ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Oct. 1976, Copyright X) 1976 American Society for Microbiology
p.
Vol. 10, No. 4
573-578
Printed in U.S.A.
Classification of R Plasmids by Incompatibility Pseudomonas aeruginosa H. SAGAI,* K. HASUDA, S. IYOBE, L. E. BRYAN, B. W. HOLLOWAY,
AND
in
S. MITSUHASHI
Department of Microbiology, School of Medicine, Gunma University, Maebashi, Japan*; Department of Medical Bacteriology, University of Alberta, Edmonton, Canada; and Department of Genetics, Monash University, Clayton, Victoria 3168, Australia Received for publication 10 March 1976
R plasmids identified in Pseudomonas aeruginosa strains isolated in our laboratories were classified by their incompatibility characters by using intraspecies conjugation in P. aeruginosa. The fertility factor, FP2, was compatible with R plasmids belonging to all groups, and was classified in a new group. From these data, the plasmids examined have been placed into seven or eight incompatibility groups. R plasmids in Enterobacteriaceae were originally classified by their fA character (18) or type of sex pili (14). More recently, the classification of R plasmids based on incompatibility has been generally used (4, 7), resulting in the recognition of more than 20 groups in the Escherichia coli system (9). In Pseudomonas aeruginosa strains, many R plasmids have been isolated (1, 2, 11-13, 15, 16), but almost all have been found to be either conjugally nontransferable or transferable at very low frequency to E. coli (1, 11, 16); therefore they cannot readily be classified in this system. Those few that are conjugally transferable to E. coli have been classified as P group (7) or C group (6, 13). Bryan et al. (1, 2, 17) proposed an incompatibility classification of plasmids detected in P. aeruginosa based on intraspecies conjugation in Pseudomonas and classified R plasmids into four incompatibility groups, P-1, P-2, P-3, and P-4. This paper deals with the classification of additional R plasmids from P. aeruginosa by incompatibility testing, using intraspecies conjugation in Pseudomonas. MATERIALS AND METHODS Bacterial strains. Two strains of P. aeruginosa, ML4262 (trp- his- ilv- met- rif ) (rifr indicates rifampin resistant) and ML4600 (trp- his-), which are isogenic except for the characters given, and two independently isolated strains, P. aeruginosa 280 (rif' met-) (1) and P. aeruginosa 5265 (1), were used as host strains for the R plasmids. R plasmids. R plasmids and their resistance markers are shown in Table 1. Drugs. Tetracycline (Tc), chloramphenicol (Cm), streptomycin (Sm), and sulfanilamide (Su) were kindly supplied by Japan Lederle Co., Sankyo Seiyaku Co., Kyowa Hakko Co., and the Dainihon Seiyaku Co., respectively. Carbenicillin (Cb), lividomycin (Lv), rifampin (Rp), and gentamicin C com-
plex (Gm) were supplied by the Fujisawa Seiyaku Co., Kowa Co., Daiichi Seiyaku Co., and Shionogi Co., respectively. Mercuric chloride (Hg; Wako Jyunyaku) was obtained commercially. Media. Nutrient broth and nutrient agar were used routinely. Nutrient broth consisted of 10 g of beef extract, 10 g of peptone, and 3 g of NaCl in 1,000 ml of distilled water. Nutrient agar consisted of nutrient broth supplemented with 1.5% agar. The transconjugants that had acquired drug resistance were selected on nutrient agar containing the appropriate selective drug together with Rp. For the assay of Su resistance or the selection of Su-resistant transconjugants, semisynthetic medium was used, which consisted of 1,000 ml of medium A (8) to which 2 g of Casamino Acids, 10 mg of tryptophan, 2 mg of nicotinic acid, 10 mg of thiamine, 2 g of glucose, and 15 g of agar were added. Medium A consisted of 8 g of Na2HPO4 * 12H20, 2 g of KH2PO4, 0.4 g of sodium citrate, 0.1 g of MgSO4 * 7H20, and 1.0 g of (NH4)2SO4 in 1,000 ml of distilled water. Incompatibility experiments. Two milliliters of a culture of recipient cells (ML4262), either carrying or not carrying an R plasmid, and 0.5 ml of donor culture (ML4600 R+) were mixed at early stationary phases of growth. The mixed culture was incubated with gentle shaking at 37°C for 2 h, and appropriate dilutions were then spread on selective plates. Five of the colonies that grew on the plates were cloned three times, using the same medium, and their drug resistance was examined. The stability of two R plasmids coexisting in the same cell was examined after subculture for at least 10 generations in nutrient broth containing no drug. Transconjugants carrying two plasmids. When transconjugant cells were concluded to carry two R plasmids, 2 ml of recipient (ML4262) culture and 0.5 ml of donor (ML4600 R,+ R+2) culture were mixed and gently shaken for 2 h. Then appropriate dilutions of the mixed culture were plated on two plates, selecting for either R, or R2, and the resistance patterns of the transconjugants that grew on each plate were examined. The percentage of transconjugants possessing only R, (or R2) as a fraction of the total 573
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SAGAI ET AL.
ANTIMICROB. AGENTS CHEMOTHER.
TABLE 1. R plasmids R plasmid Rmsl59 Rmsl61 Rms161-1a Rmsl64 Rms164-la Rmsl78 Rmsl63 Rmsl76 Rmsl48 Rms149 Rms149-la RP4 Rlb931
Rlbl30
Rlbl51 Rlb5265 FP2
Drug resistance Tc.Cm.Sm.Hg Cm.Sm.Su.Km.Hg Cm.Km.Hg Sm.Su.Gm.Hg. Su.Gm.Hg Tc.Cm.Sm.Su.Cb Tc.Cm.Su Sm Sm Sm.Su.Gm.Cb Su.Cb Tc.Km.Cb Tc.Sm.Hg Sm.Su.Gm.Hg Sm.Su.Gm.Cb Sm.Su Hg
Reference This paper This paper This paper This paper This paper Iyobe et al. (11) Iyobe et al. (11) Iyobe et al. (11) Sagai et al. (16) Sagai et al. (16) Sagai et al. (16) Datta et al. (7) Bryan et al. (1) Bryan et al. (1) Bryan et al. (2) Shahrabadi et al. (17) Holloway (10)
aRmsl61-1, Rms164-1, and Rms149-1 are mutants that have lost one or more resistance markers.
number of the transconjugants obtained by selection for R, (or R2) was calculated.
