Journal of’Genera1Microbiology (1975), 87,30 1-3 Printed in Great Britain
II
R Factors from Proteus mirabilis and P. vulgaris By R. W. H E D G E S Bacteriology Department, Royal Postgraduate Medical School, London W12 oHS (Received 23 September I 974) SUMMARY
Eighty-nine R factors were transmitted by conjugation to Escherichia coli ~ 1 from isolates of Proteus hauseri (P-mirabilis plus P. vulgaris). More than half were non-selftranmissible. The remainder included plasmids assigned to the previously defined groups F I I , A-C complex, J, N and P, as well as some not belonging to any known compatibility groups. R factors from strains isolated in India, Thailand and Japan carried plasmids whose inheritance was extremely unstable in E. coli K12. All belonged to a new compatibility group, V.
2
INTRODUCTION
Swarming Proteus isolates are regarded either as constituting a single species (Proteus hauseri) (Kauffmann, 1951) or two distinct species (P. mirabilis and P. vulgaris) (Hauser, 1885). The question of their separability was discussed by Coetzee (1972). Clearly, even if two species are distinguished they are closely related : they share antigens including phage and bacteriocin receptors (Coetzee, 1972); some, at least, of their enzymes are serologically indistinguishable (Guo & Liu, 1965; Smit & Coetzee, 1967); and they show extensive homology in DNA: DNA hybridization tests (Brenner & Falkow, 1971; Coetzee, 1972). Thus, the group may be considered as a single species or as two closely related species, perhaps constituting a subgenus (Kauffman, 1956). For the purpose of this paper nodistinction will be drawn between P. mirabilis and P. vulgaris, and strains will be referred to as ‘P. mirabilislp. vulgaris’. Several reports of natural isolates of P. mirabilislP. vulgaris carrying R factors appeared during the mid 1960s. These are listed by Coetzee (1972). This work was undertaken to compare the R factor set from P. mirabilis/P. vulgaris with those of P. rettgeri (Coetze, Datta & Hedges, 1972), P. morganii (Hedges, Datta, Coetzee & Dennison, 1973) and Providencia (Hedges, 1974). METHODS
Proteus mirabilis/P. vulgaris. About I ooo cultures, donated by laboratories from many different countries, were tested as R factor donors. Pm13 (Coetzee & Sacks, 1960; Coetzee, 1963) was selected as a standard host for compatibility determination in this species. Two strains were selected for this purpose, Pm I 3-A which carries a spontaneous mutation to nalidixic acid resistance, and Pm13-B which carries a spontaneous rifampicin resistance mutation. Escherichia coli K 1 2 strains 553-1 (F-pro met nalR), 553-2 (F-pro met rifR) and 562 (F-pro his trp lac) were used (Hedges et al. 1973). Phage was phage PRRI (Olsen & Shipley, 1973). 20-2
R. W. H E D G E S
302
Table Plasmid Flac R386 R1drd16 RI-I R I 36 ColB-Kg8 R I 24 Folac RAI RAI-I b R57b R57b-I R4oa R27 R726 R726-I R64 R144 R648 R483 R62I a R62 I a-1 R62 I a-I a R391 R387 R472 R83 I R47 I R446b RIP69 R1~135 R447b N3T R390 R16 R16-1 R724 RP4 R75 I R906 R478 R477-I Rtsr
R40I
R388 R6K PI CM $Amp
Compatibility group FI FI FII FII FII FIII FIV FV A A C C C H H H Iu C IC Iu
IY IY IY
J K L L L M M M N N N 0 0 0 P P P S S T T
w X Y Y
I.
Standard plasmids
Genetic markers*
Reference Jacob & Adelberg (1959) Dennison (I 972) Meynell & Datta (1967) Hedges et al. (1973) Lawn, Meynell, Meynell & Datta (1967)
lac+ T K A, S, C, Su T colB T lac+ T, Su c, s u A, C, Gk, Su
A, K, Su T s, T, c , s u
Hedges & Datta (1972) D a m (1974) Hedges & Datta (1971) Datta & Hedges (I 973) Datta & Hedges (1972~)
I
S, Tp, colIa T A, T, c , K A, c , K K
s, c
A
A,
c, K
s, T A, T, K S, T, G, Su A, K T A, S, T, C, Su A, S, T, Su s, s u
S, T, C, Su A, T, K TP A, S, Su T,c , K S, T, Su K A, s TP,s u A, s C A
Grindley, Grindley & Anderson
(I 972)
Datta & Olarte (1974) Hedges & Datta
(I 973)
Hedges (1974) Segregant of R62 I a-1 Coetzee et al. (1972) Hedges & Datta (1971) Hedges, Rodriguez-Lemoine & Datta (19746) Hedges et al. (1973) Witchitz & Gerbaud (1972) Hedges et al. (1973) Hedges (I 972) Coetzee et al. (1972) Datta & Olarte (1974) Hedges (1 974) Datta & Olarte (1974) Datta et al. (1971) Jobanputra & Datta (1974) Hedges, Jacob & Smith (1974~) Hedges et al. (1974b) Coetzee et al. (1972) Datta & Hedges (1972b) Kontomichalou, Mitani & Clowes (1970) Ikeda & Tomizawa (1968); Smith (1972); J. R. Scott & R. W. Hedges (unpublished); M. Yarmolinsky (unpublished).
