Vol. 57, No. 8
APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Aug. 1991, p. 2345-2350
0099-2240/91/082345-06$02.00/0 Copyright © 1991, American Society for Microbiology
Plasmids of Pseudomonas cepacia Strains of Diverse Origins EILEEN LENNON AND B. T. DECICCO* Institute for Biomolecular Studies, Department of Biology, The Catholic University
of America, Washington, D.C. 20064 Received 28 February 1991/Accepted 2 June 1991
Thirty-seven strains of Pseudomonas cepacia from clinical, pharmaceutical-industrial, and environmental origins were analyzed for the presence of plasmid DNA by a modification of the rapid alkaline extraction method of Birnboim (H. C. Birnboim, Methods Enzymol. 100:243-255, 1983). Plasmids were present in 31 strains (84%) from all sources, with no one source showing less than 75% plasmid carriage among its strains. The plasmid profiles indicated that the presence of large plasmids (146 to 222 kb) was the norm. Those strains with greater antibiotic resistance were mainly in the clinical and pharmaceutical groups and carried large plasmids (222 kb) that appeared essentially identical by restriction digest analysis. The ability for conjugative transfer was shown with the broad-host-range plasmid R751 carrying the gene for resistance to trimethoprim, one of the few antimicrobial agents effective against P. cepacia. The plasmid was transferred from Pseudomonas aeruginosa to P. cepacia strains as well as from P. cepacia transconjugants to other P. cepacia strains.
Although originally described as a phytopathogen (3), Pseudomonas cepacia is now best characterized by extreme resistance to antimicrobial agents (8), metabolic diversity (22), and the ability to survive and grow in purified waters (4). These factors have led to the emergence of P. cepacia as a problem organism in health care product contamination (6, 7, 21), nosocomial infections (5, 12, 18), and recalcitrant infections of cystic fibrosis patients (19, 23, 24). There have been several reports describing plasmids from P. cepacia isolates of clinical or plant origin. Gonzalez and Vidaver compared strains of clinical and plant origin for bacteriocin production, onion maceration, and plasmid content (11). McKevitt and Woods studied clinical isolates from cystic fibrosis patients for the presence of virulence factors and plasmids; only 11 of 48 strains in their study possessed one or more plasmids (19). Certain strains of P. cepacia have been noted to use penicillin or octopine as sole carbon and energy sources; however, no correlation to the presence of plasmid DNA was found (1, 19a). Only two groups have assigned possible resistance traits to plasmids in P. cepacia (13, 25). In general, the plasmids in the species are considered cryptic. More-extensive analyses of plasmids of P. cepacia have been hindered by problems in extraction by standard methods. Both Hirai et al. (13) and Williams et al. (25) reported difficulties in plasmid isolation. Williams et al. (26) suggested that nucleases active in EDTA or sodium dodecyl sulfate (SDS) at the concentrations used in plasmid extraction procedures were responsible for irreproducible plasmid profiles. In our initial experiments we also encountered difficulties in plasmid isolation from many of the strains included in this study. Selection and modification of some commonly employed extraction methods coupled with optimization of growth conditions led to a procedure that yielded consistent, discrete results with all 37 strains of P. cepacia included in this study. The employed P. cepacia strains included clinical isolates from human infection, environmental isolates from soil and onions, and pharmaceutical isolates from health care prod*
ucts and manufacturing sites covering a wide geographic
the presence of plasmid DNA conjunction with comparative restriction digest analyses of similarly sized plasmids to gain information on the molecular relationships among these strains of diverse origins. Conjugative transfer among P. cepacia strains and between Pseudomonas aeruginosa and P. cepacia was demonstrated with a broad-host-range plasmid encoding resistance to trimethoprim. This antimicrobial agent is one of the few active against P. cepacia. The experimental genetic exchange underscores the potential for the exchange of DNA in nature that could extend further the resistance phenotype or metabolic diversity of P. cepacia. range. An in-depth survey for among the strains was done in
MATERIALS AND METHODS
Bacterial strains. A list of the P. cepacia strains used in this study is given in Table 1. The strains are categorized by source, i.e., clinical, environmental, or industrial-pharmaceutical product isolate. The identification of all cultures was verified by either the N/F system (Flow Laboratories Inc., Roslyn, N.Y.) or the Rapid NFT system (DMS Laboratories Inc., Plainview, N.Y.). Maintenance and cultivation of bacteria. All cultures were stored at -20°C in Trypticase soy broth (BBL Microbiology Systems, Cockeysville, Md.) containing 20% glycerol. Working stock cultures were maintained at room temperature on pyruvate maintenance medium (10) which contained, per liter of deionized water, 3 g of sodium pyruvate, 0.2 g of MgSO4, 1 g of (NH4)2SO4, 0.857 g of K2HPO4, 4.77 g of KH2PO4, 0.02 g of phenol red, 0.01 g of Fe(NH4)2(SO4)2 6H20, and 15 g of agar. Cultures were grown in either 0.1% Trypticase soy broth or modified L broth, which contained (per liter) 10 g of Trypticase (BBL), 5 g of yeast extract (Difco, Detroit, Mich.), and 5 g of NaCl. Plasmid isolation. Plasmid DNA was isolated by a method based on the rapid alkaline extraction method of Birnboim (la). The isolated plasmid DNA was used for determination of plasmid profiles, transformation studies, and restriction enzyme digests. The method was modified at several steps: test organisms were not grown to saturation (instead, cultures were grown
Corresponding author. 2345
APPL. ENVIRON. MICROBIOL.
