of those recommended by Acar & Goldstein (1985) and the WHO ( Vandepitte et al. 1991 ) for Pseudomonas species: 25 μg amoxicillin, 30 μg ceftazidime, 15 μg erythromycin, 10 μg gentamicin, 5 μg novobiocin, 10 IU penicillin G, 25 μg sulphamethoxazole and 30 μg tetracycline (Oxoid). Plates were incubated at 35 °C for 24–48 h. Escherichia coli NCTC 10418 was included as a control for interpretation of results with each batch of B. cepacia isolates tested.

Minimum inhibitory concentration (MIC) was determined for piperacillin, ceftazidime, gentamicin and sulphamethoxazole by an agar dilution method using doubling dilutions of antibiotics ranging from 512 μg ml−1–1 μg ml−1. Overnight cultures of B. cepacia isolates (approximately 107 cfu ml−1) were inoculated onto the surface of the plates using a Mast multipoint inoculator (Mast Diagnostics). Inoculated plates were incubated at 35 °C for 24–48 h. Minimal inhibitory concentration was taken as the lowest concentration that inhibited growth of the isolate. Escherichia coli NCTC 10418 was included as a control.

Isolation and sizing of plasmid DNA

Isolation and size estimation of plasmid DNA was according to the methods of Rochelle et al. (1985) based on Kado & Liu (1981). The methods were chosen for their accuracy in determining the sizes of small and large plasmids, with minimal shearing of large plasmids and without the need for restriction digests of the plasmid DNA. Overnight bacterial culture in nutrient broth (1·5 ml) was pelleted at 13 000 g for 4 min, the supernatant fluid discarded and the pelleted cells mixed with lysing solution (0·15 g Tris, 1·75 g sodium dodecyl sulphate, 425 μl sodium hydroxide 2 mol l−1, in 25 ml deionized water, pH 12·6) and incubated at 65 °C for 90 min. Phenol/chloroform (150 μl) ( Sambrook et al. 1989 ) was added and an emulsion formed by vigorous shaking. The emulsion was broken by centrifuging at 13 000 g for 4 min. A 75 μl aliquot of the upper aqueous layer containing the plasmid DNA was removed gently with a wide bore pipette tip to a fresh microcentrifuge tube; 20 μl of this sample was loaded into a 0·7% agarose gel in TBE buffer in a Gibco BRL Horizon 58 gel electrophoresis tank (Life Technologies) and run at 4 V cm−1 for 90 min. The gel was stained in 0·5 g ml−1 ethidium bromide and destained under running water before photographing under u.v. transillumination.

Plasmid sizes were estimated by comparison with mobility of plasmids of known sizes, after regression analysis of the plot of log10 molecular size (in kbp) against log10 mobility (distance travelled from origin in mm). The E. coli strains V517 ( Macrina et al. 1978 ) containing eight plasmids of 55 to 2 kbp, and 39R ( Threlfall et al. 1986 ) containing four plasmids of 151 to 7 kbp, were used as size markers.

PCR ribotyping

PCR ribotyping was conducted by the method of Ryley et al. (1995) using primers from a conserved spacer region between the 16 s and 23 s Gram-negative bacterial genes. PCR products were analysed by agarose gel electrophoresis on a high resolution gel containing ‘Infinity’ agarose extender (Appligene Oncor, Durham, UK) to give a gel approximately equivalent to 3% agarose. This method was previously used to type B. cepacia isolates from CF patients in Denmark ( Ryley et al. 1996 ), and gives more rapid results than with Taq1 digestion of PCR products followed by electrophoresis. Taq DNA polymerase (at 5000 units ml−1) was supplied by Appligene Oncor. Primers were supplied by MWG Biotech (Milton Keynes, UK). Primer 1 (C1): 5′-TTG TAC ACA CCG CCG GTC A-3′ was supplied at 39·3 pmol ml−1. Primer 2 (C2): 5′-GCT ACC TTA GAT GTT TCA GTT C-3′ was supplied at 54·1 pmol ml−1. For each set of reactions, a control was set up using distilled water in place of template DNA. PCR markers (Sigma) were included in each gel.

