PLASMID
1,
346-356 (1978)
Efficient Transfer of Conjugative Plasmids by Multipoint Inoculation and Some Observations on Host Range and Prevalence of R Plasmids LARS G. BURMAN Department
of Clinical Bacteriology,
AND RUNE &TENSSON University
of Umeii, S-901 85 Urn&, Sweden
Accepted February 22, 1978 The conjugational transfer of R plasmids was demonstrated using a simple manually operated multipoint inoculator apparatus (MIP) allowing rapid inoculation and later dilution and plating of 25 mating mixtures simultaneously. Forty-five R plasmids belonging to groups F, I, N, and others originally recovered inEscherichia coli K-12 were studied in this as well as in other hosts. The semiquantitative MIP conjugation method was more efficient than conventional matings, particularly when performed in two steps employing E. co/i K-12 as intermediate host. Both as donor and as recipient, E. coli K-12 was the most “suitable” general host of the set of plamids studied, although with many plasmids the degree of expression of their transfer functions varied with the host. The expression of fertility in parental bacteria as well as factors in the new host not studied appeared to be of greater importance for the conjugational transfer of a plasmid than the host-specified restriction of plasmid deoxyribonucleic acid by the recipient strain. The MIP conjugation method was successfully used also during screening for transferable R plasmids in gram-negative bacteria present in urine and fecal specimens of humans. The use of a restrictionless mutant instead of a restricting K-12 recipient enabled the detection of additional plasmids. The labor and media-saving MIP conjugation method thus also offers efficiency and is very practical for the performance of large numbers of plasmid matings, for example, in studies of compatibilty, host range, and mobilization of plasmids, as well as for screening purposes.
Since the multipoint replicator was first described by Garrett in 1946, this tool has been used in various situations for speeding up the laboratory testing of microorganisms [see review, Ref. (IO)]. The analysis of progeny in genetic experiments, phage typing, and antibiotic susceptibility testing are situations where multipoint replicators save much labor and media by enabling the rapid inoculation of test plates with large numbers of samples of bacteria or phage. We recently adapted a simple manually operated multipoint inoculator apparatus for the demonstration on undivided test plates of the biochemical activities and motility of strains of bacteria and yeasts and, in particular, for use in recognizing members of the family Enterobacteriuceae. The method is referred 0147-619X/78/0013-0346$02.00/0 Copyright All rights
0 1978 by Academic Press, Inc. of reproduction in any form reserved.
346
to as the multipoint inoculation plate (MIP) method (7). In the present study the multipoint equipment was employed through most stages of intrageneric and intergeneric semiquantitative R plasmid conjugation experiments, using strains of Escherichia coli, Salmonella typhimurium, Serratia marcescens, and Shigella sonnei as well as other species of gram-negative bacteria. The ability of the MIP method to demonstrate R plasmid transfer appeared to be superior to that of carefully performed conventional matings, even compared to the optimized but laborious highfrequency of resistance transfer (HFRT) method of Watanabe (14). The application of the MIP principle to R plasmid conjugation thus enables a considerable reduction in the
R PLASMID TRANSFER BY MULTIPOINT
347
INOCULATION
TABLE 1 STRAINS OF GRAM-NEGATIVE BACTERIA USED
Strain
Sex
Chromosomal markers”
Source or reference
proA, trp, his proA, trp, his, nalA proA, trp, his, rpo proA, trp, his, rpo, nalA galK, galT, lac, met, thi, supE, hsr, hsm+ galK, galT, lac, met, thi, supE, hsr, hsm+, nalA, rpo
Dl is RC711 (8) Mutant of Dl (5) Mutant of Dl (5) Mutant of Dl (5) W. Arber (17) Mutant of WA802; this study
Wild type
G. Bertani (3) Mutant of C1055; this study Mutant of C1055; this study Clinical isolate; this study Mutant of UM3; this study Mutant of UM3; this study Clinical isolate; this study Mutant of UMl; this study Clinical isolate; this study Mutant of UM2; this study
E. coli K-12
Dl Dlnal Dlrif Dlnal, rif WA802 WA802na1, rif E. coli Cl055 E. coli ClOSSnal E. coli ClOSSrif S. marcescens UM3 5’. marcescens UM3nal S. marcescens UM3rif S. typhimurium UMI 5’. typhimurium UMlnal S. sonnei UM2 S. sonnei UM2 nal
FFFFFFFFFFFFFFFF-
nalA
rpo Wild type
(?) (‘7) (?) (?)
na/A rpo
(?)
nalA
Wild type
(?) Wild type (?)
nalA
a Abbreviations: gal, galactose; his, histidine; nalA, high-level nahdixic acid resistance; hsm, host-specific modification of deoxyribonucleic acid (DNA); hsr, host-specific restriction of DNA; luc, lactose; met, methionine; pro, proline; rpo, ribonucleic acid polymerase (rifampicin resistance); sup, suppression; thi, thiamine; trp, tryptophan.
time required for inoculations and in the consumption of media, without a loss of sensitivity. MATERIALS
AND METHODS
In a second series of conjugation experiments, drug-resistant strains of gram-negative bacteria isolated from urine and fecal specimens received at the Bacteriological Laboratory, Region Hospital, UmeA, were
Bacteria and R plasmids. In most of the conjugation experiments, spontaneous mutants of Escherichia coli K-12, E. coli C,
TABLE 2 SUMMARY OF 4.5 R PLASMIDS STUDIED~
Salmonella typhimurium, Serratia marcescens, or Shigella sonnei, chromosomally
resistant to rifampicin (100 pug/ml) and/or nalidixic acid (100cLg/ml),were used as donors or recipients of R plasmids. These strains are listed in Table 1. Of the 45 known R plasmids used, one type I plasmid (R64) and four type N plasmids (R15, R46, R128, and N3) were kindly provided by Naomi Datta, London. The other plasmids studied originated from the collection of this laboratory and were recently described (6). These plasmids all mediate resistance to tetracycline (and some of them also to other antibiotics) and their general characters are summarized in Table 2.
Number of plasmids determining sex pilus of typec fin”
F
I
N
+ -
9
2
1
11
Mixed
Unknown
2
1
7
2
3 6
(1Most of the plasmids were from clinical isolates of E. coli and recovered in E. coli K-12. In a few cases,
the original host of the plasmid is not known. b Fertility inhibition, i.e., the abiity to repress the fertility of the plasmid F, as determined in Ref. (6). c The plasmids were classified with regard to the type of conjugative pilus, determined as described in Ref. (6). The designation N pilus is used here despite the fact that surface appendages resembling conjugative pili have not yet been demonstrated on cells carrying type N conjugative plasmids.