LENNON AND DECICCO
2346
TABLE 1. Strains, sources, and plasmid profiles
Clinical C-175 C-393 B5912 CL-2212 CL-2213 CL-2214 CL-2215 CL-2216 CL-2218 CL-2219 CL-2220 CL-2221 CL-2222 WR#2 WR#3 WR#3-C4 Environmental 4G9
Source or location
Origin
Type and strain
Plasmid size(s) (kb)
Gums Gums Wound Human infection Human infection Human infection Human infection Human infection Human infection Human infection Human infection Human infection Human infection Human infection Human infection Antibiotic-sensitive clone of WR#3
J. Bonalski' J. Bonalski C Krueger' Merckc Merck Merck Merck Merck Merck Merck Merck Merck Merck R. Almazond R. Almazon J. Keevene
Soil Onion Soil Onion Onion Onion Forest soil Onion
J. A. Williams C. Krueger C. Krueger C. Krueger C. Krueger C. Krueger C. Krueger
48, 2.7 208, 48
ATCCf
212
Manufacturing site Manufacturing site Aqueous nasal spray Aqueous nasal spray Skin cream Skin cream Anti-inflammatory topical cream Anti-inflammatory topical cream Anti-inflammatory topical cream Antibiotic solution Contact lens solution Contact lens solution Hair conditioner
New Jersey New Jersey Puerto Rico Puerto Rico New Jersey New Jersey New Jersey New Jersey New Jersey California New Jersey New Jersey California
222, 222, 222, 222, 212 212 212, 212, 222, 150
7434 B7 64-22 30 10856 17759 25416 Pharmaceutical-industrial EL-1 EL-2 EL-D6 EL-S6
EL-Si EL-S2 EL-S3 EL-S4 EL-S5 EL-3 EL-D4 EL-D5 EL-D7
146 157 60 157, 57, 6.1, 3.6 222 222 222 6.1, 3.7, 2.7 222 222, 60 6.1, 3.7, 2.7 222 157 157
208 208 212
57 45 57 57 3.2, 2.7 3.2, 2.7 57
222
a Ciba-Geigy Corp., Summit, N.J. bStuart Pharmaceuticals, Wilmington, Del.
c Clinical Microbiology Laboratory, Merck & Co., Rahway, N.J. d Walter Reed Army Hospital, Washington, D.C. e The Catholic University of America, Washington, D.C. f American Type Culture Collection, Rockville, Md.
overnight at 30°C [with slow shaking] to an optical density at 540 nm of between 0.3 and 0.7), duplicate 1.5-ml samples instead of 0.5-ml samples were extracted, and a phenolchloroform extraction step was added after the first alcohol precipitation. This extraction procedure was repeated a second time with DNA preparations from those strains that produced a smear on gel electrophoresis, obscuring the plasmid band(s). The final pellet was dissolved in 40 >1. of TE buffer (10 mM Tris-HCl and 1 mM EDTA, pH 7.5). A 15- to 18-,ul aliquot was routinely used for separation of plasmid DNA by gel electrophoresis. Plasmid DNA for restriction enzyme digestion studies was obtained by scaling up this
procedure. Shigella flexneri 24570 and Escherichia coli V517 (obtained from D. Kopecko) were used as the source of size reference plasmids (15, 16). S. flexneri 24570 harbored plasmid species of 216.4, 157.2, 3.95, and 3.0 kb, and E. coli V517 harbored plasmid species of 54, 7.2, 5.6, 5.1, 3.9, 3.0,
2.7, and 2.1 kb. The method of isolation was as described by Birnboim (la) except for the addition of a phenol-chloroform extraction step. DNA characterization. DNA for both plasmid profiles and restriction digest analyses was separated on 0.7% agarose gels in TBE buffer (89 mM Tris-borate, 89 mM boric acid, 2 mM EDTA). Restriction enzyme digests were done according to standard methods (17). Antimicrobial susceptibility tests. Antimicrobial susceptibility was assessed by an agar diffusion method. The antimicrobial agents tested and concentrations per disc were as follows: carbenicillin, 100 ,ug; chloramphenicol, 30 pxg; erythromycin, 15 ,ug; nalidixic acid, 30 ,ug; novobiocin, 30 ,ug; rifampin, 5 ,ug; sulfamethoxazole-trimethoprim, 25 ,ug; tetracycline, 30 ,ug; amikacin, 30 jig; gentamicin, 10 ,ug; kanamycin, 50 ,ug; neomycin, 30 jig; tobramycin, 10 ,ug; streptomycin, 30 jig; spectinomycin, 30 ,ug; and polymyxin B, 300 U.