* A = ampicillin, S = streptomycin, T = tetracycline, C = chloramphenicol, K = kanamycin, Gk = gentamicin and kanamycin, G = gentamicin, Su = sulphonamides, Tp = trimethoprim.
R factors from P. mirabilis and P. vulgaris Table
2.
Drug sensitivity patterns of P. mirabilis1P. vulgaris isolates
Boston 7 -
London
No.
Percentage
I 18 I0
59 5 4
8 2
4 23 I I 0 0
6
20 I
5 3 202
I 2
I1 < I < I 0 0
3
I0 < I 2 I
303
Vancouver
y h _ _ _ _ 7
No. 324
27 25 6 I
7
0 0 2
3 5
0 0 0
0
400
*
Percentage
7
No.
Percentage
81 7 6
45
28
24
15
I0
6
I < I 2 0 0 < I I I 0 0 0 0
2
I
26
42 5
3
I
< I
30
I9 0 0 0 < I 0 0 0
0 0 0 I 0 0 0
I 60
For abbreviations see Table
Phenotype resistances* Sensitive A
su
S K
s, s u s, K K, Su A, s
A, K A, S, Su S, K, Su A, S, C, Su A, S, K, Su A, S, C, K, Su
Total I.
Plasmids are listed in Table I . Techniques were as described by Datta et al. (197i), Dennison (1972) and Coetzee et al.
R E S U L T S A N D DISCUSSION
Resistance patterns of P. mirabilislP. vulgaris isolates Approximately 1000 separate P. mirabilis/P. vulgaris isolates from a wide range of geographical soures were tested for drug resistance. Almost all P. mirabilis/P. vulgaris iso1ates:are insensitive to tetracycline and polymixin, so these drugs were not included in the present investigation. The drugs used were ampicillin, streptomycin, chloramphenicol, kanamycin, gentamicin, trimethoprim and sulphadimidine. Many of the cultures tested were selected by the bacteriologists who isolated the P. mirabiZislP. vulgaris strains as showing atypical drug resistance patterns and hence likely to carry R factors. Thus, the resistance patterns of the complete collections are not representative of the group. However, large collections of unselected isolates were available from three sources: Dr H. Gaya, Hammersmith Hospital, London; Professor M. Finland, Boston City Hospital, Massachusetts, U.S.A.; and Mrs R. Dobrey and Miss C . Malcolm, Shaughnessy Hospital, Vancouver, British Columbia, Canada. The resistance phenotypes of the cultures from these collections are listed in Table 2. The marked differences in the resistance patterns of three groups and the differences between these and the patterns reported from by other authors from other sources (e.g. Tomaschoff, 1969; Chai & Soo-Hoo, 1970; Dikyi, 1972; von Graeventiz & Nourbakhsh, 1972) indicates that, as is the case with Serratia marcescens (Hedges et al. 1974b) the P. mirabilislP. vulgaris populations of different hospitals are often distinct. This is confirmed by the differences between the specificities of R factors from the different sources (Table 3).
R. W. HEDGES
304
Table 3. R factors from P . mirabilislP. vulgaris Source Prof. M. Finland (Boston) Prof. M. Finland (Boston)
Resistances?