348
BURMANAND~~STENSSON
identified and tested for R plasmid donor ability. Transferable resistance to ampicillin (Ap), chloramphenicol (Cm), streptomycin (Sm), and tetracycline (Tc) was then studied. The wild isolates were identified as described in Ref. (7). Media and growth conditions. The liquid medium used was LB of Bertani (2), always supplemented with glucose (0.2%) and medium E (16). All plates contained PDM agar especially designed for antibiotic susceptibility testing of bacteria (AB Biodisk, Stockholm). Incubations were performed at 37°C. The absorbance of liquid cultures was determined using a Klett-Summerson colorimeter equipped with a W66 filter. Conjugation experiments. Unagitated overnight cultures (in tubes or microculture containers; see the following) of the parental strains were routinely employed because, for several R plasmids tested, such cultures yielded comparable or higher numbers of transconjugants than did agitated logarithmic-phase cultures. This was observed previously for certain R plasmids (14) and for Hfr donors (9) and was ascribed to the breakage of conjugative pili due to agitation of the donor cells cultivated in rotating flasks (9). In conventional one-step matings, 0.5 ml each of the recipient and donor cultures diluted to 5 x lo8 bacteria/ml was mixed in a test tube and incubation was continued overnight. Samples (0.1 ml) of the mating mixture or of decimal dilutions (in saline) thereof were then spread onto selective media by using a sterile glass rod. In some experiments, the following modification of the two-step conjugation method of Watanabe (14) was employed in an attempt to obtain more efficient donors (HFRT donors; see introductory section) derepressed with regard to their fertility functions. Cultures of donor (a calibrated loop carrying about 5 ~1) and intermediate host (0.05 ml) bacteria were added to a tube containing 5 ml of broth and incubated overnight. This mating mixture, potentially containing an enhanced proportion of fertility
derepressed R+ bacteria, was then used as the donor culture in the second mating step, performed as a conventional one-step conjugation experiment (see above). When applied to the well-studied, wild-type, F-like R plasmid, Rl , which has repressed transfer functions, this two-step conjugation method results in a markedly increased yield of transconjugants (about loo-fold) in comparison to the conventional one-step method. The 25-point inoculator equipment utilized here for the R plasmid transfer experiments is shown in Fig. 1. In a one-step MIP transfer experiment, each donor strain was inoculated into one of the 25 individual compartments of the microculture container to which about 0.5 ml of broth per compartment had been added in advance. The recipient strain was inoculated into a tube containing 5 ml of broth, and donor and recipient bacteria were pregrown overnight. The recipient culture was then added to an empty petri dish and, by two independent inoculator transfers, a second container with broth added was inoculated with donors and recipients. These mating mixtures were incubated overnight. Subsequent lo-* dilutions (see the following) of mating mixtures, as well as the plating of 1.5+1 volumes (see the following) of undiluted mating mixtures and dilutions thereof (25 samples per 9-cmdiameter petri plate), were then rapidly performed employing the MIP equipment. By using more microculture containers, the number of donors or recipients analyzed could be increased. Testing of additional recipients against a given set of donors was particularly easy since the 25-point inoculator could then be employed for all inoculations. To imitate HFRT conjugation experiments (see above), the MIP matings were also performed in two steps. The strain chosen as the intermediate host was then employed as the recipient in the first conjugation experiment. After overnight incubation, these mating mixtures were used as donor cultures and mated with the final recipient in a second experiment.
R PLASMID TRANSFER BY MULTIPOINT
INOCULATION
349
chromosomally resistant to nalidixic acid and rifampicin, always served as recipient. Chemicals and materials. The antibacterial agents used were from the following sources: ampicillin, AB Astra, Slidertalje, Sweden; chloramphenicol, Erco AS, Vedbaek, Denmark; nalidixic acid, Sterling-Winthrop AB, Skarholmen, Sweden; rifampicin, Sigma Chemical Co., St. Louis, Missouri; streptomycin, AB Kabi, Stockholm; oxytetracycline, Pfizer AB, Stockholm. The autoclavable 25-compartment microculture containers used were from ELESA, Milano, Italy. The 25-point inoculator was made by a local workshop and consists of 3mm-diameter stainless-steel pins pierced into a Plexiglas handle measuring 70 x 70 x 15 mm [Fig. 1 and Ref. (7)l. RESULTS Determination of Volumes Transferred the Multipoint Inoculator
6 FIG. 1. (A) Multipoint inoculator; (B) microculture container.
The selective plates usually contained 30 pg/ml of oxytetracycline for the selection of R+ clones plus nalidixic acid (30 pg/ml) and/ or rifampicin (50 pg/ml) for the contraselection of donors. When S. marcescens was the recipient, the concentration of oxytetracycline had to be increased to 300 pug/ml.For the demonstration of conjugative R plasmids in strains of gram-negative bacteria from clinical specimens, the selective plates contained either ampicillin (20 pg/ml), chloramphenicol (10 pg/ml), streptomycin (20 ,ugl ml), or oxytetracycline (30 pg/ml) plus nalidixic acid and rifampicin for the contraselection of donors. In these experiments, a doublemutant of strain Dl or WA802 (Table I),
by
For a semiquantitative estimation of the MIP transfer efficiency of a plasmid, it is necessary to know the approximate volume transferred between microculture containers or onto plates by each inoculator pin. This was established by four different methods: (i) determination of the volumes carried by pins using calibrated capillaries (Microcaps); (ii) weighing the plates just before and after replicator inoculation of 25 droplets; (iii) determination of the reduction in viable counts of bacterial suspensions upon replicator transfer between microculture containers containing 0.5 ml of saline per compartment; (iv) replicator inoculation onto plates of diluted suspensions of bacteria with known viable counts, spreading of droplets using a glass rod, and incubation and colony counting. The volume transferred between liquids was found to be about 5 ~1 per replicator pin. Since 0.5 ml of broth (for growth or mating) or saline (for dilution) is added to each microculture compartment, the multipoint transfer between culture containers results in an approximately lop2 dilution. About
350
BERMAN AND OSTENSBON
1.5 ~1 is delivered onto agar by each replicator pin. Testing of MIP Equipment for Intrageneric R Plasmid Transfer
Because of the possible negative effects on R plasmid transfer due to host-specific restriction of deoxyribonucleic acid (DNA) (4) or unknown factors influencing plasmid host range, it was considered preferable if donor and recipient strains were as closely related as possible. Since most of the plasmids studied (Table 2) were originally recovered in E. co/i K- 12and might be selected for or adapted to this host, it was first attempted to demonstrate their transfer between two almost-isogenic strains of E. coli K-12 using the MIP conjugation method. In this ideal situation, the transfer of all 44 plasmids tested was readily established by one-step MIP conjugation experiments even after lo*-fold dilutions of the mating mixtures prior to their application onto the selective plates (Expt 1, Table 3). After 104fold dilution, 3 cases of plasmid transfer escaped detection, and after 106-fold dilution 25 cases did. By analogy with the HFRT method (see above), two-step MIP matings employing an intermediate host were expected to be more efficient than one-step MIP experiments. Two steps indeed appeared to increase plasmid fertility, since another
six cases of transfer were then detectable after 106-folddilution as well as after 102-fold dilution, as before (Expts 1 and 2, Table 3). E. coli K-12 possesses a specific DNA restriction/modification system (4) capable of destroying most infecting R plasmid molecules lacking the K modification (1). Although successfully used by many workers, K-12 strains might therefore not be the most suitable recipients when screening for transferable plasmids in naturally occurring strains of gram-negative bacteria. On the other hand, E. co/i C lacks a DNA restriction/modification system (3,4) and could therefore be particularly useful in this respect. However, the transconjugant frequency obtained with E. cofi C as recipient and E. coli K-12 as donor (Expts 3 and 4, Table 3) was much less than that obtained in matings between two E. coli K-12 strains (Expts 1 and 2, Table 3). Transfer to E. cofi C could not be demonstrated with three of the plasmids, and after 104-fold dilution of the mating mixtures, only five cases of transfer were observed. The E. coli C experiments also differed from those within E. coli K-12 (Expt 2, Table 3) in that fewer plasmids were stimulated in transfer employing E. coli C as both the intermediate and the final recipient in two-step experiments (Expt 4, Table 3). If E. co/i K-12 was the intermediate host instead, an increased yield of E. coli C trans-
TABLE 3 INTFLAGENERICTRANSFEROF RPLASMIDS BY THE MIP CONJUGATION METHOD
Number of R plasmids Transferredb Expt 1 2 3 4 5 6 7
Type of MIP mating
Donor E. coli K-1%$” E. coli K-12ru E. cob K-12rif E. coli K-12rif E. coli K-12rif S. marcescens S. marcescens
nal nal
One Two One Two Two One Two
step step step step step step step
Intermediate host” R R D R
Recipient E. E. E. E. E. S. S.
coli K-12nal coli K-12nal coli Cnal coli Cd coli Cnal marcescens rf marcescens rif
Tested
-
lo-*
lo-’
10e6
44 44 44 44 45
44 44 39 41 42 39 39
44 44 36 36 39 27 34
41 42 5 5 13 10 26
19 25 0 0 0 0 0
41 41
u D, plasmidless donor; R, recipient. * The mating mixtures were applied undiluted or diluted as indicated to the selective plates. Dilutions and plating were performed using the MIP equipment (see Materials and Methods).