Macrorestriction analysis

The macrorestriction analysis (genomic fingerprinting) method used was developed from that of Grouthes et al. (1988) for the analysis of Ps. aeruginosa. Burkholderia cepacia was harvested from overnight cultures on nutrient agar using 5 ml SE buffer (0·075 mol l−1 NaCl, 0·025 mol l−1 EDTA). The cell suspension was drawn off with a pipette and turbidity adjusted to MacFarland standard three (approximately equivalent to 9 × 108 cells ml−1) with SE buffer; 500 μl of this suspension was mixed with 500 μl of 2% molten low gelling temperature agarose (Sigma Chemical Co.) maintained at 40 °C, pipetted into disposable plug moulds (BioRad) and allowed to set. Plugs were removed and twice treated for 24 h with lysis buffer ( n-lauroyl sarcosinate 1%, 0·5 mol l−1 EDTA, 500 μg ml−1 proteinase K, pH 9·5) at 56 °C. Plugs were then washed four times in TE buffer ( Sambrook et al. 1989 ). The resulting agarose-embedded genomic DNA could be stored at 4 °C for up to 2 weeks prior to use.

The embedded DNA was digested with restriction enzymes Xba I and Not I (Promega) using 50 units of enzyme per plug in 200 μl of the appropriate digestion buffer ( BioRad Laboratories 1992 ) for 14 h at 37 °C ( Sambrook et al. 1989 ).

Digested plugs were sealed in the wells of a 1% pulsed field agarose gel in 0·5 TBE buffer (Sigma) in a CHEF DR II apparatus (BioRad), and run for 21 h at 5 V cm−1 with initial pulse times of 5 s and final pulse times of 100 s in 0·5 TBE buffer at a temperature of 8 °C. Size markers of 225–2200 kbp and/or 0·1–200 kbp (Sigma), or Saccharomyces cerevisiae size markers (BioRad), were included on each gel. Gels were stained in ethidium bromide and destained in running water, then photographed under u.v. transillumination.

Results

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

In vitro maceration of onion

The degree of maceration of onion tissue was assessed as follows:

0 No maceration, discoloration or odour. Typified by sterile saline control.

1 No maceration, slight discoloration and ‘sour’ odour.

2 Patches of maceration and discoloration, ‘sour’ odour.

3 Maceration in majority of tissue. Yellow to yellow-brown discoloration. Strong ‘sour’ odour.

4 Severe maceration throughout tissue. Yellow-brown discoloration and very strong ‘sour’ odour. Typified by NCPPB 2993 (type strain and pathotype).

The levels of pathology caused by each isolate are shown in Table 1.

Table 1.  Plasmid content and sizes and onion macerating properties of Burkholderia cepacia isolates of clinical and environmental origin
IsolateSourcePlasmid content and size (kbp) Onion maceration (0–4 scale)
  • *

    ND, none detected.

NCPPB 2993Rotten onion Species type strain 2244
C1CF patient, Cardiff1341
C5CF patient, Cardiff1341
C11CF patient, Cardiff1340
C23CF patient, Cardiff139, 24, 131
C49CF patient, Cardiff2·1, 1·70
C51CF patient, CardiffND *0
C59CF patient, Cardiff200
C79CF patient, Cardiff901
C81CF patient, Cardiff400
C93CF patient, CardiffND1
C96CF patient, Cardiff109, 2·1, 1·70
C116CF patient, Cardiff144, 29·3, 2·1, 1·71
C187CF patient, Cardiff51, 4·4, 4·00
C190CF patient, CardiffND *1
C205CF patient, CardiffND *0
J543Clinical, Strasbourg901
J478Clinical, Strasbourg1521
A548CF patient, EdinburghND *2
A562CF patient, Edinburgh70, 4·53
C1858CF patient, DundeeND *1
C1860CF patient, Aberdeen185, 3·40
PW1Stream waterND *1
PW2Stream water132, 51, 31, 16, 9, 8, 3·6, 1·13
PW3River water930
PW4River water311
PW5SoilND *0
PW6Stream water313
PW7Soil132, 51, 45, 11, 7, 6·2, 3·6, 2·50
PW8Rotten onionND *1
PW9Soil>2500
PW10Stream water16, 12, 7·10
PW11SoilND *3
PW12Reservoir waterND *0
PW13RhizosphereND *1
PW14River waterND *1
PW15RhizosphereND *0
PW16Stream waterND *0
PW17Reservoir waterND *3
PW18RhizosphereND *0
PW19RhizosphereND *2
PW20Stream waterND *0
PW21Reservoir waterND *0

Plasmid size and content

One or more plasmids were detected in 16 out of 21 B. cepacia CF isolates (76%), and seven out of 21 environmental isolates (33%). Plasmids detected and their sizes are shown in Table 1.