PLASMIDS OF P. CEPACIA STRAINS OF DIVERSE ORIGINS
VOL. 57, 1991
TABLE 2. P. aeruginosa strains used in conjugation studies Strana
PU 21
Characterstic(s)
Plasmid
INC group
Resistance phenotypeb
ilv val leu Str Rif'
pMG-1
P-2
Rip 64
P-3
R751
P-1
Gm Sm Su Bor Hg Tet Cb Cm Gm Su Tm Hg Tp
PU 21
PAO 303
arg
I
2
2
A
A
It
7
A
2347
a
G. Jacoby kindly supplied these P. aeruginosa strains and plasmids. Phenotypic markers: resistance to borate (Bor), carbenicillin (Cb), chloramphenicol (Cm), gentamicin (Gm), mercuric ion (Hg), streptomycin (Sm), sulfonamides (Su), tobramycin (Tm), and trimethoprim (Tp). c Str and Rif, chromosomally resistant to streptomycin and rifampin. a
b
Conjugation studies. Descriptions of the P. aeruginosa strains and the plasmids employed for conjugation studies are listed in Table 2. Conjugative transfer of plasmid DNA from P. aeruginosa to P. cepacia and from one P. cepacia strain, WR#3-C4, to other P. cepacia strains was achieved by the solid surface mating technique as described by Bradley et al. (2). The P. aeruginosa strains were sensitive to polymyxin B. For crosses between P. aeruginosa PA0303/R751 and P. cepacia strains, the transconjugantselective medium contained polymyxin B (300 U/ml) and trimethoprim (200 ,ug/ml). For crosses between P. aeruginosa PU 21/pMG-1 or PU 21/Rip 64 and P. cepacia strains, the selective medium contained HgCl2 (50 ,ug/ml) and polymyxin B (300 U/ml). For crosses between P. cepacia WR#3C4/R751 and other P. cepacia strains, the selective medium contained neomycin sulfate (50 jxg/ml) and trimethoprim (200 ,ug/ml). Maintenance of the R751 plasmid in P. cepacia transconjugants was determined by periodic culture analysis using gel electrophoresis and growth on trimethoprim agar. RESULTS Plasmid profile. Table 1 lists the incidence and sizes of plasmids found among the strains. Plasmids were isolated from 84% of the strains tested, and the incidence of plasmid carriage was high in all groups (clinical strains, 14 of 16; pharmaceutical group, 11 of 13; environmental strains, 6 of 8). A common feature of the species was the presence of large plasmids. Of the 31 strains with plasmids, 26 had one of at least 145 kb in size. Figure 1 depicts all of the plasmid sizes that occurred among the clinical strains, which harbored from one to four plasmids ranging in size from 222 to 2.7 kb. The pharmaceutical-industrial group was next in heterogeneity of plasmids carried, although a number of these strains had similar profiles. The environmental strains had little variation in plasmid size, with five of six strains harboring plasmids of 208 to 212 kb. Strain 4G9 had smaller plasmids of 48 and 2.7 kb. A plasmid of about 48 kb also occurred in environmental strain 7434. Restriction endonuclease studies. Restriction digests of similarly sized plasmids were compared among the three groups. The environmental isolates had a number of strains with plasmids ranging in size from 208 to 212 kb, resembling similarly sized plasmids in pharmaceutical strains EL-Si, EL-S2, EL-S3, and EL-S4. EL-S3 and EL-S4 had two small plasmids that were not cut in any of the digest studies. The PstI digest (Fig. 2) shows the close relationship among the four environmental strains. The digest of strain 64-22 (lane 2, Fig. 2) had the most dissimilar pattern among the environmental class, but its close relationship to the other three strains can be discerned. The PstI digest of the 212-kb
FIG. 1. Agarose gel electrophoresis of plasmids isolated from clinical strains of P. cepacia. Lane 1, strain CL-2220; lane 2, CL-2219; lanes 3 and 9, S.flexneri molecular size markers (216, 157, 3.9, and 3.0 kb); lane 4, C-175; lane 5, WR#3; lane 6, CL-2213; lane 7, CL-2218; lane 8, CL-2212.
plasmids from the product (pharmaceutical) isolates (lanes 6 and 7, Fig. 2) resulted in a pattern with only a minimal similarity to the environmental group; however, the two pharmaceutical isolates showed patterns virtually identical to each other (Fig. 2) as well as to those of the other two pharmaceutical strains (EL-Si and EL-S4; data not shown). Digestion with PvuII yielded the same relationships among the strains (data not shown). The PstI digests of the 157-kb plasmids from clinical strains C-393, WR#3, and WR#3-C4 are compared with that of the 146-kb plasmid from C-175 in Fig. 3. WR#3 and the antibiotic-sensitive clone WR#3-C4 derived from it produced indistinguishable digest patterns (lanes 1 and 2, Fig. 3), whereas those for the plasmids from C-175 and C-393 were quite distinct from each other as well as from those for plasmids from the other strains (lanes 3 and 4, Fig. 3). EcoRI and PvuII restriction digests of 222-kb plasmids that occurred in 12 of the strains (Table 1) were also compared. Six of these strains were of clinical origin, and six were of pharmaceutical origin. All 12 digests made with each restriction enzyme showed similar, if not identical, patterns (data not shown). Antimicrobial susceptibility. As is typical of P. cepacia, all strains were resistant to polymyxin B. Normally, this species is sensitive to sulfamethoxazole-trimethoprim and chloramphenicol. Strain EL-1 showed a high degree of resistance to most antibiotics tested and was the only strain resistant to sulfamethoxazole-trimethoprim. Several other strains (C-175, EL-2, EL-D6, EL-S5, EL-S6, EL-D4, and EL-D5) in addition to EL-1 were resistant to chloramphenicol. The strains were generally sensitive to nalidixic acid and novobiocin, but these antimicrobial agents are not very useful clinically. Kanamycin had the most antibacterial
2348
APPL. ENVIRON. MICROBIOL.