R PLASMID TRANSFER BY MULTIPOINT
conjugants was obtained for many plasmids (Expt 5, Table 3). Thus, passagethrough an intermediate K-12 host increased the frequency of transconjugants, possibly as a result of transient derepression of transfer functions. This phenomenon appeared to be weak in E. coli C. Intrageneric MIP plasmid conjugation experiments were also performed in a strain of Serratia marcescens to which 41 of 44 plasmids tested could be transferred by E. cofi K-12. Although almost-isogenic strains of S. marcescens were used as parental cultures, R plasmid transfer in Serratia was rather inefficient in comparison to that in E. coli K-12; it was demonstrated for 39 of 41 plasmids, and never after 106-fold dilution of the mating mixtures (Expt 6, Table 3). An increase in transconjugant frequency was obtained in two-step intrageneric Serratia MIP matings (Expt 7, Table 3). Evaluation of MIP Equipment in Intergeneric R Plasmid Transfer
The intrageneric plasmid transfer experiments reported above indicated that the MIP mating method was useful for the convenient
351
INOCULATION
demonstration of R plasmid transfer, at least in ideal situations such as between strains of E. coli K-12. The method might, however, be less reliable when transfer is inefficient, as in experiments 3 through 7 (Table 3) and, in particular, when intergeneric transfer is looked for, e.g., in studies of plasmid host range or in the screening of wild strains of bacteria for carriage of plasmids. The MIP conjugation method was therefore further evaluated by comparing the results of MIP, conventional, and HFRT matings in situations previously found by us to be unfavorable for transfer of our R plasmids. In Table 4 (Expts l-6) the attempts to transfer plasmids from E. co/i K-12 to S. marcescens are summarized. Since the transfer frequencies obtained tended to be zero or very low, undiluted mating mixtures only were routinely applied to the selective plates (about 1.5 ,ul per MIP mating and 0.1 ml for conventional and HFRT experiments; see above and Materials and Methods). The large difference in volume plated between the MIP method and the other methods was expected to be a disadvantage to the former method. Nevertheless, the MIP method was
TABLE 4 COMPARISON
BETWEEN THE MIP METHOD AND OTHER CONJUGATION IN THE INTERGENERIC TRANSFER OF R PLASMIDS
METHODS
Number of R plasmids Expt 1 2 3 4 5 6 7 8 9 10 11 12 13 14
Donor E. E. E. E. E. E. E. E. E. E. E. E. E. E.
coli coli coli coli coli coli coli coli coli coli coli coli coli coli
K-12rif K-12rif K-12$ K-12rif K-121$ K-12rif K-12$ K-lZrif K-D-if K-12rif K-12rif K-121$ K-12rif K-12rif
Type of mating
Intermediate host”
Recipient
Tested
Transferred*
One-step MIP One-step tube Two-step MIP Two-step tube Two-step MIP Two-step tube One-step MIP One-step tube Two-step MIP Two-step tube One-step MIP One-step tube Two-step MIP Two-step tube
R R D D D D D D
S. marcescens nal S. marcescens nal S. marcescens nal S. marcescens nal S. marcescens nal S. marcescens nal S. typhimurium nal S. typhimurium nal S. typhimurium nal S. typhimurium nal S. sonnei nal S. sonnei nal S. sonnei nal S. sonnei nal
44 44 44 44 45 45 45 45 45 45 4.5 45 45 45
24 10 28 11 40 36 44 39 44 40 41 33 44 36
a D, plasmidless donor; R, recipient. b Only two plasmids gave visible S. marcescens transconjugants after overnight incubation of the selective plates. The results shown for this recipient were recorded after 36-h incubation of the plates.
352
BURMAN
AND OSTENSS~N TABLE
5
INTERGENERIC TRANSFEROF R PLASMIDS BY THE MIP METHOD Number of R plasmids Transferred” Expt 1 2 3 4 5 6 7 8 9 10
Type of MIP mating
Donor E. E. E. E. s. S. S. S. S. S.
coli K-12.$ coli K-12rif coli K-12rif coli K-12rif marcescens marcescens marcescens marcescens marcescens marcescens
‘if rif rif rif rif rif
One Two One Two One Two Two One Two Two
step step step step step step step step step step
Intermediate host”
D D R D R D
Recipient S. S. S. S. E. E. E. E. E. E.
typhimurium nal typhimurium nal sonnei nal sonnei nal coli K-12.nal coli K-12nal coli K-12nal coli Cnal coli Cnal coli Cnal
Tested
-
10-Z
10-4
10-e
45 45 45 45 39 39 39 39 39 41
44 44 41 44 34 35 37 11 14 36
38 37 16 20 26 27 21 0 3 18
18 24 0 I 11 16 14 0 0 0
0 0 0 0 0 0 0 0 0 0
D D, plasmidless donor; R, recipient. b See Table 3, footnote b.
more sensitive than the conventional methods in detecting R plasmid transfer. Using one-step experiments, the MIP method demonstrated transfer to S. marcescens in 24 of 44 cases, while only 10 of the conventional matings were fertile (Expts 1 and 2, Table 4). With the respective two-step procedure (Expts 3-6, Table 4) an improvement was obtained, in particular when E. coli K-12 was the intermediate host. The MIP method was again superior to the corresponding conventional conjugation method (HFRT) and demonstrated transfer of 40 plasmids to
still detected than after 102-fold dilution of the Shigeffa mixtures (Expts l-4, Table 5). The use of two-step methods with E. coli K12 as the intermediate host often had a positive effect also on plasmid transfer to Salmonella and Shigella (Expts 7- 14, Table 4; Expts 1-4, Table 5). The conjugational behavior of the R plasmids under study was further investigated employing S. marcescens as donor and E. coli as recipient. Despite its DNA restriction ability, E. coli K-12 (Expts 5-7, Table 5) was clearly more suitable as recipient for Serratia. these plasmids than the restrictionless E. Table 4 also includes further evaluations co/i C (Expts 8-10, Table 5). In fact, E. of the MIP conjugation method employing coli K-12 appeared to be almost as good a S. typhimurium and S. sonnei as recipients recipient as a Serratia strain nearly isogenic and E. coli K-12 as donor (Expts 7-14). to the Serratia donor used (cf. Expts 6 and Again, the MIP method revealed more cases 7, Table 3). The two-step experiments with of plasmid transfer than did the other E. coli C as recipient again showed that a marked increase in R plasmid fertility folmethods. It can also be seen in Table 4 that, using E. lowed passage through S. marcescens in many cases, but rarely when E. coli C was coli K-12 as plasmid donor, S. typhimurium appears to be the species most suitable as used as the intermediate host (Expts 9 and recipient for the R plasmids tested, followed 10, Table 5). by S. sonnei and S. marcescens. The difference between S. typhimurium and S. Recovery of R Plasmids from Clinical Isolates of Gram-Negative Bacteria sonnei in this respect was more evident when The MIP conjugation method was also diluted mating mixtures were plated (Table 5). After 104-fold dilution of the Salmonella applied to wild, drug-resistant gram-negamating mixtures, more casesof transfer were tive bacteria.