N N
1 1
Prof. M. Finland (Boston)
K(tru-)
Prof. M. Finland (Boston) Prof. M. Finland (Boston)
A,
Prof. M. Finland (Boston)
Su(tru-)
s, T
s, Su(tru-)
N
D r H. Gaya (London) Dr H. Gaya (London) Dr H. Gaya (London)
R439, R440 R436b, R437b R468, R469 R470 R704a R704b R959 R645 R665 R682, R683, R684 R70 I R702 R705, R706 R753, R754, R755, R756 R757a R757b, R758 R769, R770 R866, R867, R868 R869, R870 R871 R9or , R902, Rgo3 R904 R905 R940
I
Compatibility
A, s, K A, s
FII
D r H. Gaya (London) Miss C. Malcolm (Vancouver) Miss C. Malcolm (Vancouver)
Su(tru-) Su(tru-) s, Su(tru-)
Miss C . Malcolm (Vancouver)
K(tra-)
Prof. S. Falkow (Seattle) Dr M. Yoshikawa (Tokyo) Dr P. Kontomichalou (Athens) Dr P. Kontomichalou (Athens)
su K A(tru-) S
Dr E. J. L. Lowbury (Birmingham) Dr E. J. L. Lowbury (Birmingham) Dr F. Anderson (London) Dr F. Anderson (London) Dr F. Anderson (London) Dr F. Anderson (London) D r F. Anderson (London) Dr I. Phillips (London) Dr E. Tomaschoff (Dusseldorf) Dr E. Tomaschoff (Dusseldorf) Dr F. Sabatelli (Bloomfield, U.S.A.) D r F. Sabatelli (Bloomfield, U.S.A.) Prof. J. N. Coetzee (Pretoria)
A, T, K s, Su(tru-) K( tra-) Su(tra-) A, S, C, K, Su T, Su A, S, Su(tru-) A, S, Su(tru-) A, Su S, Su(tru-) A, S, K S, T, K, Su K
P
Prof. S. Falkow (Ching Mai, Thailand) Prof. S. Falkow (Ching Mai, Thialand) Prof. S. Falkow (Ching Mai, Thailand) Dr C. K. J. Paniker (Kerala, India) Dr M. Lachmajer (Gdansk, Poland) Dr M. Lachmajer (Gdansk, Poland) Dr J. N. Wilfert (Salt Lake City) Prof. S. Mitsuhashi (Maebashi, Japan) Prof. S. Mitsuhashi (Maebashi, Japan) Prof. S. Mitsuhashi (Maebashi, Japan) Miss M. M. Moody (Bethesda, Md., USA)
A, S, C, Su A, S, Su
V V
S, C, K, Su A(tru-) A, S, Su(tru-)
V
A, S , T , K, Su
P
s, Su(tru-)
su S, C, K, Su s, Su(tru-) s, c , s u
A-C complex T
N
FII A-C complex A-C complex
N P J
A-C complex V V
* These plasmids were transferred from P. mirabilislp. vulgaris strains before being sent to Hammersmith . t tra- = non selftransmissible (NST). For other abbreviations see Table I . j: See text. No attempt has been made to determine the incompatibility relationships of the NST R factors. -~
R factors from P. mirabilis and P. vulgaris
305
Transfer of resistance determinants from P. mirabilislp. vulgaris to Escherichia coli ~ 1 2 Natural isolates of P. mirabilislP. vulgaris showing resistance to any of the antibiotics under investigation were grown with cultures of E. coli K 1 2 553-1 or 553-2 and any Rf E. coli transcipients were purified on MacConkey agar. The transferred resistance factors are listed in Table 3. Plasmid-carrying strains of ~ 5 3 - Iand 553-2 were incubated with cultures of 562. Plasmids , which did transfer to this strain but at least which either were not transferable to ~ 6 2 or 1 0o/o of whose transcipients were not capable of transfering the resistances to further E. coli K I 2 strains, were considered to be non-selftransmissible (NST). One of the most striking features of the R factor set from P. mirabilislP. vulgaris is the remarkably high proportion of NST plasmids; of' 89 R factors transferred from P. mirabilis/P. vulgaris strains 48 were NST. This contrasts with a single NST plasmid among 105 from Providencia isolates (Hedges, 1974), 5 of 28 from Proteus rnorgmii (Hedges et al. 1973) and none of 28 from Serratia marcescens (Hedges et al. 19743). The high proportion of NST plasmids among the set transferred from P. mirabilisl P. vulgaris implies the presence of transfer factors (not, themselves, conferring drug resistances) in an appreciable proportion of P. mirabilislP. vulgaris isolates. In conformity with this conclusion, Mitsuhashi (1969) reported the detection of transfer factors in several isolates of Proteus (species not recorded). Numerous authors (for references and discussion see Coetzee, 1972) have concluded that certain R factors, which in E. coli exist as single DNA molecules (composites), dissociate in P. mirabilis/P. vulgaris strains into transfer factors (Cohen & Miller, 1970) and presumably into non-selftransmissible plasmids, carrying the resistance genes, which replicate under relaxed control. It is tempting to postulate that the NST plasmids and transfer factors are derived from such dissociated R factors. Studies on DNA homologies of such plasmids may provide evidence. Since investigation of the compatibility properties of NST plasmids has been undertaken by Smith, Humphreys & Anderson (1g74), compatibility properties of NST plasmids from P. mirabilislP. vulgaris will be described only in those cases where an NST plasmid showed interaction with self-transmissible factors. The DNA molecules of several of the NST P. mirabilis/P. vulgaris plasmids have been investigated (Barth & Grinter, I 974; S. Falkow, personal communication). They have been found to be small (molecular weight less than ro6 daltons), and similar to those described by Milliken & Clowes (1973), Smith et al. (1974) and Barth & Grinter (1974). R n K a , an NST plasmid determining a p-lactamase very similar to that of the E. coli chromosome, is an exception. This is a plasmid of molecular weight 6-05 x 10' whose replication is subject to stringent control. Its properties have been described by M. M. Bobrovski and others (unpublished). Properties of the sev- t ransm issible plasm ids R494, R842, R843 and R704a. These plasmids are all members of group FII. The first three are apparently identical R factors, all from Hammersmith Hospital. They have surface exclusion of the same specificity as R444 (Hedges et al. 1973). R704a, from The West Middlesex Hospital, London, closely resembles R I .