LENNON AND DECICCO 1
FIG. 2. PstI
digest of plasmids in the size
2-
range from 208 to 212
product isolates (lanes 6 and 7). Lane 1, PsuI digest of X DNA molecular weight markers (14.0, 11.5, 5.1, 4.7, 4.5, 2.8, 2.4, 2.1, and 2.0); lane 2, 64-22; lane 3, 30; lane 4, 25416; lane 5, 10856; lane 6, EL-S2; lane 7, EL-S3 (A, uncut small plasmid). kb from environmental strains (lanes 2 to 5) and
activity of all the aminoglycosides. Although minimally effective against the clinical isolates, it was active against all of the environmental strains and all but four of the pharmaceutical isolates (EL-D6, EL-S6, EL-S5, and EL-D5). Two strains, WR#3-C4 and EL-D7, were sensitive to most antibiotics. WR#3-C4, sensitive
clone
Keeven;
EL-D7
as
noted in Table 1,
of strain was
a
WR#3
that
was
was
product isolate
an
antibiotic-
isolated
that
by J.
lacked
any
plasmid. Conjugation. Plasmid R751 was transferred from P. aeruginosa to five P. cepacia strains (Table 3). The P. cepacia recipient strains were specifically chosen to be representative of the different groups of origin. Strains EL-D7, WR#3-C4, and 7434 were all sensitive to neomycin sulfate; therefore, when each acquired the plasmid R751, it could be used as a plasmid donor to other P. cepacia strains which were resistant to neomycin. Transconjugants were selected by acquisition of trimethoprim resistance. The transconjugant strain P. cepacia WR#3-C4/R751 was used as the donor strain for the transfer of plasmid R751 to strains 17759, C-175, and CL-2212. WR#3-C4 was chosen because it could be counterselected and because it had a resident plasmid of a discernibly different size from R751, which might be mobilized; however, the 157-kb plasmid was not transferred to any of the three strains that acquired R751. The transfer frequencies for these crosses are given in Table 3. EcoRI digests of plasmid DNA from the intraspecies transconjugant 17759 and from the interspecies transconjugant EL-D7 were compared with that of R751 from P. aeruginosa (Fig. 4). The
FIG. 3. PstI digest of the 157-kb plasmid from strains WR#3, WR#3-C4, and C-393 (lanes 1, 2, and 3, respectively) compared with that of the 147-kb plasmid from strain C-175 (lane 4). Lane 5, PstI digest of X DNA (14.0, 11.5, 5.1, 4.7, 4.5, 2.8, 2.4, 2.1, 2.0, and 1.7 kb).
restriction fragment patterns were indistinguishable in all cases. The transconjugants acquired both the trimethoprim resistance trait characteristic of plasmid R751 and a plasmid identical in size and in EcoRI restriction digest profile. There was no observed conjugation between P. aeruginosa harboring either pMG-1 or Rip 64 and P. cepacia.
demonstrable
DISCUSSION As had been reported by others (13, 25, 26), we also encountered difficulties in plasmid isolation from P. cepacia TABLE 3. Conjugative transfer for R751
Recipient
Transfer
Donor
P. cepacia strain
frequencya
P. aeruginosa PAO 303/R751
EL-S6 C-175 WR#3-C4 EL-D7 7434 17759 CL-2212 C-175
7 x 1 x 8 x 4 x 2 x 3 x 2 x 2 x
P. cepacia WR#3-C4/R751
10-4 10-4 10-7
10-7 10-7 10-5 10-6 10-6
a Calculated as the number of transconjugants per donor CFU per milliliter at the beginning of mating (2).
VOL. 57, 1991
PLASMIDS OF P. CEPACIA STRAINS OF DIVERSE ORIGINS
FIG. 4. EcoRI digest of R751. Lane 1, interspecies transconjugant EL-D7; lane 2, intraspecies transconjugant 17759; lane 3, P.
aeruginosa PAO 303; lane 4, 7.4, 5.8, 4.9, and 3.5 kb).