R PLASMID
TRANSFER
BY MULTIPOINT TABLE
353
INOCULATION
6
DETECTIONOF TRANSFERABLERESISTANCETO TETRACYCLINE AMONGGRAM-NEGATIVE URINARY BACTERIA Number of donors of tetracycline resistancec Type of MIP
Expt
mating One Two One Two
1
2 3 4
Intermediate host”
step step step step
R R
coli coli colt’ coli
K-12, K-12, K-12, K-12,
Pseudomonas
Proteus
(171
(381
(12)
(1)
Total cases of transfer
hsr+, hsm+ hsr+, hsm+ hsr-, hsm+
10 14
hsr-, hsm+
11
3 4 4 4
3 3 4 4
0 0 0 0
16 21 16 19
Recipientb E. E. E. E.
Klebsiella Enterobacter
E. coli
8
a R, recipient. b For abbreviations, see Table 1. The hsr+ strain is Dlnal, rif. The hsr- strain is W802na1, rif, c The numbers in parentheses refer to the number of strains analyzed.
A set of 113 drug-resistant urinary pathogens belonging to E. coli, Klebsiella, Enterobacter, Proteus, and Pseudomonas was used as donors and the E. coli K-12 strains Dlnal, rif, and WA802na1, rif, were the recipients. WA802 is a restrictionless mutant and was therefore expected to be ideal as an all-round plasmid recipient. Since WA802 is able to modify DNA, the further transfer of plasmids from this strain to other K-12 laboratory strains should still be unrestricted. As seen in Table 6, two-step matings using the recipient strain also as the intermediate host were more effective than one-step matings, at least for the Tc plasmids carried by the wild E. coli strains. The use of strain WA802 as recipient offered no advantage
over the standard K-12 strain Dl. However, an opposite situation was found for Ap plasmids (Table 7). One-step experiments were, in most cases, equal to two-step matings, but the restrictionless recipient WA802 enabled the detection of many additional cases of Ap transfer. Finally, fecal specimens of 100 healthy individuals were studied with regard to bacterial resistance to ampicillin, chloramphenicol, streptomycin, and tetracycline. The drugresistant clones of aerobic gram-negative bacteria selected were subject to two-step MIP conjugation experiments employing strain Dlnal, rif, as the recipient and intermediate host. The transconjugants obtained were tested in each case for resistance against the three drugs not selected for by using
TABLE
7
DETECTIONOF TRANSFERABLERESISTANCETO AMPICILLIN AMONGGRAM-NEGATIVE URINARY BACTERIA Number of donors of ampicillin resistancec Expt 1 2 3 4
Type of MIP mating One Two One Two
Intermediate host=
step step step step
a R, recipient. b See Table 6, footnote b. c See Table 6, footnote c.
R R
Recipien@ E. coli K-12, hsr+, E. coli K-12, hsr+, E. coli K-12, hsr-, E. coli K-12, hsr-,
hsm+ hsm+ hsm+ hsm+
E. coli
Proteus
(35)
(10)
Total cases of transfer
20 20 28 29
3 5 4 6
23 25 32 35
354
BURMAN AND OSTENSSON TABLE 8
mids also could be performed in parallel using the same equipment. It was of particular DRUG RESISTANCE IN GRAM-NEGATIVE FECAL BACTERIA OF 100 HEALTHY SUBJECTS interest to compare the efficiency of MIP matings with that of established conjugation Number of subjects” with methods. Employing a set of 45 R plasmids (Table 2), this issue was therefore studied Drug-resistant Transferable in greater detail. Resistance to bacteria resistance Whereas transfer between two almost-isoAmpicillin 45 21 genie strains ofE. coli K-12 was easily demChloramphenicol 22 16 onstrated by the MIP method with all R plasStreptomycin 30 8 mids studied (Table 3) this process was less Tetracycline 45 20 efficient when other pairs of parental strains One or more drugs 58 23 were used (Tables 3,4, and 5). Nevertheless, a Of 100 fecal specimens studied, 2 did not yield the sensitivity of the MIP conjugation method growth of aerobic gram-negative bacteria. was found to be greater than that of conventional matings in all situations tested, the multipoint inoculator. Gram-negative fecal despite the 50- to loo-fold difference in the bacteria resistant to one or several of the amount of mating mixture plated. Without drugs tested were found in 58 subjects, 23 of much extra effort, the efficiency of the MIP whom carried strains with conjugative R plas- transfer of many plasmids could be increased mids (Table 8). Similar fecal R plasmid car- by performing the matings as a two-step prorier rates were observed in Sweden by other cedure. Thereby the MIP method apparently workers using conventional conjugation became superior to the optimized but laborious two-step mating method (HFRT) of methods (12). As seen in Table 8, resistances to ampicil- Watanabe [see above and Ref. (14)]. One possible explanation for the supelin and tetracycline were the most common; riority of the MIP conjugation method is that they were found in 45 subjects and were transferable in 21 and 20 subjects, respec- small inocula of donor and recipient bacteria tively. Although chloramphenicol is very are used; hence growth takes place in the mating mixtures. Many wild-type R plasmids rarely used in human medicine today, transferable resistance to this drug was surprisingly common (in 16 subjects). Among the TABLE 9 23 R plasmid-carrying subjects, 40 separate SEPARATE PATTERNS OF TRANSFERABLE DRUG transferable drug-resistance patterns were RESISTANCE IN GRAM-NEGATIVE FECAL observed (Table 9). Ap was mediated by 31 BACTERIA OF 100 SUBJECTS plasmids, whereas Tc was carried by 27, Number of separate Cm by 25, and Sm by 8 plasmids. Patterna
DISCUSSION
Ap, Cm, Tc
patterns observed 10
6 Ap, Tc The chief aim of the present investiga6 Tc tion was to evaluate a simple manually op4 Ap, Cm, Sm, Tc erated multipoint inoculation apparatus for 4 Ap, Cm, Sm the performance of R plasmid transfer ex4 AP, Cm 3 periments. Since this equipment is very pracAP 2 Cm tical, e.g., for the identification of gram-nega1 Cm, Tc tive bacteria (7) and for large-scale antibiotic susceptibility testing of bacteria using the a Abbreviations: Ap, ampicillin; Cm, chlorampheniagar dilution method, it would be extra con- col; Sm, streptomycin; Tc, tetracycline. No other antivenient if screening of the strains for R plas- biotics were studied.
R PLASMID
TRANSFER
BY MULTIPOINT
are repressed with regard to fertility; consequently, their transfer to other host cells might lead to a temporary depression of their fertility functions and thus a more efficient propagation of the plasmid among the recipient bacteria. This provision for amplification is employed in the lirst step of the HFRT method and may be active also in MIP matings. The fact that the two-step MIP matings often were even more efficient than the one-step experiments supports this hypothesis. However, for reasons we do not know, the degree of increased fertility thus obtained was dependent not only on the plasmid in question but also on the plasmid host (strong in E. cofi K-12, moderate in S. murcescens, and poor in E. coli C). The choice of parental strains greatly influenced the transfer of our laboratory plasmids. E. coli K-12 was a more efficient donor than S. marcescens and also seemed to be the most suitable recipient for the set of plasmids studied, followed by S. typhimurium, S. sonnei, E. coli C, and S. marcescens. The apparent superiority of E. co/i K-12 in these respects and the surprisingly inefficient matings with E. coli C could be related to the degree of improved transfer observed in two-step experiments (often marked in K-12, and poor or absent in C). Furthermore, the envelope lipopolysaccharide (LPS) of E. cofi K- 12 contains very little O-antigen side chains (13). Since Salmonella mutants, having lost the polysaccharide portion of their LPS, show improved R plasmid recipient ability (II ,15), an analogous situation in E. cofi could contribute to the unusual recipient ability of the K-12 strains. Finally, the laboratory plasmids studied were originally recovered in E. co/i K-12. Thus, it is possible that they are selected for or adapted to K-12 as host. S. marcescens appeared to be a particularly unsuitable host for most of the R plasmids studied. Several plasmids were not received or retained at all by S. marcescens, and all but two of the plasmids caused greatly reduced host growth rates on the selective plates where loss of the plasmids was prevented. The recipient ability of S.