Antibiotic susceptibility

All B. cepacia isolates from CF patients and the environment were found to be resistant to penicillin G, amoxicillin, erythro- mycin and novobiocin; 76% of environmental isolates (16 of 21) and 90% of CF isolates (19 of 21) were also found to be resistant to tetracycline. Resistance levels to piperacillin, gentamicin, ceftazidime and sulphamethoxazole varied more between the environmental and CF isolates and are shown in Table 2. The MIC values for these antibiotics were generally higher amongst the CF isolates than amongst environmental isolates.

Table 2.  Antibiotic resistance profiles and MIC range of Burkholderia cepacia isolates
CeftazidimeGentamicinPiperacillinSulphamethoxazole
Source of isolate Resistant isolates (%) MIC range μg ml−1Resistant isolates (%) MIC range μg ml−1Resistant isolates (%) MIC range μg ml−1Resistant isolates (%) MIC range μg ml−1
Environment141–32291–25601–4704–256
CF patients504–328132–>512801–256134–>512

PCR ribotyping

Different patterns of PCR products were found for all 21 environmental isolates, with between two and eight bands ranging in size from 700 to 2500 bp occurring. This indicated 21 ribotypes that differed from all the CF isolates tested and also the species type strain ( Fig. 1 illustrates the range of results). All the CF isolates from the Cardiff centre, with the exception of C93, gave an identical or near identical pattern with a band at around 800 bp and two other bands at around 2000 and 2500 bp. The same pattern of products was found to occur with B. cepacia isolates from the Strasbourg (J543) and Dundee (C1858) CF centres. The other isolates from these centres differed in pattern from the Cardiff CF isolates, all the environmental isolates and the type strain NCPPB 2993 (ACTC 25416).

image

Figure 1. PCR ribotyping of Burkholderia cepacia strains. Composite photograph, lanes numbered left to right. Lanes 1 and 20 are size markers; lanes 2–13 are environmental isolates; lanes 14–19 are CF isolates

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Macrorestriction analysis

Following digestion with Xba I, around 40 fragments of 1–200 kbp were found by PFGE. Each of the 21 environmental B. cepacia isolates produced a unique restriction pattern that differed from all the patterns obtained with isolates from CF patients ( Fig. 2). All Cardiff CF B. cepacia isolates, again with the exception of C93, produced patterns that were identical or near identical. The Strasbourg CF isolate J543 and the Scottish CF isolate C1858 also produced this digest pattern. Digestion with Not I produced the same similarities and differences between isolates.

image

Figure 2. Macrorestriction analysis of Burkholderia cepacia strains. Composite photograph, lanes numbered left to right. Lanes 18 and 19 are size markers; lanes 1–9 are CF isolates; lanes 10–17 are environmental isolates

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Discussion

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

All environmental isolates tested in this study were found to differ genetically from each other, indicating that there is great genetic variability in the environmental population of B. cepacia in South Wales. High levels of variability have previously been described in environmental populations of B. cepacia ( Wise et al. 1996 ). Sixty-five unique electropherotypes (ETs) were found by multilocus enzyme electrophoresis in 217 B. cepacia isolates collected from a 5 km stretch of stream over a period of 32 d. It is also notable that genetic differences were found in isolates obtained from sites close together. Examples of this are PW1, PW2 and PW6, isolated over a 100 m stretch of stream, and PW5 and PW7, isolated from soil samples taken a few metres apart. The 12 environmental isolates from botanical gardens described by Butler et al. (1995) gave 11 different profiles by macrorestriction analysis, though it was notable that isolates from the same source differed in profile. This evidence strongly indicates high genetic variability amongst environmental populations of B. cepacia. More detailed genetic and phenotypic investigation, such as the use of fatty acid methyl ester analysis (FAME), would allow the genomovar status of the isolates obtained from the environment in this study to be determined.

No identity was found between the environmental B. cepacia isolates from South Wales and the B. cepacia isolates from patients attending the Cardiff CF centre, indicating that the environmental isolates differ genetically from those infecting CF patients. The environmental isolates were also found to differ from those obtained from other CF centres, from the species type strain NCPPB 2993 (ACTC 25416), and also from other environmental isolates obtained from the National Collection of Industrial and Marine Bacteria (NCIMB) and the National Collection of Plant Pathogenic Bacteria (NCPPB). The differences would suggest that environmental B. cepacia isolates may form a separate population to those infecting CF patients.