DNA molecular size markers (24.8,
using standard methodology. Initially the Kado and Liiu (14) procedure was used for plasmid extraction, but the results were not consistent. The rapid alkaline extraction procedure of Bimboim (la) was used with more success. The plasmids from some strains were isolated with little modification to the method, while others consistently remained recalcitrant and smeared on the gels. The procedure was therefore modified by the addition of a phenol-chloroform extraction step. In addition, several minor changes were made, including increasing the concentration of lysozyme and lengthening the time of treatment with alkaline SDS. Besides extraction procedure changes it was observed that better results were obtained if growth was not to saturation. This was suggested because plasmid isolation from slow-growing strains (e.g., EL-S6 and EL-D6) always yielded discrete bands whereas some of the faster-growing strains gave less-reproducible results. Therefore, the method was adjusted by extracting from overnight cultures with optical densities at 540 nm of
APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Aug. 1991, p. 2345-2350
0099-2240/91/082345-06$02.00/0 Copyright © 1991, American Society for Microbiology
Plasmids of Pseudomonas cepacia Strains of Diverse Origins EILEEN LENNON AND B. T. DECICCO* Institute for Biomolecular Studies, Department of Biology, The Catholic University
of America, Washington, D.C. 20064 Received 28 February 1991/Accepted 2 June 1991
Thirty-seven strains of Pseudomonas cepacia from clinical, pharmaceutical-industrial, and environmental origins were analyzed for the presence of plasmid DNA by a modification of the rapid alkaline extraction method of Birnboim (H. C. Birnboim, Methods Enzymol. 100:243-255, 1983). Plasmids were present in 31 strains (84%) from all sources, with no one source showing less than 75% plasmid carriage among its strains. The plasmid profiles indicated that the presence of large plasmids (146 to 222 kb) was the norm. Those strains with greater antibiotic resistance were mainly in the clinical and pharmaceutical groups and carried large plasmids (222 kb) that appeared essentially identical by restriction digest analysis. The ability for conjugative transfer was shown with the broad-host-range plasmid R751 carrying the gene for resistance to trimethoprim, one of the few antimicrobial agents effective against P. cepacia. The plasmid was transferred from Pseudomonas aeruginosa to P. cepacia strains as well as from P. cepacia transconjugants to other P. cepacia strains.
Although originally described as a phytopathogen (3), Pseudomonas cepacia is now best characterized by extreme resistance to antimicrobial agents (8), metabolic diversity (22), and the ability to survive and grow in purified waters (4). These factors have led to the emergence of P. cepacia as a problem organism in health care product contamination (6, 7, 21), nosocomial infections (5, 12, 18), and recalcitrant infections of cystic fibrosis patients (19, 23, 24). There have been several reports describing plasmids from P. cepacia isolates of clinical or plant origin. Gonzalez and Vidaver compared strains of clinical and plant origin for bacteriocin production, onion maceration, and plasmid content (11). McKevitt and Woods studied clinical isolates from cystic fibrosis patients for the presence of virulence factors and plasmids; only 11 of 48 strains in their study possessed one or more plasmids (19). Certain strains of P. cepacia have been noted to use penicillin or octopine as sole carbon and energy sources; however, no correlation to the presence of plasmid DNA was found (1, 19a). Only two groups have assigned possible resistance traits to plasmids in P. cepacia (13, 25). In general, the plasmids in the species are considered cryptic. More-extensive analyses of plasmids of P. cepacia have been hindered by problems in extraction by standard methods. Both Hirai et al. (13) and Williams et al. (25) reported difficulties in plasmid isolation. Williams et al. (26) suggested that nucleases active in EDTA or sodium dodecyl sulfate (SDS) at the concentrations used in plasmid extraction procedures were responsible for irreproducible plasmid profiles. In our initial experiments we also encountered difficulties in plasmid isolation from many of the strains included in this study. Selection and modification of some commonly employed extraction methods coupled with optimization of growth conditions led to a procedure that yielded consistent, discrete results with all 37 strains of P. cepacia included in this study. The employed P. cepacia strains included clinical isolates from human infection, environmental isolates from soil and onions, and pharmaceutical isolates from health care prod*
ucts and manufacturing sites covering a wide geographic
the presence of plasmid DNA conjunction with comparative restriction digest analyses of similarly sized plasmids to gain information on the molecular relationships among these strains of diverse origins. Conjugative transfer among P. cepacia strains and between Pseudomonas aeruginosa and P. cepacia was demonstrated with a broad-host-range plasmid encoding resistance to trimethoprim. This antimicrobial agent is one of the few active against P. cepacia. The experimental genetic exchange underscores the potential for the exchange of DNA in nature that could extend further the resistance phenotype or metabolic diversity of P. cepacia. range. An in-depth survey for among the strains was done in
MATERIALS AND METHODS
Bacterial strains. A list of the P. cepacia strains used in this study is given in Table 1. The strains are categorized by source, i.e., clinical, environmental, or industrial-pharmaceutical product isolate. The identification of all cultures was verified by either the N/F system (Flow Laboratories Inc., Roslyn, N.Y.) or the Rapid NFT system (DMS Laboratories Inc., Plainview, N.Y.). Maintenance and cultivation of bacteria. All cultures were stored at -20°C in Trypticase soy broth (BBL Microbiology Systems, Cockeysville, Md.) containing 20% glycerol. Working stock cultures were maintained at room temperature on pyruvate maintenance medium (10) which contained, per liter of deionized water, 3 g of sodium pyruvate, 0.2 g of MgSO4, 1 g of (NH4)2SO4, 0.857 g of K2HPO4, 4.77 g of KH2PO4, 0.02 g of phenol red, 0.01 g of Fe(NH4)2(SO4)2 6H20, and 15 g of agar. Cultures were grown in either 0.1% Trypticase soy broth or modified L broth, which contained (per liter) 10 g of Trypticase (BBL), 5 g of yeast extract (Difco, Detroit, Mich.), and 5 g of NaCl. Plasmid isolation. Plasmid DNA was isolated by a method based on the rapid alkaline extraction method of Birnboim (la). The isolated plasmid DNA was used for determination of plasmid profiles, transformation studies, and restriction enzyme digests. The method was modified at several steps: test organisms were not grown to saturation (instead, cultures were grown
Corresponding author. 2345
APPL. ENVIRON. MICROBIOL.