INOCULATION
355
marcescens was not improved by using an almost-isogenic Serratia strain as plasmid donor. Nevertheless, passage through Serratia as the intermediate recipient improved the fertility of matings involving Serratiu, as well as other genera, as the donors and final recipients. Although the transfer efficiency and host range of various plasmids were not the primary questions at issue in this investigation, we obtained results suggesting that the expression of fertility in parental bacteria as well as other factors (vegetative replication?) in the new host may be of greater importance than the host specificity of DNA to the outcome of R plasmid matings. The MIP method of plasmid transfer and testing of transconjugants was useful not only in the experiments with certain plasmids present in laboratory strains but also in the detection and studies of conjugative R plasmids in wild strains of bacteria. In this situation, we expected a restrictionless K-12 mutant to be particularly useful as recipient, since degradation of foreign DNA may lead to a lOO-fold reduction in transfer frequency with a restricting K-12 recipient (I). The use of such a mutant, however, increased the yield of Ap transconjugants but did not increase the yield of Tc transconjugants. Thus, plasmid-specific modification/restriction systems may be as important as host-specific systems in this context. On the other hand, two-step mating seemed to offer less advantage with Ap plasmids. It should be emphasized finally that MIP matings also represent a minimum estimate of plasmid transfer. Mating mixtures containing about lo2 transconjugants per milliliter may give either positive or negative MIP results, and lower yields of transconjugants lead to a falsely negative outcome unless larger volumes of the undiluted mixtures are plated. Thus, occasional MIP matings may alternate between fertile and negative upon repetition. In conclusion, the labor and media-saving MIP conjugation method described and evaluated here appears to be more efficient than conventional methods. The MIP method
356
BURMANANDOSTENSSON
is particularly useful in large-scale R plasmid screening programs, in investigations of host range and mobilization of plasmids, and in studies of other aspects of plasmid biology where large numbers of plasmid transfer experiments are necessary (e.g., in compatibility grouping of plasmids). ACKNOWLEDGMENTS We thank Naomi Datta for gifts of plasmids. This work was supported by Swedish Medical Research Council Grants 4508 and 5217.
REFERENCES 1. ARBER, W., AND MORSE,M. L. (1965). Host specificity of DNA produced by Escherichiu coli. VI. Effects of bacterial conjugation. Genetics 51, 137- 148. 2. BERTANI, G. (1951). Studies on lysogenesis. I. The mode of phage liberation by Iysogenic Escherichiu coli. J. Bacterial.
62, 293-300.
3. BERTANI, G., AND WEIGLE, J. J. (1953). Hostcontrolled variation in bacterial viruses. J. Bacferiol. 65, 113-121. 4. BOYER, H. W. (1971). DNA restriction and modification mechanisms in bacteria. Annu. Rev. Microbial. 25, 153-176. 5. BURMAN, L. G. (1975). Amplification of sex repressor function of one J;+ R-factor during coli. J. anaerobic growth of Escherichia Bacterial. 123, 265-27 1. 6. BURMAN, L. G. (1977). Expression of R-plasmid functions during anaerobic growth of an Escherichia K-12 host. J. Bacterial. 131,69-75. 7. BURMAN, L. G., AND GSTENSSON,R. (1978). Time and media saving testing and identification of microorganisms by multipoint inoculation onto agar plates. J. Clin. Microbial. (in press).
8. CLOWES,R. C., AND ROWLEY, D. (1954). Some observations on linkage effects in genetic recombination in Escherichia co/i K-12. J. Gen. Microbial.
6, 250-258.
9. CURTISS,R., III, AND STALLIONS, D. R. (1967). Energy requirements for specific pair formation during conjugation in Escherichiu co/i K-12. J. Bacterial.
94, 490-492.
IO. HARTMAN, P. A. (1968). Miniaturized microbiological methods. In “Advances in Applied Microbiology” (W. W. Umbreit, Ed.), Vol. 1. Academic press, New York. Il. JARHOLMEN,H., AND KEMP, G. (1969). Association of increased recipient ability for R factors and reduced virulence among variants of Salmonella cholerasuis var. kunzendorf. J. Bacterial. 97, 962-963. 12. JONSSON,M. (1974). On the persistence of R-factors in the gram-negative aerobic flora of the human intestine. Stand. J. Infect. Dis. 6, 339-344. 13. L~~DERLITZ,O., STAUB,A. M., AND WESTPHAL,0. (1966). Immunochemistry of 0 and R antigens of Salmonella and related Enterobacteriaceae. Bacterial. Rev. 30, 192-251. 14. WATANABE, T. (1963). Episome-mediated drug resistance in Enterobacteriaceae. VI. High-frequency resistance transfer system in Escherichiu coli. J. Bacferiol.
85, 788-794.
15. WATANABE,T., ARAI, T., AND HATTORI, T. (1970). Effect of cell wall polysaccharide on the mating ability of Salmonella typhimurium. Nature (London) 225, 70-71. 16. VOGEL, H. J., AND BONNER, D. M. (1956). Acetylornithinase of Escherichia co/i: Partial purification and some properties. J. Biol. Chem. 218, 97-106. 17. WOOD, W. B. (1966). Host specificity of DNA produced by Escherichia coli: Bacterial mutations affecting the restriction and modification of DNA. J. Mol. Biol. 16, 118-133.
1,
346-356 (1978)
Efficient Transfer of Conjugative Plasmids by Multipoint Inoculation and Some Observations on Host Range and Prevalence of R Plasmids LARS G. BURMAN Department
of Clinical Bacteriology,
AND RUNE &TENSSON University
of Umeii, S-901 85 Urn&, Sweden
Accepted February 22, 1978 The conjugational transfer of R plasmids was demonstrated using a simple manually operated multipoint inoculator apparatus (MIP) allowing rapid inoculation and later dilution and plating of 25 mating mixtures simultaneously. Forty-five R plasmids belonging to groups F, I, N, and others originally recovered inEscherichia coli K-12 were studied in this as well as in other hosts. The semiquantitative MIP conjugation method was more efficient than conventional matings, particularly when performed in two steps employing E. co/i K-12 as intermediate host. Both as donor and as recipient, E. coli K-12 was the most “suitable” general host of the set of plamids studied, although with many plasmids the degree of expression of their transfer functions varied with the host. The expression of fertility in parental bacteria as well as factors in the new host not studied appeared to be of greater importance for the conjugational transfer of a plasmid than the host-specified restriction of plasmid deoxyribonucleic acid by the recipient strain. The MIP conjugation method was successfully used also during screening for transferable R plasmids in gram-negative bacteria present in urine and fecal specimens of humans. The use of a restrictionless mutant instead of a restricting K-12 recipient enabled the detection of additional plasmids. The labor and media-saving MIP conjugation method thus also offers efficiency and is very practical for the performance of large numbers of plasmid matings, for example, in studies of compatibilty, host range, and mobilization of plasmids, as well as for screening purposes.