306
R. W. H E D G E S
Table 4. Reduction of rate of transfer of P group plasmids bjj R factors of group la The recipient was always ~ 6 2 . Drug used in selection*
Donor
Efficiency of transfer
T K K T T K K T K K
*
For abbreviations see Table
I x IO-~ I
x IO-~
I x IO-~
2 x IO-~
7x IO-~ 9x IO-~ I x IO-~ I x IO-~
4 X 10-3 7 X 10-5 I.
In that their R factor set includes FII plasmids, P. mirabilis/P. vulgaris resembles P. rnorganii. R22Kb, R393, R701, R759a, R760a, R761a and R771a. Proteus mirabilislP. vulgaris isolates carrying R factors of group N have been obtained from Athens, London, Boston and New Jersey. Plasmids of this group were, thus, well represented in the R factor set from P. rnirabilis/P. vulgaris. They were abundant among plasmids from P. rettgeri and P. morganii and were also observed in isolates of Providencia. Plac, R665, R704b and R871. Plasmids of the A-C complex (Hedges, 1974) were transferred from P. rnirabi1islP. vulgaris isolates from Germany, U.K., U.S.A. and Canada. They therefore seem widespread in this species, although much less abundant than in Providencia. It may be significant that three of the A-C plasmids came from sources which had supplied Providencia isolates carrying A-C plasmids (West Middlesex Hospital, London; Shaughnessy Hospital, Vancouver, Canada ; University of Utah Medical Center, Salt Lake City, U.S.A.). R436a, R437a, R438, R439, R440, R702 and R940. Plasmids of group P were reported in P. mirabilis/P. vulgaris by Roe, Jones & Lowbury (1971). The properties of R702 have been described by Hedges & Jacob (1974). Although plasmids of this group have been detected in a wide range of genera, P. mirabilislP. vulgaris is the first species in which plasmids with three distinct resistance patterns have been observed. As with RP4 (Datta et al. 1971) the efficiency of transfer of these plasmids was reduced by the presence of R64 (or other members of compatibility group Ia) in the donor (Table 4). RtsI, This plasmid, transferred from an isolate of P. mirabilis/P. vulgaris (Terawaki, Takayasu & Akiba, 1967),is one of the prototypes of group T. Other plasmids of this group have been detected only in P. rettgeri (Coetzee et al. 1972). R705 and R706. These two plasmids, conferring resistance to kanamycin, are apparently identical with R factors such as R39I and R748, transferred from P. rettgeri (Coetzee et al. 1972) and Providencia (Hedges, I974), respectively. All came from strains isolated in Pretoria. All are provisionally assigned to group J, but because of identity of genetic markers direct testing has not been possible. R753, R754, R755, R757a, R769, R770, R901, R902, Rgo3 and R905. These R factors were all transmissible into E. coli K 1 2 strains but were very unstably inherited, often being lost during replication of an R+ E. coli culture unless selection against R- segregants was
R factors from P. mirabilis and P. vulgaris
307
Table 5. Compatibility properties of representative plasmids of group V
-
The selective medium was MacConkey aga.r+rifampicin+ kanamycin. The donor was P ~3AI (R769-I), a segregant of R769 which had lost the chloramphenicol resistance determinant (i.e. it conferred resistance to streptomycin, kanamycin and sulphonamides).