RESULTS Classification of R plasmids by incompatibility tests. Incompatibility testing of R plasmids isolated from P. aeruginosa strains in Japan (11) and West Germany (16) and from RP4 (7) was performed. Rmsl59 was transferred to the recipient strain at a frequency of 10-2, but its transfer frequency was decreased when the recipient strain carried Rmsl61, Rmsl61-1, Rmsl64, or Rmsl64-1 (Table 2). In these four cases, all resistances conferred by the resident R plasmids were eliminated from the transconjugants by the newly transferred Rmsl59. In reciprocal transfer experiments, the transfer of Rmsl64 or Rmsl64-1 was slightly reduced by Rmsl59, and neither plasmid could coexist with Rmsl59. When Rmsl59, Rmsl64, or Rmsl61 was transferred to the Rmsl78+ recipient, Rmsl78 was eliminated by all three, although the transfer frequency of Rmsl59 or Rmsl64 was not reduced. In reciprocal transfers, Rmsl78 coexisted with Rmsl59 or Rmsl64 but not with Rmsl61, although the transfer frequency of Rmsl78 was reduced by the presence of either one of the three plasmids in the recipient (Table 2). If Rmsl78 coexists with either Rmsl59 or Rmsl64 separately in a cell, the two plasmids may be transferred independently from the transconjugant. But retransfer of R plasmids from five clones of ML4262 that had acquired the drug resistances of both Rmsl78 and Rmsl59 (or Rmsl64) showed that resistance markers specific for each of the plasmids did not subsequently segregate in 20 transconjugants, even when selected for either Cb, an Rmsl78 marker, or Hg, an Rmsl59 (or Rmsl64)
marker. These data suggested that Rmsl59 or Rmsl64 coexisted with Rmsl78 after the formation of a recombinant between both plasmids. Therefore, we concluded that Rmsl78 did not coexist with Rmsl59 or Rmsl64 as an independent plasmid; i.e., it was not compatible with Rmsl59 or Rmsl64. On the other hand, Rmsl59 coexisted stably with each of three plasmids, Rmsl49, Rmsl63, and RP4, and Rmsl64-1 coexisted with Rmsl48 in the same host cell. Therefore, we concluded that Rmsl59, Rmsl61, Rmsl64, and Rmsl78 form a single incompatibility group, different from each of the plasmids Rmsl63, Rmsl49, Rmsl48, and RP4. Rmsl63 was transferred to recipient cells carrying either Rmsl59, Rmsl64, Rmsl76, Rmsl49, Rmsl48, or RP4. In all cases except Rmsl76, the transfer frequency of Rmsl63 was not reduced by the resident R plasmid and Rmsl63 coexisted with the resident plasmid in the recipient. When Rmsl76 was transferred to a recipient carrying Rmsl63, the transfer frequency was reduced, and Rmsl63 was excluded by Rmsl76. Therefore, Rmsl63 and Rmsl76 form a single incompatibility group, different from the group of Rmsl59 and from each of the plasmids Rmsl48, Rmsl49, and RP4. The transfer frequency of Rmsl49 was not reduced by the presence of Rmsl59, Rmsl63, or RP4 but was reduced slightly by Rmsl49-1, and Rmsl49 coexisted with each of the plasmids Rmsl59, Rmsl63, and RP4. When the transconjugants concluded to carry both Rmsl49 and Rmsl49-1 were examined by retransfer experiments, the resistance pattern of all transconjugants thus obtained was Sm.Su.Gm and Cb, and the markers of Rmsl49-1 (Su.Cb) were not obtained even when selection was made for Su or Cb. Therefore we concluded that Rmsl49 eliminated Rmsl49-1. Rmsl49-1 coexisted with Rmsl48. In reciprocal experiments, Rmsl48 coexisted with Rmsl64-1, Rmsl63, RP4, and Rmsl49-1, and RP4 coexisted stably with Rmsl59, Rmsl63, Rmsl49, and Rmsl48 (Table 2). In these cases, the transfer frequency of Rmsl48 or RP4 was not reduced by the resident R plasmid in the recipient strains. From the results described above, we therefore conclude that there are five groups, consisting of (i) Rmsl59, Rmsl61, Rmsl64, and Rmsl78, (ii) Rmsl63 and Rmsl76, (iii) Rmsl48, (iv) Rmsl49, and (v) RP4. Retransfer of R plasmids from the transconjugants. Incompatibility testing was carried out to determine whether two plasmids coexisted in the same host. It is possible, however, that two incompatible R plasmids may appear to coexist in a cell after recombination
VOL. 10, 1976
INCOMPATIBILITY OF P. AERUGINOSA R PLASMIDS
575
TABLE 2. Incompatibility testing of R plasmids a Donor (ML4600) carrying:
Recipient (ML4262)
Selection
Frequency
Drug resistance of transconjugants
carrying: Ratiob
RmslS9
Tc
Possessinga:
4.2 x 10-2 4.1 x 10-2 5.8 x 10-2
Rmsl64 Rmsl64-1 Rmsl6l Rmsl6l-1 Rmsl78
Sm Hg Tc Tc Tc Sm Hg
1.2 3.1 6.0 9.0 4.5
10-3 10-4 10-4 10-4 x 10-2
5/5 5/5 5/5 5/5 5/5
Rmsl59 only Rmsl59 only Rmsl59 only Rmsl59 only Rmsl59 only Rmsl59 only
Rmsl59 Rmsl6l Rmsl78
Gm Gm Gm Gm
2.2 2.1 1.3 1.7
x x x x
10-' 10-2 10-3 10-'
5/5 5/5 5/5
Rmsl64 only Rmsl64 only Rmsl64 only
Rmsl59
Gm Gm
7.8 x 10-2 4.5 x 10-3
5/5
Rmsl64-1 only
Rmsl64 Rmsl78
Lv Lv Lv
9.3 x 10-3 2.3 x 10-7 7.8 x 10-6
5/5 5/5
Rmsl61 only
Rmsl59 Rmsl64 Rmsl6l
Tc Cb Cb Cb Tc
1.3 x 1.0 x 1.7 x 1.2 x 4.0 x
10-4 10-6 10-6
5/5 5/5 5/5
Both Both Rmsl78 only
Rmsl59
Rmsl63 Rmsl49 RP4
Sm Tc Sm
6.1 x 10-2 3.7 x 10-2 5.8 x 10-2
5/5 5/5 5/5
Both Both Both
Rmsl64-1
Rmsl48
Gm
5.3 x 10-2
5/5
Both
Tc Su
3.8 8.2 2.5 7.7 3.2 4.9 3.1 3.6
10-2 10-2 10-2 10-2 10-7 10-2 x 10-3 x 10-2
5/5 5/5 5/5 5/5 5/5
5/5
Both Both Rmsl63 only Both Both Both
5/5 5/5
Both Rmsl76 only
Rmsl64
Rmsl64-1 Rmsl6l
Rmsl78
Rmsl63 Rmsl59 Rmsl64 Rmsl76 Rmsl49 Rmsl48 RP4 Rmsl76
Su
Tc Tc Tc Tc Su
x x x x
Rmsl61 only
10-2 10-2
x x x x x x
Rmsl64-1 Rmsl63
Sm
1.1 x 10-2 2.6 x 10-2 6.2 x 10-6
Rmsl59 Rmsl63 Rmsl49-1 RP4
Gm Cb Cb Cb Gm Gm
6.0 x 10-6 4.0 x 10-6 2.9 x 10-6 5.4 x 10-6 3.2 x 10-7 5.1 x 10-6
5/5 5/5
Both Both
5/5
Both
Rmsl48
Cb Cb
5.2 x 10-6 5.1 x 10-6
5/5
Both
Rmsl49
Rmsl49-1
Sm Sm
576
SAGAI ET AL.