LENNON AND DECICCO
2346
TABLE 1. Strains, sources, and plasmid profiles
Clinical C-175 C-393 B5912 CL-2212 CL-2213 CL-2214 CL-2215 CL-2216 CL-2218 CL-2219 CL-2220 CL-2221 CL-2222 WR#2 WR#3 WR#3-C4 Environmental 4G9
Source or location
Origin
Type and strain
Plasmid size(s) (kb)
Gums Gums Wound Human infection Human infection Human infection Human infection Human infection Human infection Human infection Human infection Human infection Human infection Human infection Human infection Antibiotic-sensitive clone of WR#3
J. Bonalski' J. Bonalski C Krueger' Merckc Merck Merck Merck Merck Merck Merck Merck Merck Merck R. Almazond R. Almazon J. Keevene
Soil Onion Soil Onion Onion Onion Forest soil Onion
J. A. Williams C. Krueger C. Krueger C. Krueger C. Krueger C. Krueger C. Krueger
48, 2.7 208, 48
ATCCf
212
Manufacturing site Manufacturing site Aqueous nasal spray Aqueous nasal spray Skin cream Skin cream Anti-inflammatory topical cream Anti-inflammatory topical cream Anti-inflammatory topical cream Antibiotic solution Contact lens solution Contact lens solution Hair conditioner
New Jersey New Jersey Puerto Rico Puerto Rico New Jersey New Jersey New Jersey New Jersey New Jersey California New Jersey New Jersey California
222, 222, 222, 222, 212 212 212, 212, 222, 150
7434 B7 64-22 30 10856 17759 25416 Pharmaceutical-industrial EL-1 EL-2 EL-D6 EL-S6
EL-Si EL-S2 EL-S3 EL-S4 EL-S5 EL-3 EL-D4 EL-D5 EL-D7
146 157 60 157, 57, 6.1, 3.6 222 222 222 6.1, 3.7, 2.7 222 222, 60 6.1, 3.7, 2.7 222 157 157
208 208 212
57 45 57 57 3.2, 2.7 3.2, 2.7 57
222
a Ciba-Geigy Corp., Summit, N.J. bStuart Pharmaceuticals, Wilmington, Del.
c Clinical Microbiology Laboratory, Merck & Co., Rahway, N.J. d Walter Reed Army Hospital, Washington, D.C. e The Catholic University of America, Washington, D.C. f American Type Culture Collection, Rockville, Md.
overnight at 30°C [with slow shaking] to an optical density at 540 nm of between 0.3 and 0.7), duplicate 1.5-ml samples instead of 0.5-ml samples were extracted, and a phenolchloroform extraction step was added after the first alcohol precipitation. This extraction procedure was repeated a second time with DNA preparations from those strains that produced a smear on gel electrophoresis, obscuring the plasmid band(s). The final pellet was dissolved in 40 >1. of TE buffer (10 mM Tris-HCl and 1 mM EDTA, pH 7.5). A 15- to 18-,ul aliquot was routinely used for separation of plasmid DNA by gel electrophoresis. Plasmid DNA for restriction enzyme digestion studies was obtained by scaling up this
procedure. Shigella flexneri 24570 and Escherichia coli V517 (obtained from D. Kopecko) were used as the source of size reference plasmids (15, 16). S. flexneri 24570 harbored plasmid species of 216.4, 157.2, 3.95, and 3.0 kb, and E. coli V517 harbored plasmid species of 54, 7.2, 5.6, 5.1, 3.9, 3.0,
2.7, and 2.1 kb. The method of isolation was as described by Birnboim (la) except for the addition of a phenol-chloroform extraction step. DNA characterization. DNA for both plasmid profiles and restriction digest analyses was separated on 0.7% agarose gels in TBE buffer (89 mM Tris-borate, 89 mM boric acid, 2 mM EDTA). Restriction enzyme digests were done according to standard methods (17). Antimicrobial susceptibility tests. Antimicrobial susceptibility was assessed by an agar diffusion method. The antimicrobial agents tested and concentrations per disc were as follows: carbenicillin, 100 ,ug; chloramphenicol, 30 pxg; erythromycin, 15 ,ug; nalidixic acid, 30 ,ug; novobiocin, 30 ,ug; rifampin, 5 ,ug; sulfamethoxazole-trimethoprim, 25 ,ug; tetracycline, 30 ,ug; amikacin, 30 jig; gentamicin, 10 ,ug; kanamycin, 50 ,ug; neomycin, 30 jig; tobramycin, 10 ,ug; streptomycin, 30 jig; spectinomycin, 30 ,ug; and polymyxin B, 300 U.