Recipient PmI 3-B Pm13-B(R753) PmI 3-B(Rgog)
Frequency of transfer *
<
II
R Factors from Proteus mirabilis and P. vulgaris By R. W. H E D G E S Bacteriology Department, Royal Postgraduate Medical School, London W12 oHS (Received 23 September I 974) SUMMARY
Eighty-nine R factors were transmitted by conjugation to Escherichia coli ~ 1 from isolates of Proteus hauseri (P-mirabilis plus P. vulgaris). More than half were non-selftranmissible. The remainder included plasmids assigned to the previously defined groups F I I , A-C complex, J, N and P, as well as some not belonging to any known compatibility groups. R factors from strains isolated in India, Thailand and Japan carried plasmids whose inheritance was extremely unstable in E. coli K12. All belonged to a new compatibility group, V.
2
INTRODUCTION
Swarming Proteus isolates are regarded either as constituting a single species (Proteus hauseri) (Kauffmann, 1951) or two distinct species (P. mirabilis and P. vulgaris) (Hauser, 1885). The question of their separability was discussed by Coetzee (1972). Clearly, even if two species are distinguished they are closely related : they share antigens including phage and bacteriocin receptors (Coetzee, 1972); some, at least, of their enzymes are serologically indistinguishable (Guo & Liu, 1965; Smit & Coetzee, 1967); and they show extensive homology in DNA: DNA hybridization tests (Brenner & Falkow, 1971; Coetzee, 1972). Thus, the group may be considered as a single species or as two closely related species, perhaps constituting a subgenus (Kauffman, 1956). For the purpose of this paper nodistinction will be drawn between P. mirabilis and P. vulgaris, and strains will be referred to as ‘P. mirabilislp. vulgaris’. Several reports of natural isolates of P. mirabilislP. vulgaris carrying R factors appeared during the mid 1960s. These are listed by Coetzee (1972). This work was undertaken to compare the R factor set from P. mirabilis/P. vulgaris with those of P. rettgeri (Coetze, Datta & Hedges, 1972), P. morganii (Hedges, Datta, Coetzee & Dennison, 1973) and Providencia (Hedges, 1974). METHODS
Proteus mirabilis/P. vulgaris. About I ooo cultures, donated by laboratories from many different countries, were tested as R factor donors. Pm13 (Coetzee & Sacks, 1960; Coetzee, 1963) was selected as a standard host for compatibility determination in this species. Two strains were selected for this purpose, Pm I 3-A which carries a spontaneous mutation to nalidixic acid resistance, and Pm13-B which carries a spontaneous rifampicin resistance mutation. Escherichia coli K 1 2 strains 553-1 (F-pro met nalR), 553-2 (F-pro met rifR) and 562 (F-pro his trp lac) were used (Hedges et al. 1973). Phage was phage PRRI (Olsen & Shipley, 1973). 20-2
R. W. H E D G E S
302
Table Plasmid Flac R386 R1drd16 RI-I R I 36 ColB-Kg8 R I 24 Folac RAI RAI-I b R57b R57b-I R4oa R27 R726 R726-I R64 R144 R648 R483 R62I a R62 I a-1 R62 I a-I a R391 R387 R472 R83 I R47 I R446b RIP69 R1~135 R447b N3T R390 R16 R16-1 R724 RP4 R75 I R906 R478 R477-I Rtsr
R40I
R388 R6K PI CM $Amp
Compatibility group FI FI FII FII FII FIII FIV FV A A C C C H H H Iu C IC Iu
IY IY IY
J K L L L M M M N N N 0 0 0 P P P S S T T
w X Y Y
I.
Standard plasmids
Genetic markers*
Reference Jacob & Adelberg (1959) Dennison (I 972) Meynell & Datta (1967) Hedges et al. (1973) Lawn, Meynell, Meynell & Datta (1967)
lac+ T K A, S, C, Su T colB T lac+ T, Su c, s u A, C, Gk, Su
A, K, Su T s, T, c , s u
Hedges & Datta (1972) D a m (1974) Hedges & Datta (1971) Datta & Hedges (I 973) Datta & Hedges (1972~)
I
S, Tp, colIa T A, T, c , K A, c , K K
s, c
A
A,
c, K
s, T A, T, K S, T, G, Su A, K T A, S, T, C, Su A, S, T, Su s, s u
S, T, C, Su A, T, K TP A, S, Su T,c , K S, T, Su K A, s TP,s u A, s C A
Grindley, Grindley & Anderson
(I 972)
Datta & Olarte (1974) Hedges & Datta
(I 973)
Hedges (1974) Segregant of R62 I a-1 Coetzee et al. (1972) Hedges & Datta (1971) Hedges, Rodriguez-Lemoine & Datta (19746) Hedges et al. (1973) Witchitz & Gerbaud (1972) Hedges et al. (1973) Hedges (I 972) Coetzee et al. (1972) Datta & Olarte (1974) Hedges (1 974) Datta & Olarte (1974) Datta et al. (1971) Jobanputra & Datta (1974) Hedges, Jacob & Smith (1974~) Hedges et al. (1974b) Coetzee et al. (1972) Datta & Hedges (1972b) Kontomichalou, Mitani & Clowes (1970) Ikeda & Tomizawa (1968); Smith (1972); J. R. Scott & R. W. Hedges (unpublished); M. Yarmolinsky (unpublished).