ANTIMICROB. AGENTS CHEMOTHER. TABLE 2-Continued Drug resistance of transconju-
Donor (ML4600) carrying:
Recipient (ML4262) carrying:
Rmsl48
Selection
gants
Frequency Ratio"
Possessinga:
Rmsl64-1 Rmsl63 Rmsl49-1 RP4
Sm Sm
1.7 x 10-3 4.7 x 10-3
Sm Sm Sm
2.1 x 10-3 1.4 x 10-3 2.5 x 10-3
5/5 5/5 5/5 5/5
Both Both Both Both
Lv
Rmsl59 Rmsl63 Rmsl49 Rmsl48
Cb Lv Lv Lv Cb
1.4 1.3 1.2 7.7 6.3 2.1
5/5 5/5 5/5 5/5
Both Both Both Both
RP4
x 10-1 x
x x x x
10-1 10-' 10-2 10-2 10-1
" See text for details. b Nominator, number of transconjugants possessing the indicated R plasmid(s); denominator, number of transconjugants examined.
between the plasmids, as was seen in the case of Rmsl59 and Rmsl78 and of Rmsl64 and Rmsl78. In these cases, the two original plasmids were not transferred separately from the transconjugants. Retransfer of plasmids from the transconjugants that were concluded to carry two plasmids was considered necessary to confirm the separate existence of both plasmids in the cell. The data of retransfer experiments are shown in Table 3. In all combinations of the five groups, two plasmids were retransferred separately from the transconjugants, indicating that both plasmids existed separately in the transconjugants and thus confirming the five incompatibility groups of P. aeruginosa R plasmids. Comparison of incompatibility groups with other plasmids. It was reported that Rlbl3O, Rlb931, and other plasmids form one incompatibility group, P-2 (1), and that Rlbl51 belongs to a separate group, P-3 (2). The P-3 group was identical with the C group (17) that was classified in the E. coli system (6). We carried out further incompatibility testing of all plasmids that were classified by the tests described above, using Rlbl3O, Rlb931, Rlbl51, and FP2. FP2 is an extrachromosomal and transferable genetic element in P. aeruginosa and has been used as a sex factor for genetic studies (10). The transfer frequency of Rlbl3O was reduced by the presence of Rmsl59 in the recipient but not by any other plasmid including FP2, and Rlbl3O did not coexist with Rmsl59 (Table 4). Rlb931, a member of incompatibility group P-2, did not coexist with Rmsl64. Therefore it can be concluded that Rmsl59, Rmsl61, Rmsl64, and Rmsl78 belong to the P-2 group (Table 4).
TABLE 3. Retransfer of the R plasmids from transconjugants Donor (ML4600) carrying both plasmidsa
R,
R2
Rmsl63
Rmsl64
Rms149
Rms159
Selection for:
% of cells carrying
R, or Drug R2
R,
R, R2 R, R2
Rmsl64-1
Rmsl48
Rmsl59
RP4
R,
Rmsl63
Rmsl49
R,
Rmsl48
Rmsl63
R2 R, R2
Rmsl63
RP4
R,
Rmsl49-1
Rms148
R,
Rmsl49
RP4
RP4
Rmsl48
R,
R2
R2
R2 R2
R, R2
R, R2
Tc Cm Cb Tc Hg Sm Sm Cb Tc Sm Sm Tc Cm Lv Cb Sm Sm Tc Cb Sm
only":
R2
80 93 10 100 100
100
100 56 93 8
100 100 100 100
5 100 15
100 100 100
aML4262 was used as the recipient. Ratio of cells carrying only one type of R plasmid to total number of transconjugants obtained when selected for either R, or R2.
The transfer frequency of Rlbl51 was not affected by the presence of any of the resident plasmids, Rmsl59, Rmsl63, Rmsl49, Rmsl48, RP4, and FP2, in the recipient, and Rlbl51 coexisted with Rmsl61-1, Rmsl63, Rmsl49-1, Rmsl48, RP4, and FP2 (Table 4). Retransfer experiments showed that all transconjugants that apparently carried two plasmids subse-
INCOMPATIBILITY OF P. AERUGINOSA R PLASMIDS
VOL. 10, 1976
DISCUSSION
TABLE 4. Incompatibility testing of R plasmids Donor
Drug resistance of transconju-
.
(ML4600O) Recipient carry- carrying: tionFrqec
Selec-Frqec
ing:
ing: Rlbl3O
Rmsl59 Rmsl63 Rmsl49 Rms148 RP4 FP2
Rlb931 Rmsl64
Rlbl51 Rms161-1 Rms163 Rmsl49-1
Rmsl48 RP4 FP2 FP2
Rmsl78 Rmsl63 Rmsl49 Rmsl48 RP4 R}bl51
gat
gat Ratio
Ratio
Sm Gm Hg Gm Sm Hg Gm Sm Sm Tc Tc Sm Gm Gm Sm Sm Gm Sm Sm Hg Hg Hg Hg Hg Hg Hg
9.2 6.9 7.0 1.2 1.9 2.7 2.8 1.7 1.9 3.2 4.1 2.8 8.1 9.9 3.0 2.8 7.8 3.2 3.1 3.0 1.7 7.6 4.2 2.8 3.1 8.2
~~~~~~Possess~~~~~~~inga:
x 10-1 x 10-'
x x x x x x x x x x x x x x x x x x x x x x x x
10-' 10-3 10-' 10-1 10-' 10-1 10-'
10-1 10-2 10-' 10-1 10-1 10-1 10-'
10-1 10-1 10-1 10-2 10-2 10-3 10-3 10-3 10-3 10-3
5/5 5/5 5/5 5/5 5/5
Rlbl3O only Both Both Both Both
5/5 Rlb931 only
5/5 5/5 5/5 5/5 5/5 5/5
Both Both Bothb
5/5 5/5 5/5 5/5 5/5 5/5
Both Both Both Both Both Both
Bothe Both Both
See text. bIncompatibility testing was done with Sm, Gm, and Cb resistance. Both R plasmids specify Cb resistance, but Rms149-1 specifies a higher level of Cb resistance (800 Ag/ ml) than Rlbl51 (200 Ag/ml) in ML4262. c Both plasmids specify Sm resistance, but Rmsl48 specifies a higher level of resistance than Rlbl51. a
quently transferred each plasmid independently (Table 5), and Rlbl51 was concluded to be compatible with each of Rmsl61-1, Rmsl63, Rmsl49-1, Rmsl48, RP4, and FP2. Shahrabadi et al. (17) reported that Rlb5265 was compatible with the R plasmids that were classified into P-1, P-2, and P-3 groups and was assigned to the P-4 group. However, because Rlb5265 was not transferable to the test strains, ML4262 and ML4600, P. aeruginosa 280 (rif met-) (1) was used as the recipient, and its incompatibility was tested by the method of Bryan et al. (1). The transfer frequency of Rlb5265 was not reduced by the resident plasmids, RP4, Rlb931, RlbMlM, Rmsl63, Rmsl48, and FP2, and Rlb5265 coexisted stably with RP4, Rlb93,1, Rlbl51, Rmsl63, and FP2. The coexistence of Rlb5265 and Rmsl48 could not be examined because the single resistance marker of Rmsl48 was masked by those of Rlb5265. The incompatibility testing of Rlb5265 and Rmsl49 was impossible due to nontransferability of Rmsl49 to 280 (rifr met-).