Since the multipoint replicator was first described by Garrett in 1946, this tool has been used in various situations for speeding up the laboratory testing of microorganisms [see review, Ref. (IO)]. The analysis of progeny in genetic experiments, phage typing, and antibiotic susceptibility testing are situations where multipoint replicators save much labor and media by enabling the rapid inoculation of test plates with large numbers of samples of bacteria or phage. We recently adapted a simple manually operated multipoint inoculator apparatus for the demonstration on undivided test plates of the biochemical activities and motility of strains of bacteria and yeasts and, in particular, for use in recognizing members of the family Enterobacteriuceae. The method is referred 0147-619X/78/0013-0346$02.00/0 Copyright All rights
0 1978 by Academic Press, Inc. of reproduction in any form reserved.
346
to as the multipoint inoculation plate (MIP) method (7). In the present study the multipoint equipment was employed through most stages of intrageneric and intergeneric semiquantitative R plasmid conjugation experiments, using strains of Escherichia coli, Salmonella typhimurium, Serratia marcescens, and Shigella sonnei as well as other species of gram-negative bacteria. The ability of the MIP method to demonstrate R plasmid transfer appeared to be superior to that of carefully performed conventional matings, even compared to the optimized but laborious highfrequency of resistance transfer (HFRT) method of Watanabe (14). The application of the MIP principle to R plasmid conjugation thus enables a considerable reduction in the
R PLASMID TRANSFER BY MULTIPOINT
347
INOCULATION
TABLE 1 STRAINS OF GRAM-NEGATIVE BACTERIA USED
Strain
Sex
Chromosomal markers”
Source or reference
proA, trp, his proA, trp, his, nalA proA, trp, his, rpo proA, trp, his, rpo, nalA galK, galT, lac, met, thi, supE, hsr, hsm+ galK, galT, lac, met, thi, supE, hsr, hsm+, nalA, rpo
Dl is RC711 (8) Mutant of Dl (5) Mutant of Dl (5) Mutant of Dl (5) W. Arber (17) Mutant of WA802; this study
Wild type
G. Bertani (3) Mutant of C1055; this study Mutant of C1055; this study Clinical isolate; this study Mutant of UM3; this study Mutant of UM3; this study Clinical isolate; this study Mutant of UMl; this study Clinical isolate; this study Mutant of UM2; this study
E. coli K-12
Dl Dlnal Dlrif Dlnal, rif WA802 WA802na1, rif E. coli Cl055 E. coli ClOSSnal E. coli ClOSSrif S. marcescens UM3 5’. marcescens UM3nal S. marcescens UM3rif S. typhimurium UMI 5’. typhimurium UMlnal S. sonnei UM2 S. sonnei UM2 nal
FFFFFFFFFFFFFFFF-
nalA
rpo Wild type
(?) (‘7) (?) (?)
na/A rpo
(?)
nalA
Wild type
(?) Wild type (?)
nalA
a Abbreviations: gal, galactose; his, histidine; nalA, high-level nahdixic acid resistance; hsm, host-specific modification of deoxyribonucleic acid (DNA); hsr, host-specific restriction of DNA; luc, lactose; met, methionine; pro, proline; rpo, ribonucleic acid polymerase (rifampicin resistance); sup, suppression; thi, thiamine; trp, tryptophan.
time required for inoculations and in the consumption of media, without a loss of sensitivity. MATERIALS
AND METHODS
In a second series of conjugation experiments, drug-resistant strains of gram-negative bacteria isolated from urine and fecal specimens received at the Bacteriological Laboratory, Region Hospital, UmeA, were
Bacteria and R plasmids. In most of the conjugation experiments, spontaneous mutants of Escherichia coli K-12, E. coli C,
TABLE 2 SUMMARY OF 4.5 R PLASMIDS STUDIED~
Salmonella typhimurium, Serratia marcescens, or Shigella sonnei, chromosomally
resistant to rifampicin (100 pug/ml) and/or nalidixic acid (100cLg/ml),were used as donors or recipients of R plasmids. These strains are listed in Table 1. Of the 45 known R plasmids used, one type I plasmid (R64) and four type N plasmids (R15, R46, R128, and N3) were kindly provided by Naomi Datta, London. The other plasmids studied originated from the collection of this laboratory and were recently described (6). These plasmids all mediate resistance to tetracycline (and some of them also to other antibiotics) and their general characters are summarized in Table 2.
Number of plasmids determining sex pilus of typec fin”
F
I
N
+ -
9
2
1
11
Mixed
Unknown
2
1
7
2
3 6
(1Most of the plasmids were from clinical isolates of E. coli and recovered in E. coli K-12. In a few cases,
the original host of the plasmid is not known. b Fertility inhibition, i.e., the abiity to repress the fertility of the plasmid F, as determined in Ref. (6). c The plasmids were classified with regard to the type of conjugative pilus, determined as described in Ref. (6). The designation N pilus is used here despite the fact that surface appendages resembling conjugative pili have not yet been demonstrated on cells carrying type N conjugative plasmids.
348
BURMANAND~~STENSSON
identified and tested for R plasmid donor ability. Transferable resistance to ampicillin (Ap), chloramphenicol (Cm), streptomycin (Sm), and tetracycline (Tc) was then studied. The wild isolates were identified as described in Ref. (7). Media and growth conditions. The liquid medium used was LB of Bertani (2), always supplemented with glucose (0.2%) and medium E (16). All plates contained PDM agar especially designed for antibiotic susceptibility testing of bacteria (AB Biodisk, Stockholm). Incubations were performed at 37°C. The absorbance of liquid cultures was determined using a Klett-Summerson colorimeter equipped with a W66 filter. Conjugation experiments. Unagitated overnight cultures (in tubes or microculture containers; see the following) of the parental strains were routinely employed because, for several R plasmids tested, such cultures yielded comparable or higher numbers of transconjugants than did agitated logarithmic-phase cultures. This was observed previously for certain R plasmids (14) and for Hfr donors (9) and was ascribed to the breakage of conjugative pili due to agitation of the donor cells cultivated in rotating flasks (9). In conventional one-step matings, 0.5 ml each of the recipient and donor cultures diluted to 5 x lo8 bacteria/ml was mixed in a test tube and incubation was continued overnight. Samples (0.1 ml) of the mating mixture or of decimal dilutions (in saline) thereof were then spread onto selective media by using a sterile glass rod. In some experiments, the following modification of the two-step conjugation method of Watanabe (14) was employed in an attempt to obtain more efficient donors (HFRT donors; see introductory section) derepressed with regard to their fertility functions. Cultures of donor (a calibrated loop carrying about 5 ~1) and intermediate host (0.05 ml) bacteria were added to a tube containing 5 ml of broth and incubated overnight. This mating mixture, potentially containing an enhanced proportion of fertility
derepressed R+ bacteria, was then used as the donor culture in the second mating step, performed as a conventional one-step conjugation experiment (see above). When applied to the well-studied, wild-type, F-like R plasmid, Rl , which has repressed transfer functions, this two-step conjugation method results in a markedly increased yield of transconjugants (about loo-fold) in comparison to the conventional one-step method. The 25-point inoculator equipment utilized here for the R plasmid transfer experiments is shown in Fig. 1. In a one-step MIP transfer experiment, each donor strain was inoculated into one of the 25 individual compartments of the microculture container to which about 0.5 ml of broth per compartment had been added in advance. The recipient strain was inoculated into a tube containing 5 ml of broth, and donor and recipient bacteria were pregrown overnight. The recipient culture was then added to an empty petri dish and, by two independent inoculator transfers, a second container with broth added was inoculated with donors and recipients. These mating mixtures were incubated overnight. Subsequent lo-* dilutions (see the following) of mating mixtures, as well as the plating of 1.5+1 volumes (see the following) of undiluted mating mixtures and dilutions thereof (25 samples per 9-cmdiameter petri plate), were then rapidly performed employing the MIP equipment. By using more microculture containers, the number of donors or recipients analyzed could be increased. Testing of additional recipients against a given set of donors was particularly easy since the 25-point inoculator could then be employed for all inoculations. To imitate HFRT conjugation experiments (see above), the MIP matings were also performed in two steps. The strain chosen as the intermediate host was then employed as the recipient in the first conjugation experiment. After overnight incubation, these mating mixtures were used as donor cultures and mated with the final recipient in a second experiment.