* A = ampicillin, S = streptomycin, T = tetracycline, C = chloramphenicol, K = kanamycin, Gk = gentamicin and kanamycin, G = gentamicin, Su = sulphonamides, Tp = trimethoprim.
R factors from P. mirabilis and P. vulgaris Table
2.
Drug sensitivity patterns of P. mirabilis1P. vulgaris isolates
Boston 7 -
London
No.
Percentage
I 18 I0
59 5 4
8 2
4 23 I I 0 0
6
20 I
5 3 202
I 2
I1 < I < I 0 0
3
I0 < I 2 I
303
Vancouver
y h _ _ _ _ 7
No. 324
27 25 6 I
7
0 0 2
3 5
0 0 0
0
400
*
Percentage
7
No.
Percentage
81 7 6
45
28
24
15
I0
6
I < I 2 0 0 < I I I 0 0 0 0
2
I
26
42 5
3
I
< I
30
I9 0 0 0 < I 0 0 0
0 0 0 I 0 0 0
I 60
For abbreviations see Table
Phenotype resistances* Sensitive A
su
S K
s, s u s, K K, Su A, s
A, K A, S, Su S, K, Su A, S, C, Su A, S, K, Su A, S, C, K, Su
Total I.
Plasmids are listed in Table I . Techniques were as described by Datta et al. (197i), Dennison (1972) and Coetzee et al.
R E S U L T S A N D DISCUSSION
Resistance patterns of P. mirabilislP. vulgaris isolates Approximately 1000 separate P. mirabilis/P. vulgaris isolates from a wide range of geographical soures were tested for drug resistance. Almost all P. mirabilis/P. vulgaris iso1ates:are insensitive to tetracycline and polymixin, so these drugs were not included in the present investigation. The drugs used were ampicillin, streptomycin, chloramphenicol, kanamycin, gentamicin, trimethoprim and sulphadimidine. Many of the cultures tested were selected by the bacteriologists who isolated the P. mirabiZislP. vulgaris strains as showing atypical drug resistance patterns and hence likely to carry R factors. Thus, the resistance patterns of the complete collections are not representative of the group. However, large collections of unselected isolates were available from three sources: Dr H. Gaya, Hammersmith Hospital, London; Professor M. Finland, Boston City Hospital, Massachusetts, U.S.A.; and Mrs R. Dobrey and Miss C . Malcolm, Shaughnessy Hospital, Vancouver, British Columbia, Canada. The resistance phenotypes of the cultures from these collections are listed in Table 2. The marked differences in the resistance patterns of three groups and the differences between these and the patterns reported from by other authors from other sources (e.g. Tomaschoff, 1969; Chai & Soo-Hoo, 1970; Dikyi, 1972; von Graeventiz & Nourbakhsh, 1972) indicates that, as is the case with Serratia marcescens (Hedges et al. 1974b) the P. mirabilislP. vulgaris populations of different hospitals are often distinct. This is confirmed by the differences between the specificities of R factors from the different sources (Table 3).
R. W. HEDGES
304
Table 3. R factors from P . mirabilislP. vulgaris Source Prof. M. Finland (Boston) Prof. M. Finland (Boston)
Resistances?