577
The data of the incompatibility groups are summarized in Table 6 and show additions to the classification of Bryan et al., (1). Rmsl48 and Rmsl49 are shown as P-7 and P-8 group plasmids, respectively, although we could not perform complete incompatibility testing because of nontransferability of Rmsl49 and TABLE 5. Retransfer ofplasmids from transconjugants Donor (ML4600) carrying eithera:
R,
R2
Rmsl61-1
Rlbl51
Selection for either:
R,R2or Drug
% of cells carrying onlyb:
R,
R2
R,
Lv 77 Sm 100 Rmsl63 Rlbl51 92 R, Tc R2 Sm 100 Rlbl51 Rmsl49-1 100 R, Sm R2 Cbc 3 Rlbl51 Rmsl48 100 R, Cb R2 Smd 27 RP4 Rlbl51 93 R, Tc 93 R2 Sm FP2 68 Rlbl51 R, Hg 100 R2 Sm FP2 86 Rmsl78 R, Hg R2 Tc 100 FP2 Rmsl63 100 R, Hg R2 Tc 100 FP2 100 Rmsl49 R, Hg R2 Sm 100 FP2 Rmsl48e 1.5 R, Hg R2 Sm 100 FP2 RP4 100 R, Hg 100 R2 Tc a ML4262 was used as the recipient. b Ratio of cells carrying only one type of R plasmid to total number of transconjugants obtained when selected for either R, or R2. c 300 .g of Cb per ml was used for selection of Rmsl49-1. d 100 jg of Sm per ml was used for selection of Rmsl48. ' ML4262 was used as the host strain of R plasmids (see text). R2
TABLE 6. Summarized data of the incompatibility grouping of R plasmids in P. aeruginosa R plasmid Group P-i RP4 P-2 Rlb931, Rlbl3O, Rlb680, Rlb290, Rlb546, Rlb442, Rlb454, Rlb3108, Rlb55, Rlbl3, Rsu38, Rms159, Rms161, Rmsl64, Rms178, Rmsl69, Rmsl70, Rmsl71, Rmsl72, Rmsl73, Rmsl74, Rmsl62, Rmsl46, Rmsl96, Rmsl97, Rmsl98, Rmsl99 P-3 RIbl51 P-4 Rlb5265 P-5 Rmsl63, Rmsl76 FP2 P-6 P-7 Rmsl48 Rmsl49 P-8
578
SAGAI ET AL.
Rlb5265 and because of masking of Rmsl48specified streptomycin resistance by Rlb5265. Almost all of the R plasmids demonstrated from P. aeruginosa in Japan have been classified into the P-2 group (Table 6) except two plasmids, Rmsl63 and Rmsl76 (11). A similar prevalence of the P-2 group has been observed among R plasmids detected in isolates of P. aeruginosa in North America (2, 17). Transfer of P-2 group R plasmids by conjugation to E. coli strains has not been observed (1, 11, 17). These results suggest that the P-2 plasmids are representative of conjugative R plasmids in P. aeruginosa and that the origin of R plasmids in P. aeruginosa may be different from that of plasmids identified in species of the Enterobacteriaceae. The P-5 plasmids, Rmsl63 and Rmsl76, were detected inP. aeruginosa strains of the same clinic (11), and both P-7 and P-8 plasmids were isolated from the same strain in Germany (16). These groups are seen to be rare types in P. aeruginosa. We are collecting many R plasmids in an attempt to extend the P-4, P-6, P-7, and P-8 incompatibility groups and to clarify the relationship between the P-4 and the P-7 and P-8 groups.
LITERATURE CITED 1. Bryan, L. E., S. D. Semeka, M. H. Van Den Elzen, J. E. Kinnear, and R. E. S. Whitehouse. 1973. Characteristics of R931 and other Pseudomonas aeruginosa R factors. Antimicrob.. Agents Chemother. 3:625-637. 2. Bryan, L. E., M. S. Shahrabadi, and H. M. Van Den Elzen. 1974. Gentamicin resistance in Pseudomonas aeruginosa R-factor-mediated resistance. Antimicrob. Agents Chemother. 6:191-199. 3. Coetzee, J. N., N. Datta, and R. W. Hedges. 1972. R factors from Proteus rettgeri. J. Gen. Microbiol. 72:543-552. 4. Datta, N. 1974. Epidemiology and classification of plasmids, p. 9-15. In D. Schlessinger (ed.), Microbiology- 1974. American Society for Microbiology, Washington, D.C.
ANTIMICROB. AGENTS CHEMOTHER. 5. Datta, N., and R. W. Hedges. 1971. Compatibility groups among fi- R factors. Nature (London) 234:222223. 6. Datta, N., and R. W. Hedges. 1972. R factors identified in Paris, some conferring gentamicin resistance, constitute a new compatibility group. Ann. Inst. Pasteur Paris 129:849-852. 7. Datta, N., R. W. Hedges, E. J. Shaw, R. B. Sykes, and M. H. Richmond. 1971. Properties of an R factor from Pseudomonas aeruginosa. J. Bacteriol. 108:12441249. 8. Davis, B. D., and E. S. Mingioli. 1960. Mutants of Escherichia coli requiring methionine or vitamin B12. J. Bacteriol. 60:17-29. 9. Hedges, R. W., V. Rodriguez-Lemoine, and N. Datta. 1975. R factors from Serratia marcescens. J. Gen. Microbiol. 86:88-92. 10. Holloway, B. W. 1969. Genetics ofPseudomonas. Bacteriol. Rev. 33:419-443. 11. lyobe, S., K. Hasuda, A. Fuse, and S. Mitsuhashi. 1974. Demonstration of R factors from Pseudomonas aeruginosa. Antimicrob. Agents Chemother. 5:547552. 12. Kawakami, Y., F. Mikoshiba, S. Nagasaki, H. Matsumoto, and T. Tazaki. 1972. Prevalence of Pseudomonas aeruginosa strains possessing R factor in a hospital. J. Antibiot. 25:607-609. 13. Kontomichalou, P., and E. Papachristou. 1975. Multiresistance plasmids in Pseudomonas aeruginosa highly resistant to gentamicin, p. 329-348. In S. Mitsuhashi and H. Hashimoto (ed.), Microbial drug resistance. University of Tokyo Press, Tokyo. 14. Lawn, A. M., G. G. Meynell, E. Meynell, and N. Datta. 1967. Sex pili and the classification of sex factors in theEnterobacteriaceae. Nature (London) 216:343-346. 15. Roe, E., B. J. Jones, and E. J. L. Lowbury. 1971. Transfer of antibiotic resistance between Pseudomonas aeruginosa, Escherichia coli, and other gramnegative bacilli in burns. Lancet 1:149-152. 16. Sagai, H., V. Krcmery, K. Hasuda, S. Iyobe, H. Knothe, and S. Mitauhashi. 1975. R factor mediated resistance to aminoglycoside antibiotics in Pseudomonas aeruginosa. Jpn. J. Microbiol. 19:427-432. 17. Shahrabadi, M. S., L. E. Bryan, and H. M. Van Den Elzen. 1975. Further properties of P-2 R-factors of Pseudomonas aeruginosa and their relationship to other plasmids. Can. J. Microbiol. 21:592-605. 18. Watanabe, T., H. Nishida, C. Ogata, T. Arai, and S. Sato. 1964. Episome-mediated transfer of drug resistance in Enterobacteriaceae. VII. Two types of naturally occurring R factors. J. Bacteriol. 88:716-726.
p.