R PLASMID TRANSFER BY MULTIPOINT
INOCULATION
349
chromosomally resistant to nalidixic acid and rifampicin, always served as recipient. Chemicals and materials. The antibacterial agents used were from the following sources: ampicillin, AB Astra, Slidertalje, Sweden; chloramphenicol, Erco AS, Vedbaek, Denmark; nalidixic acid, Sterling-Winthrop AB, Skarholmen, Sweden; rifampicin, Sigma Chemical Co., St. Louis, Missouri; streptomycin, AB Kabi, Stockholm; oxytetracycline, Pfizer AB, Stockholm. The autoclavable 25-compartment microculture containers used were from ELESA, Milano, Italy. The 25-point inoculator was made by a local workshop and consists of 3mm-diameter stainless-steel pins pierced into a Plexiglas handle measuring 70 x 70 x 15 mm [Fig. 1 and Ref. (7)l. RESULTS Determination of Volumes Transferred the Multipoint Inoculator
6 FIG. 1. (A) Multipoint inoculator; (B) microculture container.
The selective plates usually contained 30 pg/ml of oxytetracycline for the selection of R+ clones plus nalidixic acid (30 pg/ml) and/ or rifampicin (50 pg/ml) for the contraselection of donors. When S. marcescens was the recipient, the concentration of oxytetracycline had to be increased to 300 pug/ml.For the demonstration of conjugative R plasmids in strains of gram-negative bacteria from clinical specimens, the selective plates contained either ampicillin (20 pg/ml), chloramphenicol (10 pg/ml), streptomycin (20 ,ugl ml), or oxytetracycline (30 pg/ml) plus nalidixic acid and rifampicin for the contraselection of donors. In these experiments, a doublemutant of strain Dl or WA802 (Table I),
by
For a semiquantitative estimation of the MIP transfer efficiency of a plasmid, it is necessary to know the approximate volume transferred between microculture containers or onto plates by each inoculator pin. This was established by four different methods: (i) determination of the volumes carried by pins using calibrated capillaries (Microcaps); (ii) weighing the plates just before and after replicator inoculation of 25 droplets; (iii) determination of the reduction in viable counts of bacterial suspensions upon replicator transfer between microculture containers containing 0.5 ml of saline per compartment; (iv) replicator inoculation onto plates of diluted suspensions of bacteria with known viable counts, spreading of droplets using a glass rod, and incubation and colony counting. The volume transferred between liquids was found to be about 5 ~1 per replicator pin. Since 0.5 ml of broth (for growth or mating) or saline (for dilution) is added to each microculture compartment, the multipoint transfer between culture containers results in an approximately lop2 dilution. About
350
BERMAN AND OSTENSBON
1.5 ~1 is delivered onto agar by each replicator pin. Testing of MIP Equipment for Intrageneric R Plasmid Transfer
Because of the possible negative effects on R plasmid transfer due to host-specific restriction of deoxyribonucleic acid (DNA) (4) or unknown factors influencing plasmid host range, it was considered preferable if donor and recipient strains were as closely related as possible. Since most of the plasmids studied (Table 2) were originally recovered in E. co/i K- 12and might be selected for or adapted to this host, it was first attempted to demonstrate their transfer between two almost-isogenic strains of E. coli K-12 using the MIP conjugation method. In this ideal situation, the transfer of all 44 plasmids tested was readily established by one-step MIP conjugation experiments even after lo*-fold dilutions of the mating mixtures prior to their application onto the selective plates (Expt 1, Table 3). After 104fold dilution, 3 cases of plasmid transfer escaped detection, and after 106-fold dilution 25 cases did. By analogy with the HFRT method (see above), two-step MIP matings employing an intermediate host were expected to be more efficient than one-step MIP experiments. Two steps indeed appeared to increase plasmid fertility, since another
six cases of transfer were then detectable after 106-folddilution as well as after 102-fold dilution, as before (Expts 1 and 2, Table 3). E. coli K-12 possesses a specific DNA restriction/modification system (4) capable of destroying most infecting R plasmid molecules lacking the K modification (1). Although successfully used by many workers, K-12 strains might therefore not be the most suitable recipients when screening for transferable plasmids in naturally occurring strains of gram-negative bacteria. On the other hand, E. co/i C lacks a DNA restriction/modification system (3,4) and could therefore be particularly useful in this respect. However, the transconjugant frequency obtained with E. cofi C as recipient and E. coli K-12 as donor (Expts 3 and 4, Table 3) was much less than that obtained in matings between two E. coli K-12 strains (Expts 1 and 2, Table 3). Transfer to E. cofi C could not be demonstrated with three of the plasmids, and after 104-fold dilution of the mating mixtures, only five cases of transfer were observed. The E. coli C experiments also differed from those within E. coli K-12 (Expt 2, Table 3) in that fewer plasmids were stimulated in transfer employing E. coli C as both the intermediate and the final recipient in two-step experiments (Expt 4, Table 3). If E. co/i K-12 was the intermediate host instead, an increased yield of E. coli C trans-
TABLE 3 INTFLAGENERICTRANSFEROF RPLASMIDS BY THE MIP CONJUGATION METHOD
Number of R plasmids Transferredb Expt 1 2 3 4 5 6 7
Type of MIP mating
Donor E. coli K-1%$” E. coli K-12ru E. cob K-12rif E. coli K-12rif E. coli K-12rif S. marcescens S. marcescens
nal nal
One Two One Two Two One Two
step step step step step step step
Intermediate host” R R D R
Recipient E. E. E. E. E. S. S.
coli K-12nal coli K-12nal coli Cnal coli Cd coli Cnal marcescens rf marcescens rif
Tested
-
lo-*
lo-’
10e6
44 44 44 44 45
44 44 39 41 42 39 39
44 44 36 36 39 27 34
41 42 5 5 13 10 26
19 25 0 0 0 0 0
41 41
u D, plasmidless donor; R, recipient. * The mating mixtures were applied undiluted or diluted as indicated to the selective plates. Dilutions and plating were performed using the MIP equipment (see Materials and Methods).