N N
1 1
Prof. M. Finland (Boston)
K(tru-)
Prof. M. Finland (Boston) Prof. M. Finland (Boston)
A,
Prof. M. Finland (Boston)
Su(tru-)
s, T
s, Su(tru-)
N
D r H. Gaya (London) Dr H. Gaya (London) Dr H. Gaya (London)
R439, R440 R436b, R437b R468, R469 R470 R704a R704b R959 R645 R665 R682, R683, R684 R70 I R702 R705, R706 R753, R754, R755, R756 R757a R757b, R758 R769, R770 R866, R867, R868 R869, R870 R871 R9or , R902, Rgo3 R904 R905 R940
I
Compatibility
A, s, K A, s
FII
D r H. Gaya (London) Miss C. Malcolm (Vancouver) Miss C. Malcolm (Vancouver)
Su(tru-) Su(tru-) s, Su(tru-)
Miss C . Malcolm (Vancouver)
K(tra-)
Prof. S. Falkow (Seattle) Dr M. Yoshikawa (Tokyo) Dr P. Kontomichalou (Athens) Dr P. Kontomichalou (Athens)
su K A(tru-) S
Dr E. J. L. Lowbury (Birmingham) Dr E. J. L. Lowbury (Birmingham) Dr F. Anderson (London) Dr F. Anderson (London) Dr F. Anderson (London) Dr F. Anderson (London) D r F. Anderson (London) Dr I. Phillips (London) Dr E. Tomaschoff (Dusseldorf) Dr E. Tomaschoff (Dusseldorf) Dr F. Sabatelli (Bloomfield, U.S.A.) D r F. Sabatelli (Bloomfield, U.S.A.) Prof. J. N. Coetzee (Pretoria)
A, T, K s, Su(tru-) K( tra-) Su(tra-) A, S, C, K, Su T, Su A, S, Su(tru-) A, S, Su(tru-) A, Su S, Su(tru-) A, S, K S, T, K, Su K
P
Prof. S. Falkow (Ching Mai, Thailand) Prof. S. Falkow (Ching Mai, Thialand) Prof. S. Falkow (Ching Mai, Thailand) Dr C. K. J. Paniker (Kerala, India) Dr M. Lachmajer (Gdansk, Poland) Dr M. Lachmajer (Gdansk, Poland) Dr J. N. Wilfert (Salt Lake City) Prof. S. Mitsuhashi (Maebashi, Japan) Prof. S. Mitsuhashi (Maebashi, Japan) Prof. S. Mitsuhashi (Maebashi, Japan) Miss M. M. Moody (Bethesda, Md., USA)
A, S, C, Su A, S, Su
V V
S, C, K, Su A(tru-) A, S, Su(tru-)
V
A, S , T , K, Su
P
s, Su(tru-)
su S, C, K, Su s, Su(tru-) s, c , s u
A-C complex T
N
FII A-C complex A-C complex
N P J
A-C complex V V
* These plasmids were transferred from P. mirabilislp. vulgaris strains before being sent to Hammersmith . t tra- = non selftransmissible (NST). For other abbreviations see Table I . j: See text. No attempt has been made to determine the incompatibility relationships of the NST R factors. -~
R factors from P. mirabilis and P. vulgaris
305
Transfer of resistance determinants from P. mirabilislp. vulgaris to Escherichia coli ~ 1 2 Natural isolates of P. mirabilislP. vulgaris showing resistance to any of the antibiotics under investigation were grown with cultures of E. coli K 1 2 553-1 or 553-2 and any Rf E. coli transcipients were purified on MacConkey agar. The transferred resistance factors are listed in Table 3. Plasmid-carrying strains of ~ 5 3 - Iand 553-2 were incubated with cultures of 562. Plasmids , which did transfer to this strain but at least which either were not transferable to ~ 6 2 or 1 0o/o of whose transcipients were not capable of transfering the resistances to further E. coli K I 2 strains, were considered to be non-selftransmissible (NST). One of the most striking features of the R factor set from P. mirabilislP. vulgaris is the remarkably high proportion of NST plasmids; of' 89 R factors transferred from P. mirabilis/P. vulgaris strains 48 were NST. This contrasts with a single NST plasmid among 105 from Providencia isolates (Hedges, 1974), 5 of 28 from Proteus rnorgmii (Hedges et al. 1973) and none of 28 from Serratia marcescens (Hedges et al. 19743). The high proportion of NST plasmids among the set transferred from P. mirabilisl P. vulgaris implies the presence of transfer factors (not, themselves, conferring drug resistances) in an appreciable proportion of P. mirabilislP. vulgaris isolates. In conformity with this conclusion, Mitsuhashi (1969) reported the detection of transfer factors in several isolates of Proteus (species not recorded). Numerous authors (for references and discussion see Coetzee, 1972) have concluded that certain R factors, which in E. coli exist as single DNA molecules (composites), dissociate in P. mirabilis/P. vulgaris strains into transfer factors (Cohen & Miller, 1970) and presumably into non-selftransmissible plasmids, carrying the resistance genes, which replicate under relaxed control. It is tempting to postulate that the NST plasmids and transfer factors are derived from such dissociated R factors. Studies on DNA homologies of such plasmids may provide evidence. Since investigation of the compatibility properties of NST plasmids has been undertaken by Smith, Humphreys & Anderson (1g74), compatibility properties of NST plasmids from P. mirabilislP. vulgaris will be described only in those cases where an NST plasmid showed interaction with self-transmissible factors. The DNA molecules of several of the NST P. mirabilis/P. vulgaris plasmids have been investigated (Barth & Grinter, I 974; S. Falkow, personal communication). They have been found to be small (molecular weight less than ro6 daltons), and similar to those described by Milliken & Clowes (1973), Smith et al. (1974) and Barth & Grinter (1974). R n K a , an NST plasmid determining a p-lactamase very similar to that of the E. coli chromosome, is an exception. This is a plasmid of molecular weight 6-05 x 10' whose replication is subject to stringent control. Its properties have been described by M. M. Bobrovski and others (unpublished). Properties of the sev- t ransm issible plasm ids R494, R842, R843 and R704a. These plasmids are all members of group FII. The first three are apparently identical R factors, all from Hammersmith Hospital. They have surface exclusion of the same specificity as R444 (Hedges et al. 1973). R704a, from The West Middlesex Hospital, London, closely resembles R I .