Vol. 10, No. 4
573-578
Printed in U.S.A.
Classification of R Plasmids by Incompatibility Pseudomonas aeruginosa H. SAGAI,* K. HASUDA, S. IYOBE, L. E. BRYAN, B. W. HOLLOWAY,
AND
in
S. MITSUHASHI
Department of Microbiology, School of Medicine, Gunma University, Maebashi, Japan*; Department of Medical Bacteriology, University of Alberta, Edmonton, Canada; and Department of Genetics, Monash University, Clayton, Victoria 3168, Australia Received for publication 10 March 1976
R plasmids identified in Pseudomonas aeruginosa strains isolated in our laboratories were classified by their incompatibility characters by using intraspecies conjugation in P. aeruginosa. The fertility factor, FP2, was compatible with R plasmids belonging to all groups, and was classified in a new group. From these data, the plasmids examined have been placed into seven or eight incompatibility groups. R plasmids in Enterobacteriaceae were originally classified by their fA character (18) or type of sex pili (14). More recently, the classification of R plasmids based on incompatibility has been generally used (4, 7), resulting in the recognition of more than 20 groups in the Escherichia coli system (9). In Pseudomonas aeruginosa strains, many R plasmids have been isolated (1, 2, 11-13, 15, 16), but almost all have been found to be either conjugally nontransferable or transferable at very low frequency to E. coli (1, 11, 16); therefore they cannot readily be classified in this system. Those few that are conjugally transferable to E. coli have been classified as P group (7) or C group (6, 13). Bryan et al. (1, 2, 17) proposed an incompatibility classification of plasmids detected in P. aeruginosa based on intraspecies conjugation in Pseudomonas and classified R plasmids into four incompatibility groups, P-1, P-2, P-3, and P-4. This paper deals with the classification of additional R plasmids from P. aeruginosa by incompatibility testing, using intraspecies conjugation in Pseudomonas. MATERIALS AND METHODS Bacterial strains. Two strains of P. aeruginosa, ML4262 (trp- his- ilv- met- rif ) (rifr indicates rifampin resistant) and ML4600 (trp- his-), which are isogenic except for the characters given, and two independently isolated strains, P. aeruginosa 280 (rif' met-) (1) and P. aeruginosa 5265 (1), were used as host strains for the R plasmids. R plasmids. R plasmids and their resistance markers are shown in Table 1. Drugs. Tetracycline (Tc), chloramphenicol (Cm), streptomycin (Sm), and sulfanilamide (Su) were kindly supplied by Japan Lederle Co., Sankyo Seiyaku Co., Kyowa Hakko Co., and the Dainihon Seiyaku Co., respectively. Carbenicillin (Cb), lividomycin (Lv), rifampin (Rp), and gentamicin C com-
plex (Gm) were supplied by the Fujisawa Seiyaku Co., Kowa Co., Daiichi Seiyaku Co., and Shionogi Co., respectively. Mercuric chloride (Hg; Wako Jyunyaku) was obtained commercially. Media. Nutrient broth and nutrient agar were used routinely. Nutrient broth consisted of 10 g of beef extract, 10 g of peptone, and 3 g of NaCl in 1,000 ml of distilled water. Nutrient agar consisted of nutrient broth supplemented with 1.5% agar. The transconjugants that had acquired drug resistance were selected on nutrient agar containing the appropriate selective drug together with Rp. For the assay of Su resistance or the selection of Su-resistant transconjugants, semisynthetic medium was used, which consisted of 1,000 ml of medium A (8) to which 2 g of Casamino Acids, 10 mg of tryptophan, 2 mg of nicotinic acid, 10 mg of thiamine, 2 g of glucose, and 15 g of agar were added. Medium A consisted of 8 g of Na2HPO4 * 12H20, 2 g of KH2PO4, 0.4 g of sodium citrate, 0.1 g of MgSO4 * 7H20, and 1.0 g of (NH4)2SO4 in 1,000 ml of distilled water. Incompatibility experiments. Two milliliters of a culture of recipient cells (ML4262), either carrying or not carrying an R plasmid, and 0.5 ml of donor culture (ML4600 R+) were mixed at early stationary phases of growth. The mixed culture was incubated with gentle shaking at 37°C for 2 h, and appropriate dilutions were then spread on selective plates. Five of the colonies that grew on the plates were cloned three times, using the same medium, and their drug resistance was examined. The stability of two R plasmids coexisting in the same cell was examined after subculture for at least 10 generations in nutrient broth containing no drug. Transconjugants carrying two plasmids. When transconjugant cells were concluded to carry two R plasmids, 2 ml of recipient (ML4262) culture and 0.5 ml of donor (ML4600 R,+ R+2) culture were mixed and gently shaken for 2 h. Then appropriate dilutions of the mixed culture were plated on two plates, selecting for either R, or R2, and the resistance patterns of the transconjugants that grew on each plate were examined. The percentage of transconjugants possessing only R, (or R2) as a fraction of the total 573
574
SAGAI ET AL.
ANTIMICROB. AGENTS CHEMOTHER.
TABLE 1. R plasmids R plasmid Rmsl59 Rmsl61 Rms161-1a Rmsl64 Rms164-la Rmsl78 Rmsl63 Rmsl76 Rmsl48 Rms149 Rms149-la RP4 Rlb931
Rlbl30
Rlbl51 Rlb5265 FP2
Drug resistance Tc.Cm.Sm.Hg Cm.Sm.Su.Km.Hg Cm.Km.Hg Sm.Su.Gm.Hg. Su.Gm.Hg Tc.Cm.Sm.Su.Cb Tc.Cm.Su Sm Sm Sm.Su.Gm.Cb Su.Cb Tc.Km.Cb Tc.Sm.Hg Sm.Su.Gm.Hg Sm.Su.Gm.Cb Sm.Su Hg
Reference This paper This paper This paper This paper This paper Iyobe et al. (11) Iyobe et al. (11) Iyobe et al. (11) Sagai et al. (16) Sagai et al. (16) Sagai et al. (16) Datta et al. (7) Bryan et al. (1) Bryan et al. (1) Bryan et al. (2) Shahrabadi et al. (17) Holloway (10)
aRmsl61-1, Rms164-1, and Rms149-1 are mutants that have lost one or more resistance markers.
number of the transconjugants obtained by selection for R, (or R2) was calculated.