PLASMIDS OF P. CEPACIA STRAINS OF DIVERSE ORIGINS
VOL. 57, 1991
TABLE 2. P. aeruginosa strains used in conjugation studies Strana
PU 21
Characterstic(s)
Plasmid
INC group
Resistance phenotypeb
ilv val leu Str Rif'
pMG-1
P-2
Rip 64
P-3
R751
P-1
Gm Sm Su Bor Hg Tet Cb Cm Gm Su Tm Hg Tp
PU 21
PAO 303
arg
I
2
2
A
A
It
7
A
2347
a
G. Jacoby kindly supplied these P. aeruginosa strains and plasmids. Phenotypic markers: resistance to borate (Bor), carbenicillin (Cb), chloramphenicol (Cm), gentamicin (Gm), mercuric ion (Hg), streptomycin (Sm), sulfonamides (Su), tobramycin (Tm), and trimethoprim (Tp). c Str and Rif, chromosomally resistant to streptomycin and rifampin. a
b
Conjugation studies. Descriptions of the P. aeruginosa strains and the plasmids employed for conjugation studies are listed in Table 2. Conjugative transfer of plasmid DNA from P. aeruginosa to P. cepacia and from one P. cepacia strain, WR#3-C4, to other P. cepacia strains was achieved by the solid surface mating technique as described by Bradley et al. (2). The P. aeruginosa strains were sensitive to polymyxin B. For crosses between P. aeruginosa PA0303/R751 and P. cepacia strains, the transconjugantselective medium contained polymyxin B (300 U/ml) and trimethoprim (200 ,ug/ml). For crosses between P. aeruginosa PU 21/pMG-1 or PU 21/Rip 64 and P. cepacia strains, the selective medium contained HgCl2 (50 ,ug/ml) and polymyxin B (300 U/ml). For crosses between P. cepacia WR#3C4/R751 and other P. cepacia strains, the selective medium contained neomycin sulfate (50 jxg/ml) and trimethoprim (200 ,ug/ml). Maintenance of the R751 plasmid in P. cepacia transconjugants was determined by periodic culture analysis using gel electrophoresis and growth on trimethoprim agar. RESULTS Plasmid profile. Table 1 lists the incidence and sizes of plasmids found among the strains. Plasmids were isolated from 84% of the strains tested, and the incidence of plasmid carriage was high in all groups (clinical strains, 14 of 16; pharmaceutical group, 11 of 13; environmental strains, 6 of 8). A common feature of the species was the presence of large plasmids. Of the 31 strains with plasmids, 26 had one of at least 145 kb in size. Figure 1 depicts all of the plasmid sizes that occurred among the clinical strains, which harbored from one to four plasmids ranging in size from 222 to 2.7 kb. The pharmaceutical-industrial group was next in heterogeneity of plasmids carried, although a number of these strains had similar profiles. The environmental strains had little variation in plasmid size, with five of six strains harboring plasmids of 208 to 212 kb. Strain 4G9 had smaller plasmids of 48 and 2.7 kb. A plasmid of about 48 kb also occurred in environmental strain 7434. Restriction endonuclease studies. Restriction digests of similarly sized plasmids were compared among the three groups. The environmental isolates had a number of strains with plasmids ranging in size from 208 to 212 kb, resembling similarly sized plasmids in pharmaceutical strains EL-Si, EL-S2, EL-S3, and EL-S4. EL-S3 and EL-S4 had two small plasmids that were not cut in any of the digest studies. The PstI digest (Fig. 2) shows the close relationship among the four environmental strains. The digest of strain 64-22 (lane 2, Fig. 2) had the most dissimilar pattern among the environmental class, but its close relationship to the other three strains can be discerned. The PstI digest of the 212-kb
FIG. 1. Agarose gel electrophoresis of plasmids isolated from clinical strains of P. cepacia. Lane 1, strain CL-2220; lane 2, CL-2219; lanes 3 and 9, S.flexneri molecular size markers (216, 157, 3.9, and 3.0 kb); lane 4, C-175; lane 5, WR#3; lane 6, CL-2213; lane 7, CL-2218; lane 8, CL-2212.
plasmids from the product (pharmaceutical) isolates (lanes 6 and 7, Fig. 2) resulted in a pattern with only a minimal similarity to the environmental group; however, the two pharmaceutical isolates showed patterns virtually identical to each other (Fig. 2) as well as to those of the other two pharmaceutical strains (EL-Si and EL-S4; data not shown). Digestion with PvuII yielded the same relationships among the strains (data not shown). The PstI digests of the 157-kb plasmids from clinical strains C-393, WR#3, and WR#3-C4 are compared with that of the 146-kb plasmid from C-175 in Fig. 3. WR#3 and the antibiotic-sensitive clone WR#3-C4 derived from it produced indistinguishable digest patterns (lanes 1 and 2, Fig. 3), whereas those for the plasmids from C-175 and C-393 were quite distinct from each other as well as from those for plasmids from the other strains (lanes 3 and 4, Fig. 3). EcoRI and PvuII restriction digests of 222-kb plasmids that occurred in 12 of the strains (Table 1) were also compared. Six of these strains were of clinical origin, and six were of pharmaceutical origin. All 12 digests made with each restriction enzyme showed similar, if not identical, patterns (data not shown). Antimicrobial susceptibility. As is typical of P. cepacia, all strains were resistant to polymyxin B. Normally, this species is sensitive to sulfamethoxazole-trimethoprim and chloramphenicol. Strain EL-1 showed a high degree of resistance to most antibiotics tested and was the only strain resistant to sulfamethoxazole-trimethoprim. Several other strains (C-175, EL-2, EL-D6, EL-S5, EL-S6, EL-D4, and EL-D5) in addition to EL-1 were resistant to chloramphenicol. The strains were generally sensitive to nalidixic acid and novobiocin, but these antimicrobial agents are not very useful clinically. Kanamycin had the most antibacterial
2348
APPL. ENVIRON. MICROBIOL.