306
R. W. H E D G E S
Table 4. Reduction of rate of transfer of P group plasmids bjj R factors of group la The recipient was always ~ 6 2 . Drug used in selection*
Donor
Efficiency of transfer
T K K T T K K T K K
*
For abbreviations see Table
I x IO-~ I
x IO-~
I x IO-~
2 x IO-~
7x IO-~ 9x IO-~ I x IO-~ I x IO-~
4 X 10-3 7 X 10-5 I.
In that their R factor set includes FII plasmids, P. mirabilis/P. vulgaris resembles P. rnorganii. R22Kb, R393, R701, R759a, R760a, R761a and R771a. Proteus mirabilislP. vulgaris isolates carrying R factors of group N have been obtained from Athens, London, Boston and New Jersey. Plasmids of this group were, thus, well represented in the R factor set from P. rnirabilis/P. vulgaris. They were abundant among plasmids from P. rettgeri and P. morganii and were also observed in isolates of Providencia. Plac, R665, R704b and R871. Plasmids of the A-C complex (Hedges, 1974) were transferred from P. rnirabi1islP. vulgaris isolates from Germany, U.K., U.S.A. and Canada. They therefore seem widespread in this species, although much less abundant than in Providencia. It may be significant that three of the A-C plasmids came from sources which had supplied Providencia isolates carrying A-C plasmids (West Middlesex Hospital, London; Shaughnessy Hospital, Vancouver, Canada ; University of Utah Medical Center, Salt Lake City, U.S.A.). R436a, R437a, R438, R439, R440, R702 and R940. Plasmids of group P were reported in P. mirabilis/P. vulgaris by Roe, Jones & Lowbury (1971). The properties of R702 have been described by Hedges & Jacob (1974). Although plasmids of this group have been detected in a wide range of genera, P. mirabilislP. vulgaris is the first species in which plasmids with three distinct resistance patterns have been observed. As with RP4 (Datta et al. 1971) the efficiency of transfer of these plasmids was reduced by the presence of R64 (or other members of compatibility group Ia) in the donor (Table 4). RtsI, This plasmid, transferred from an isolate of P. mirabilis/P. vulgaris (Terawaki, Takayasu & Akiba, 1967),is one of the prototypes of group T. Other plasmids of this group have been detected only in P. rettgeri (Coetzee et al. 1972). R705 and R706. These two plasmids, conferring resistance to kanamycin, are apparently identical with R factors such as R39I and R748, transferred from P. rettgeri (Coetzee et al. 1972) and Providencia (Hedges, I974), respectively. All came from strains isolated in Pretoria. All are provisionally assigned to group J, but because of identity of genetic markers direct testing has not been possible. R753, R754, R755, R757a, R769, R770, R901, R902, Rgo3 and R905. These R factors were all transmissible into E. coli K 1 2 strains but were very unstably inherited, often being lost during replication of an R+ E. coli culture unless selection against R- segregants was
R factors from P. mirabilis and P. vulgaris
307
Table 5. Compatibility properties of representative plasmids of group V
-
The selective medium was MacConkey aga.r+rifampicin+ kanamycin. The donor was P ~3AI (R769-I), a segregant of R769 which had lost the chloramphenicol resistance determinant (i.e. it conferred resistance to streptomycin, kanamycin and sulphonamides).
Recipient PmI 3-B Pm13-B(R753) PmI 3-B(Rgog)
Frequency of transfer *
<