RESULTS Classification of R plasmids by incompatibility tests. Incompatibility testing of R plasmids isolated from P. aeruginosa strains in Japan (11) and West Germany (16) and from RP4 (7) was performed. Rmsl59 was transferred to the recipient strain at a frequency of 10-2, but its transfer frequency was decreased when the recipient strain carried Rmsl61, Rmsl61-1, Rmsl64, or Rmsl64-1 (Table 2). In these four cases, all resistances conferred by the resident R plasmids were eliminated from the transconjugants by the newly transferred Rmsl59. In reciprocal transfer experiments, the transfer of Rmsl64 or Rmsl64-1 was slightly reduced by Rmsl59, and neither plasmid could coexist with Rmsl59. When Rmsl59, Rmsl64, or Rmsl61 was transferred to the Rmsl78+ recipient, Rmsl78 was eliminated by all three, although the transfer frequency of Rmsl59 or Rmsl64 was not reduced. In reciprocal transfers, Rmsl78 coexisted with Rmsl59 or Rmsl64 but not with Rmsl61, although the transfer frequency of Rmsl78 was reduced by the presence of either one of the three plasmids in the recipient (Table 2). If Rmsl78 coexists with either Rmsl59 or Rmsl64 separately in a cell, the two plasmids may be transferred independently from the transconjugant. But retransfer of R plasmids from five clones of ML4262 that had acquired the drug resistances of both Rmsl78 and Rmsl59 (or Rmsl64) showed that resistance markers specific for each of the plasmids did not subsequently segregate in 20 transconjugants, even when selected for either Cb, an Rmsl78 marker, or Hg, an Rmsl59 (or Rmsl64)
marker. These data suggested that Rmsl59 or Rmsl64 coexisted with Rmsl78 after the formation of a recombinant between both plasmids. Therefore, we concluded that Rmsl78 did not coexist with Rmsl59 or Rmsl64 as an independent plasmid; i.e., it was not compatible with Rmsl59 or Rmsl64. On the other hand, Rmsl59 coexisted stably with each of three plasmids, Rmsl49, Rmsl63, and RP4, and Rmsl64-1 coexisted with Rmsl48 in the same host cell. Therefore, we concluded that Rmsl59, Rmsl61, Rmsl64, and Rmsl78 form a single incompatibility group, different from each of the plasmids Rmsl63, Rmsl49, Rmsl48, and RP4. Rmsl63 was transferred to recipient cells carrying either Rmsl59, Rmsl64, Rmsl76, Rmsl49, Rmsl48, or RP4. In all cases except Rmsl76, the transfer frequency of Rmsl63 was not reduced by the resident R plasmid and Rmsl63 coexisted with the resident plasmid in the recipient. When Rmsl76 was transferred to a recipient carrying Rmsl63, the transfer frequency was reduced, and Rmsl63 was excluded by Rmsl76. Therefore, Rmsl63 and Rmsl76 form a single incompatibility group, different from the group of Rmsl59 and from each of the plasmids Rmsl48, Rmsl49, and RP4. The transfer frequency of Rmsl49 was not reduced by the presence of Rmsl59, Rmsl63, or RP4 but was reduced slightly by Rmsl49-1, and Rmsl49 coexisted with each of the plasmids Rmsl59, Rmsl63, and RP4. When the transconjugants concluded to carry both Rmsl49 and Rmsl49-1 were examined by retransfer experiments, the resistance pattern of all transconjugants thus obtained was Sm.Su.Gm and Cb, and the markers of Rmsl49-1 (Su.Cb) were not obtained even when selection was made for Su or Cb. Therefore we concluded that Rmsl49 eliminated Rmsl49-1. Rmsl49-1 coexisted with Rmsl48. In reciprocal experiments, Rmsl48 coexisted with Rmsl64-1, Rmsl63, RP4, and Rmsl49-1, and RP4 coexisted stably with Rmsl59, Rmsl63, Rmsl49, and Rmsl48 (Table 2). In these cases, the transfer frequency of Rmsl48 or RP4 was not reduced by the resident R plasmid in the recipient strains. From the results described above, we therefore conclude that there are five groups, consisting of (i) Rmsl59, Rmsl61, Rmsl64, and Rmsl78, (ii) Rmsl63 and Rmsl76, (iii) Rmsl48, (iv) Rmsl49, and (v) RP4. Retransfer of R plasmids from the transconjugants. Incompatibility testing was carried out to determine whether two plasmids coexisted in the same host. It is possible, however, that two incompatible R plasmids may appear to coexist in a cell after recombination
VOL. 10, 1976
INCOMPATIBILITY OF P. AERUGINOSA R PLASMIDS
575
TABLE 2. Incompatibility testing of R plasmids a Donor (ML4600) carrying:
Recipient (ML4262)
Selection
Frequency
Drug resistance of transconjugants
carrying: Ratiob
RmslS9
Tc
Possessinga:
4.2 x 10-2 4.1 x 10-2 5.8 x 10-2
Rmsl64 Rmsl64-1 Rmsl6l Rmsl6l-1 Rmsl78
Sm Hg Tc Tc Tc Sm Hg
1.2 3.1 6.0 9.0 4.5
10-3 10-4 10-4 10-4 x 10-2
5/5 5/5 5/5 5/5 5/5
Rmsl59 only Rmsl59 only Rmsl59 only Rmsl59 only Rmsl59 only Rmsl59 only
Rmsl59 Rmsl6l Rmsl78
Gm Gm Gm Gm
2.2 2.1 1.3 1.7
x x x x
10-' 10-2 10-3 10-'
5/5 5/5 5/5
Rmsl64 only Rmsl64 only Rmsl64 only
Rmsl59
Gm Gm
7.8 x 10-2 4.5 x 10-3
5/5
Rmsl64-1 only
Rmsl64 Rmsl78
Lv Lv Lv
9.3 x 10-3 2.3 x 10-7 7.8 x 10-6
5/5 5/5
Rmsl61 only
Rmsl59 Rmsl64 Rmsl6l
Tc Cb Cb Cb Tc
1.3 x 1.0 x 1.7 x 1.2 x 4.0 x
10-4 10-6 10-6
5/5 5/5 5/5
Both Both Rmsl78 only
Rmsl59
Rmsl63 Rmsl49 RP4
Sm Tc Sm
6.1 x 10-2 3.7 x 10-2 5.8 x 10-2
5/5 5/5 5/5
Both Both Both
Rmsl64-1
Rmsl48
Gm
5.3 x 10-2
5/5
Both
Tc Su
3.8 8.2 2.5 7.7 3.2 4.9 3.1 3.6
10-2 10-2 10-2 10-2 10-7 10-2 x 10-3 x 10-2
5/5 5/5 5/5 5/5 5/5
5/5
Both Both Rmsl63 only Both Both Both
5/5 5/5
Both Rmsl76 only
Rmsl64
Rmsl64-1 Rmsl6l
Rmsl78
Rmsl63 Rmsl59 Rmsl64 Rmsl76 Rmsl49 Rmsl48 RP4 Rmsl76
Su
Tc Tc Tc Tc Su
x x x x
Rmsl61 only
10-2 10-2
x x x x x x
Rmsl64-1 Rmsl63
Sm
1.1 x 10-2 2.6 x 10-2 6.2 x 10-6
Rmsl59 Rmsl63 Rmsl49-1 RP4
Gm Cb Cb Cb Gm Gm
6.0 x 10-6 4.0 x 10-6 2.9 x 10-6 5.4 x 10-6 3.2 x 10-7 5.1 x 10-6
5/5 5/5
Both Both
5/5
Both
Rmsl48
Cb Cb
5.2 x 10-6 5.1 x 10-6
5/5
Both
Rmsl49
Rmsl49-1
Sm Sm
576
SAGAI ET AL.