LENNON AND DECICCO 1
FIG. 2. PstI
digest of plasmids in the size
2-
range from 208 to 212
product isolates (lanes 6 and 7). Lane 1, PsuI digest of X DNA molecular weight markers (14.0, 11.5, 5.1, 4.7, 4.5, 2.8, 2.4, 2.1, and 2.0); lane 2, 64-22; lane 3, 30; lane 4, 25416; lane 5, 10856; lane 6, EL-S2; lane 7, EL-S3 (A, uncut small plasmid). kb from environmental strains (lanes 2 to 5) and
activity of all the aminoglycosides. Although minimally effective against the clinical isolates, it was active against all of the environmental strains and all but four of the pharmaceutical isolates (EL-D6, EL-S6, EL-S5, and EL-D5). Two strains, WR#3-C4 and EL-D7, were sensitive to most antibiotics. WR#3-C4, sensitive
clone
Keeven;
EL-D7
as
noted in Table 1,
of strain was
a
WR#3
that
was
was
product isolate
an
antibiotic-
isolated
that
by J.
lacked
any
plasmid. Conjugation. Plasmid R751 was transferred from P. aeruginosa to five P. cepacia strains (Table 3). The P. cepacia recipient strains were specifically chosen to be representative of the different groups of origin. Strains EL-D7, WR#3-C4, and 7434 were all sensitive to neomycin sulfate; therefore, when each acquired the plasmid R751, it could be used as a plasmid donor to other P. cepacia strains which were resistant to neomycin. Transconjugants were selected by acquisition of trimethoprim resistance. The transconjugant strain P. cepacia WR#3-C4/R751 was used as the donor strain for the transfer of plasmid R751 to strains 17759, C-175, and CL-2212. WR#3-C4 was chosen because it could be counterselected and because it had a resident plasmid of a discernibly different size from R751, which might be mobilized; however, the 157-kb plasmid was not transferred to any of the three strains that acquired R751. The transfer frequencies for these crosses are given in Table 3. EcoRI digests of plasmid DNA from the intraspecies transconjugant 17759 and from the interspecies transconjugant EL-D7 were compared with that of R751 from P. aeruginosa (Fig. 4). The
FIG. 3. PstI digest of the 157-kb plasmid from strains WR#3, WR#3-C4, and C-393 (lanes 1, 2, and 3, respectively) compared with that of the 147-kb plasmid from strain C-175 (lane 4). Lane 5, PstI digest of X DNA (14.0, 11.5, 5.1, 4.7, 4.5, 2.8, 2.4, 2.1, 2.0, and 1.7 kb).
restriction fragment patterns were indistinguishable in all cases. The transconjugants acquired both the trimethoprim resistance trait characteristic of plasmid R751 and a plasmid identical in size and in EcoRI restriction digest profile. There was no observed conjugation between P. aeruginosa harboring either pMG-1 or Rip 64 and P. cepacia.
demonstrable
DISCUSSION As had been reported by others (13, 25, 26), we also encountered difficulties in plasmid isolation from P. cepacia TABLE 3. Conjugative transfer for R751
Recipient
Transfer
Donor
P. cepacia strain
frequencya
P. aeruginosa PAO 303/R751
EL-S6 C-175 WR#3-C4 EL-D7 7434 17759 CL-2212 C-175
7 x 1 x 8 x 4 x 2 x 3 x 2 x 2 x
P. cepacia WR#3-C4/R751
10-4 10-4 10-7
10-7 10-7 10-5 10-6 10-6
a Calculated as the number of transconjugants per donor CFU per milliliter at the beginning of mating (2).
VOL. 57, 1991
PLASMIDS OF P. CEPACIA STRAINS OF DIVERSE ORIGINS
FIG. 4. EcoRI digest of R751. Lane 1, interspecies transconjugant EL-D7; lane 2, intraspecies transconjugant 17759; lane 3, P.
aeruginosa PAO 303; lane 4, 7.4, 5.8, 4.9, and 3.5 kb).
DNA molecular size markers (24.8,
using standard methodology. Initially the Kado and Liiu (14) procedure was used for plasmid extraction, but the results were not consistent. The rapid alkaline extraction procedure of Bimboim (la) was used with more success. The plasmids from some strains were isolated with little modification to the method, while others consistently remained recalcitrant and smeared on the gels. The procedure was therefore modified by the addition of a phenol-chloroform extraction step. In addition, several minor changes were made, including increasing the concentration of lysozyme and lengthening the time of treatment with alkaline SDS. Besides extraction procedure changes it was observed that better results were obtained if growth was not to saturation. This was suggested because plasmid isolation from slow-growing strains (e.g., EL-S6 and EL-D6) always yielded discrete bands whereas some of the faster-growing strains gave less-reproducible results. Therefore, the method was adjusted by extracting from overnight cultures with optical densities at 540 nm of