With the techniques used in this study, identity between all but one of the Cardiff CF isolates was shown. These isolates can be further subdivided by Taq I digestion of the PCR products ( Ryley et al. 1995 ). Nevertheless, the identity shown in this study between these strains, and colleagues from Scotland and Strasbourg, both by PCR ribotyping and macrorestriction analysis, would indicate that they are closely related if not identical. The presence of a common strain amongst these centres would indicate both a common initial source, and strong evidence of person-to-person transmission ( Govan et al. 1996 ).

Phenotypic differences in antibiotic resistance and the ability to cause onion maceration both between CF and environmental isolates, and within the populations, were also observed ( Tables 1 and 2). Levels of antibiotic resistance were generally higher amongst CF isolates, as found by Butler et al. (1995) . However, it is not clear whether such differences are fundamentally related to the biology of the strains themselves, or are due to the development of acquired resistance amongst the CF isolates which will have received considerable exposure to such drugs. Maceration of onion tissue was more common and generally more severe by environmental isolates, but was not exclusively caused by these isolates.

Plasmids were found in 55% of B. cepacia isolates, though they were more commonly found amongst the CF isolates (76% of isolates) than in environmental isolates (33% of isolates). Plasmids in excess of 100 kbp were harboured by both clinical and environmental isolates. Lennon & DeCicco (1991) suggested that large plasmids were associated with clinical isolates and high levels of antibiotic resistance but in this study, there was no evidence of large plasmids being more frequently harboured in CF isolates; indeed, many of the largest plasmids were found in environmental isolates. Strains which appeared identical by ribotyping and genomic fingerprinting were nevertheless found to differ in their plasmid content, e.g. the Cardiff CF isolates. The transfer, acquisition and assimilation of plasmid-borne genes is extensively documented, and the fact that closely related strains differ in their plasmid content could reflect differences in selective pressure, such as antibiotic treatment regimes, and differences in the transferable plasmid-bearing lung microflora of other species in CF patients. It is not known if the plasmids are sufficiently stable to be an aid in strain identification; the plasmids in our collection are as yet cryptic.

Three main points emerge from this study. First, the environmental population is genetically very diverse. There is also considerable phenotypic diversity amongst these strains. Secondly, the clinical population of B. cepacia is genetically much more homogeneous and is distinct from the environmental population. The final point that can be made is that the variation both between and within environmental and clinical groups may be the result of B. cepacia being a complex formed by five genomic species, with the clinical population being predominantly formed by B. cepacia genomovar III, B. vietnamensis and B. multivorans, while many environmental isolates, particularly phytopathogenic isolates, fall into genomovar I ( Govan et al. 1996 ; Revets et al. 1996 ; Vandamme et al. 1997 ). However, it would appear that such a grouping is not rigid, with genomovar I being described in CF patients and other clinical sources and some strains predominantly found in CF patients also occurring in soil ( Vandamme et al. 1997 ). Although this study suggests that environmentally occurring B. cepacia forms a separate population to that infecting CF patients, it is limited to only 21 environmental isolates and 21 clinical isolates. A more extensive study, particularly including a wider range of less related CF isolates, would be needed to confirm the validity of these findings. The apparent acquisition of B. cepacia from the environment by CF patients ( Cazzola et al. 1996 ), the close genetic relationship between the species type strain and the ET12 CF epidemic strain ( Johnson et al. 1994 ), the ability of some CF strains to cause onion maceration, and the overlap of genomic species between the environment and CF patients ( Vandamme et al. 1997 ), suggest that a greater understanding of relationships between clinical and environmental B. cepacia is needed. The occurrence of genomic species infecting CF patients in the environment and the ability of primarily environmental genomovars to infect man needs further investigation. The use of molecular typing techniques such as PCR and PFGE, together with animal models such as the cftrm1HGU mouse which develops lung disease when challenged with B. cepacia ( Davidson et al. 1995 ), may allow both a greater understanding of the relationship between clinical and environmental isolates of B. cepacia and their potential pathogenicity. Such information may answer the question of whether environmentally occurring B. cepacia pose a risk of infection to CF patients.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

The authors wish to thank Mr Alan Paull, PHLS, Cardiff and Professor J.R.W. Govan, University of Edinburgh for the kind gift of the clinical isolates of B. cepacia, Dr Henry Ryley, University of Wales College of Medicine for help and advice in conducting the PCR ribotyping, and the Faculty of Community Health Sciences, UWIC for supporting the work.

References

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
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Footnotes
  • *

    Present address: Institute for Animal Health, Compton Laboratory, Compton, Newbury, RG20 7NN, UK.