ANTIMICROB. AGENTS CHEMOTHER. TABLE 2-Continued Drug resistance of transconju-
Donor (ML4600) carrying:
Recipient (ML4262) carrying:
Rmsl48
Selection
gants
Frequency Ratio"
Possessinga:
Rmsl64-1 Rmsl63 Rmsl49-1 RP4
Sm Sm
1.7 x 10-3 4.7 x 10-3
Sm Sm Sm
2.1 x 10-3 1.4 x 10-3 2.5 x 10-3
5/5 5/5 5/5 5/5
Both Both Both Both
Lv
Rmsl59 Rmsl63 Rmsl49 Rmsl48
Cb Lv Lv Lv Cb
1.4 1.3 1.2 7.7 6.3 2.1
5/5 5/5 5/5 5/5
Both Both Both Both
RP4
x 10-1 x
x x x x
10-1 10-' 10-2 10-2 10-1
" See text for details. b Nominator, number of transconjugants possessing the indicated R plasmid(s); denominator, number of transconjugants examined.
between the plasmids, as was seen in the case of Rmsl59 and Rmsl78 and of Rmsl64 and Rmsl78. In these cases, the two original plasmids were not transferred separately from the transconjugants. Retransfer of plasmids from the transconjugants that were concluded to carry two plasmids was considered necessary to confirm the separate existence of both plasmids in the cell. The data of retransfer experiments are shown in Table 3. In all combinations of the five groups, two plasmids were retransferred separately from the transconjugants, indicating that both plasmids existed separately in the transconjugants and thus confirming the five incompatibility groups of P. aeruginosa R plasmids. Comparison of incompatibility groups with other plasmids. It was reported that Rlbl3O, Rlb931, and other plasmids form one incompatibility group, P-2 (1), and that Rlbl51 belongs to a separate group, P-3 (2). The P-3 group was identical with the C group (17) that was classified in the E. coli system (6). We carried out further incompatibility testing of all plasmids that were classified by the tests described above, using Rlbl3O, Rlb931, Rlbl51, and FP2. FP2 is an extrachromosomal and transferable genetic element in P. aeruginosa and has been used as a sex factor for genetic studies (10). The transfer frequency of Rlbl3O was reduced by the presence of Rmsl59 in the recipient but not by any other plasmid including FP2, and Rlbl3O did not coexist with Rmsl59 (Table 4). Rlb931, a member of incompatibility group P-2, did not coexist with Rmsl64. Therefore it can be concluded that Rmsl59, Rmsl61, Rmsl64, and Rmsl78 belong to the P-2 group (Table 4).
TABLE 3. Retransfer of the R plasmids from transconjugants Donor (ML4600) carrying both plasmidsa
R,
R2
Rmsl63
Rmsl64
Rms149
Rms159
Selection for:
% of cells carrying
R, or Drug R2
R,
R, R2 R, R2
Rmsl64-1
Rmsl48
Rmsl59
RP4
R,
Rmsl63
Rmsl49
R,
Rmsl48
Rmsl63
R2 R, R2
Rmsl63
RP4
R,
Rmsl49-1
Rms148
R,
Rmsl49
RP4
RP4
Rmsl48
R,
R2
R2
R2 R2
R, R2
R, R2
Tc Cm Cb Tc Hg Sm Sm Cb Tc Sm Sm Tc Cm Lv Cb Sm Sm Tc Cb Sm
only":
R2
80 93 10 100 100
100
100 56 93 8
100 100 100 100
5 100 15
100 100 100
aML4262 was used as the recipient. Ratio of cells carrying only one type of R plasmid to total number of transconjugants obtained when selected for either R, or R2.
The transfer frequency of Rlbl51 was not affected by the presence of any of the resident plasmids, Rmsl59, Rmsl63, Rmsl49, Rmsl48, RP4, and FP2, in the recipient, and Rlbl51 coexisted with Rmsl61-1, Rmsl63, Rmsl49-1, Rmsl48, RP4, and FP2 (Table 4). Retransfer experiments showed that all transconjugants that apparently carried two plasmids subse-
INCOMPATIBILITY OF P. AERUGINOSA R PLASMIDS
VOL. 10, 1976
DISCUSSION
TABLE 4. Incompatibility testing of R plasmids Donor
Drug resistance of transconju-
.
(ML4600O) Recipient carry- carrying: tionFrqec
Selec-Frqec
ing:
ing: Rlbl3O
Rmsl59 Rmsl63 Rmsl49 Rms148 RP4 FP2
Rlb931 Rmsl64
Rlbl51 Rms161-1 Rms163 Rmsl49-1
Rmsl48 RP4 FP2 FP2
Rmsl78 Rmsl63 Rmsl49 Rmsl48 RP4 R}bl51
gat
gat Ratio
Ratio
Sm Gm Hg Gm Sm Hg Gm Sm Sm Tc Tc Sm Gm Gm Sm Sm Gm Sm Sm Hg Hg Hg Hg Hg Hg Hg
9.2 6.9 7.0 1.2 1.9 2.7 2.8 1.7 1.9 3.2 4.1 2.8 8.1 9.9 3.0 2.8 7.8 3.2 3.1 3.0 1.7 7.6 4.2 2.8 3.1 8.2
~~~~~~Possess~~~~~~~inga:
x 10-1 x 10-'
x x x x x x x x x x x x x x x x x x x x x x x x
10-' 10-3 10-' 10-1 10-' 10-1 10-'
10-1 10-2 10-' 10-1 10-1 10-1 10-'
10-1 10-1 10-1 10-2 10-2 10-3 10-3 10-3 10-3 10-3
5/5 5/5 5/5 5/5 5/5
Rlbl3O only Both Both Both Both
5/5 Rlb931 only
5/5 5/5 5/5 5/5 5/5 5/5
Both Both Bothb
5/5 5/5 5/5 5/5 5/5 5/5
Both Both Both Both Both Both
Bothe Both Both
See text. bIncompatibility testing was done with Sm, Gm, and Cb resistance. Both R plasmids specify Cb resistance, but Rms149-1 specifies a higher level of Cb resistance (800 Ag/ ml) than Rlbl51 (200 Ag/ml) in ML4262. c Both plasmids specify Sm resistance, but Rmsl48 specifies a higher level of resistance than Rlbl51. a
quently transferred each plasmid independently (Table 5), and Rlbl51 was concluded to be compatible with each of Rmsl61-1, Rmsl63, Rmsl49-1, Rmsl48, RP4, and FP2. Shahrabadi et al. (17) reported that Rlb5265 was compatible with the R plasmids that were classified into P-1, P-2, and P-3 groups and was assigned to the P-4 group. However, because Rlb5265 was not transferable to the test strains, ML4262 and ML4600, P. aeruginosa 280 (rif met-) (1) was used as the recipient, and its incompatibility was tested by the method of Bryan et al. (1). The transfer frequency of Rlb5265 was not reduced by the resident plasmids, RP4, Rlb931, RlbMlM, Rmsl63, Rmsl48, and FP2, and Rlb5265 coexisted stably with RP4, Rlb93,1, Rlbl51, Rmsl63, and FP2. The coexistence of Rlb5265 and Rmsl48 could not be examined because the single resistance marker of Rmsl48 was masked by those of Rlb5265. The incompatibility testing of Rlb5265 and Rmsl49 was impossible due to nontransferability of Rmsl49 to 280 (rifr met-).
577
The data of the incompatibility groups are summarized in Table 6 and show additions to the classification of Bryan et al., (1). Rmsl48 and Rmsl49 are shown as P-7 and P-8 group plasmids, respectively, although we could not perform complete incompatibility testing because of nontransferability of Rmsl49 and TABLE 5. Retransfer ofplasmids from transconjugants Donor (ML4600) carrying eithera:
R,
R2
Rmsl61-1
Rlbl51
Selection for either:
R,R2or Drug
% of cells carrying onlyb:
R,
R2
R,
Lv 77 Sm 100 Rmsl63 Rlbl51 92 R, Tc R2 Sm 100 Rlbl51 Rmsl49-1 100 R, Sm R2 Cbc 3 Rlbl51 Rmsl48 100 R, Cb R2 Smd 27 RP4 Rlbl51 93 R, Tc 93 R2 Sm FP2 68 Rlbl51 R, Hg 100 R2 Sm FP2 86 Rmsl78 R, Hg R2 Tc 100 FP2 Rmsl63 100 R, Hg R2 Tc 100 FP2 100 Rmsl49 R, Hg R2 Sm 100 FP2 Rmsl48e 1.5 R, Hg R2 Sm 100 FP2 RP4 100 R, Hg 100 R2 Tc a ML4262 was used as the recipient. b Ratio of cells carrying only one type of R plasmid to total number of transconjugants obtained when selected for either R, or R2. c 300 .g of Cb per ml was used for selection of Rmsl49-1. d 100 jg of Sm per ml was used for selection of Rmsl48. ' ML4262 was used as the host strain of R plasmids (see text). R2
TABLE 6. Summarized data of the incompatibility grouping of R plasmids in P. aeruginosa R plasmid Group P-i RP4 P-2 Rlb931, Rlbl3O, Rlb680, Rlb290, Rlb546, Rlb442, Rlb454, Rlb3108, Rlb55, Rlbl3, Rsu38, Rms159, Rms161, Rmsl64, Rms178, Rmsl69, Rmsl70, Rmsl71, Rmsl72, Rmsl73, Rmsl74, Rmsl62, Rmsl46, Rmsl96, Rmsl97, Rmsl98, Rmsl99 P-3 RIbl51 P-4 Rlb5265 P-5 Rmsl63, Rmsl76 FP2 P-6 P-7 Rmsl48 Rmsl49 P-8
578
SAGAI ET AL.
Rlb5265 and because of masking of Rmsl48specified streptomycin resistance by Rlb5265. Almost all of the R plasmids demonstrated from P. aeruginosa in Japan have been classified into the P-2 group (Table 6) except two plasmids, Rmsl63 and Rmsl76 (11). A similar prevalence of the P-2 group has been observed among R plasmids detected in isolates of P. aeruginosa in North America (2, 17). Transfer of P-2 group R plasmids by conjugation to E. coli strains has not been observed (1, 11, 17). These results suggest that the P-2 plasmids are representative of conjugative R plasmids in P. aeruginosa and that the origin of R plasmids in P. aeruginosa may be different from that of plasmids identified in species of the Enterobacteriaceae. The P-5 plasmids, Rmsl63 and Rmsl76, were detected inP. aeruginosa strains of the same clinic (11), and both P-7 and P-8 plasmids were isolated from the same strain in Germany (16). These groups are seen to be rare types in P. aeruginosa. We are collecting many R plasmids in an attempt to extend the P-4, P-6, P-7, and P-8 incompatibility groups and to clarify the relationship between the P-4 and the P-7 and P-8 groups.
LITERATURE CITED 1. Bryan, L. E., S. D. Semeka, M. H. Van Den Elzen, J. E. Kinnear, and R. E. S. Whitehouse. 1973. Characteristics of R931 and other Pseudomonas aeruginosa R factors. Antimicrob.. Agents Chemother. 3:625-637. 2. Bryan, L. E., M. S. Shahrabadi, and H. M. Van Den Elzen. 1974. Gentamicin resistance in Pseudomonas aeruginosa R-factor-mediated resistance. Antimicrob. Agents Chemother. 6:191-199. 3. Coetzee, J. N., N. Datta, and R. W. Hedges. 1972. R factors from Proteus rettgeri. J. Gen. Microbiol. 72:543-552. 4. Datta, N. 1974. Epidemiology and classification of plasmids, p. 9-15. In D. Schlessinger (ed.), Microbiology- 1974. American Society for Microbiology, Washington, D.C.
ANTIMICROB. AGENTS CHEMOTHER. 5. Datta, N., and R. W. Hedges. 1971. Compatibility groups among fi- R factors. Nature (London) 234:222223. 6. Datta, N., and R. W. Hedges. 1972. R factors identified in Paris, some conferring gentamicin resistance, constitute a new compatibility group. Ann. Inst. Pasteur Paris 129:849-852. 7. Datta, N., R. W. Hedges, E. J. Shaw, R. B. Sykes, and M. H. Richmond. 1971. Properties of an R factor from Pseudomonas aeruginosa. J. Bacteriol. 108:12441249. 8. Davis, B. D., and E. S. Mingioli. 1960. Mutants of Escherichia coli requiring methionine or vitamin B12. J. Bacteriol. 60:17-29. 9. Hedges, R. W., V. Rodriguez-Lemoine, and N. Datta. 1975. R factors from Serratia marcescens. J. Gen. Microbiol. 86:88-92. 10. Holloway, B. W. 1969. Genetics ofPseudomonas. Bacteriol. Rev. 33:419-443. 11. lyobe, S., K. Hasuda, A. Fuse, and S. Mitsuhashi. 1974. Demonstration of R factors from Pseudomonas aeruginosa. Antimicrob. Agents Chemother. 5:547552. 12. Kawakami, Y., F. Mikoshiba, S. Nagasaki, H. Matsumoto, and T. Tazaki. 1972. Prevalence of Pseudomonas aeruginosa strains possessing R factor in a hospital. J. Antibiot. 25:607-609. 13. Kontomichalou, P., and E. Papachristou. 1975. Multiresistance plasmids in Pseudomonas aeruginosa highly resistant to gentamicin, p. 329-348. In S. Mitsuhashi and H. Hashimoto (ed.), Microbial drug resistance. University of Tokyo Press, Tokyo. 14. Lawn, A. M., G. G. Meynell, E. Meynell, and N. Datta. 1967. Sex pili and the classification of sex factors in theEnterobacteriaceae. Nature (London) 216:343-346. 15. Roe, E., B. J. Jones, and E. J. L. Lowbury. 1971. Transfer of antibiotic resistance between Pseudomonas aeruginosa, Escherichia coli, and other gramnegative bacilli in burns. Lancet 1:149-152. 16. Sagai, H., V. Krcmery, K. Hasuda, S. Iyobe, H. Knothe, and S. Mitauhashi. 1975. R factor mediated resistance to aminoglycoside antibiotics in Pseudomonas aeruginosa. Jpn. J. Microbiol. 19:427-432. 17. Shahrabadi, M. S., L. E. Bryan, and H. M. Van Den Elzen. 1975. Further properties of P-2 R-factors of Pseudomonas aeruginosa and their relationship to other plasmids. Can. J. Microbiol. 21:592-605. 18. Watanabe, T., H. Nishida, C. Ogata, T. Arai, and S. Sato. 1964. Episome-mediated transfer of drug resistance in Enterobacteriaceae. VII. Two types of naturally occurring R factors. J. Bacteriol. 88:716-726.