R PLASMID TRANSFER BY MULTIPOINT
conjugants was obtained for many plasmids (Expt 5, Table 3). Thus, passagethrough an intermediate K-12 host increased the frequency of transconjugants, possibly as a result of transient derepression of transfer functions. This phenomenon appeared to be weak in E. coli C. Intrageneric MIP plasmid conjugation experiments were also performed in a strain of Serratia marcescens to which 41 of 44 plasmids tested could be transferred by E. cofi K-12. Although almost-isogenic strains of S. marcescens were used as parental cultures, R plasmid transfer in Serratia was rather inefficient in comparison to that in E. coli K-12; it was demonstrated for 39 of 41 plasmids, and never after 106-fold dilution of the mating mixtures (Expt 6, Table 3). An increase in transconjugant frequency was obtained in two-step intrageneric Serratia MIP matings (Expt 7, Table 3). Evaluation of MIP Equipment in Intergeneric R Plasmid Transfer
The intrageneric plasmid transfer experiments reported above indicated that the MIP mating method was useful for the convenient
351
INOCULATION
demonstration of R plasmid transfer, at least in ideal situations such as between strains of E. coli K-12. The method might, however, be less reliable when transfer is inefficient, as in experiments 3 through 7 (Table 3) and, in particular, when intergeneric transfer is looked for, e.g., in studies of plasmid host range or in the screening of wild strains of bacteria for carriage of plasmids. The MIP conjugation method was therefore further evaluated by comparing the results of MIP, conventional, and HFRT matings in situations previously found by us to be unfavorable for transfer of our R plasmids. In Table 4 (Expts l-6) the attempts to transfer plasmids from E. co/i K-12 to S. marcescens are summarized. Since the transfer frequencies obtained tended to be zero or very low, undiluted mating mixtures only were routinely applied to the selective plates (about 1.5 ,ul per MIP mating and 0.1 ml for conventional and HFRT experiments; see above and Materials and Methods). The large difference in volume plated between the MIP method and the other methods was expected to be a disadvantage to the former method. Nevertheless, the MIP method was
TABLE 4 COMPARISON
BETWEEN THE MIP METHOD AND OTHER CONJUGATION IN THE INTERGENERIC TRANSFER OF R PLASMIDS
METHODS
Number of R plasmids Expt 1 2 3 4 5 6 7 8 9 10 11 12 13 14
Donor E. E. E. E. E. E. E. E. E. E. E. E. E. E.
coli coli coli coli coli coli coli coli coli coli coli coli coli coli
K-12rif K-12rif K-12$ K-12rif K-121$ K-12rif K-12$ K-lZrif K-D-if K-12rif K-12rif K-121$ K-12rif K-12rif
Type of mating
Intermediate host”
Recipient
Tested
Transferred*
One-step MIP One-step tube Two-step MIP Two-step tube Two-step MIP Two-step tube One-step MIP One-step tube Two-step MIP Two-step tube One-step MIP One-step tube Two-step MIP Two-step tube
R R D D D D D D
S. marcescens nal S. marcescens nal S. marcescens nal S. marcescens nal S. marcescens nal S. marcescens nal S. typhimurium nal S. typhimurium nal S. typhimurium nal S. typhimurium nal S. sonnei nal S. sonnei nal S. sonnei nal S. sonnei nal
44 44 44 44 45 45 45 45 45 45 4.5 45 45 45
24 10 28 11 40 36 44 39 44 40 41 33 44 36
a D, plasmidless donor; R, recipient. b Only two plasmids gave visible S. marcescens transconjugants after overnight incubation of the selective plates. The results shown for this recipient were recorded after 36-h incubation of the plates.
352
BURMAN
AND OSTENSS~N TABLE
5
INTERGENERIC TRANSFEROF R PLASMIDS BY THE MIP METHOD Number of R plasmids Transferred” Expt 1 2 3 4 5 6 7 8 9 10
Type of MIP mating
Donor E. E. E. E. s. S. S. S. S. S.
coli K-12.$ coli K-12rif coli K-12rif coli K-12rif marcescens marcescens marcescens marcescens marcescens marcescens
‘if rif rif rif rif rif
One Two One Two One Two Two One Two Two
step step step step step step step step step step
Intermediate host”
D D R D R D
Recipient S. S. S. S. E. E. E. E. E. E.
typhimurium nal typhimurium nal sonnei nal sonnei nal coli K-12.nal coli K-12nal coli K-12nal coli Cnal coli Cnal coli Cnal
Tested
-
10-Z
10-4
10-e
45 45 45 45 39 39 39 39 39 41
44 44 41 44 34 35 37 11 14 36
38 37 16 20 26 27 21 0 3 18
18 24 0 I 11 16 14 0 0 0
0 0 0 0 0 0 0 0 0 0
D D, plasmidless donor; R, recipient. b See Table 3, footnote b.
more sensitive than the conventional methods in detecting R plasmid transfer. Using one-step experiments, the MIP method demonstrated transfer to S. marcescens in 24 of 44 cases, while only 10 of the conventional matings were fertile (Expts 1 and 2, Table 4). With the respective two-step procedure (Expts 3-6, Table 4) an improvement was obtained, in particular when E. coli K-12 was the intermediate host. The MIP method was again superior to the corresponding conventional conjugation method (HFRT) and demonstrated transfer of 40 plasmids to
still detected than after 102-fold dilution of the Shigeffa mixtures (Expts l-4, Table 5). The use of two-step methods with E. coli K12 as the intermediate host often had a positive effect also on plasmid transfer to Salmonella and Shigella (Expts 7- 14, Table 4; Expts 1-4, Table 5). The conjugational behavior of the R plasmids under study was further investigated employing S. marcescens as donor and E. coli as recipient. Despite its DNA restriction ability, E. coli K-12 (Expts 5-7, Table 5) was clearly more suitable as recipient for Serratia. these plasmids than the restrictionless E. Table 4 also includes further evaluations co/i C (Expts 8-10, Table 5). In fact, E. of the MIP conjugation method employing coli K-12 appeared to be almost as good a S. typhimurium and S. sonnei as recipients recipient as a Serratia strain nearly isogenic and E. coli K-12 as donor (Expts 7-14). to the Serratia donor used (cf. Expts 6 and Again, the MIP method revealed more cases 7, Table 3). The two-step experiments with of plasmid transfer than did the other E. coli C as recipient again showed that a marked increase in R plasmid fertility folmethods. It can also be seen in Table 4 that, using E. lowed passage through S. marcescens in many cases, but rarely when E. coli C was coli K-12 as plasmid donor, S. typhimurium appears to be the species most suitable as used as the intermediate host (Expts 9 and recipient for the R plasmids tested, followed 10, Table 5). by S. sonnei and S. marcescens. The difference between S. typhimurium and S. Recovery of R Plasmids from Clinical Isolates of Gram-Negative Bacteria sonnei in this respect was more evident when The MIP conjugation method was also diluted mating mixtures were plated (Table 5). After 104-fold dilution of the Salmonella applied to wild, drug-resistant gram-negamating mixtures, more casesof transfer were tive bacteria.
R PLASMID
TRANSFER
BY MULTIPOINT TABLE
353
INOCULATION
6
DETECTIONOF TRANSFERABLERESISTANCETO TETRACYCLINE AMONGGRAM-NEGATIVE URINARY BACTERIA Number of donors of tetracycline resistancec Type of MIP
Expt
mating One Two One Two
1
2 3 4
Intermediate host”
step step step step
R R
coli coli colt’ coli
K-12, K-12, K-12, K-12,
Pseudomonas
Proteus
(171
(381
(12)
(1)
Total cases of transfer
hsr+, hsm+ hsr+, hsm+ hsr-, hsm+
10 14
hsr-, hsm+
11
3 4 4 4
3 3 4 4
0 0 0 0
16 21 16 19
Recipientb E. E. E. E.
Klebsiella Enterobacter
E. coli
8
a R, recipient. b For abbreviations, see Table 1. The hsr+ strain is Dlnal, rif. The hsr- strain is W802na1, rif, c The numbers in parentheses refer to the number of strains analyzed.
A set of 113 drug-resistant urinary pathogens belonging to E. coli, Klebsiella, Enterobacter, Proteus, and Pseudomonas was used as donors and the E. coli K-12 strains Dlnal, rif, and WA802na1, rif, were the recipients. WA802 is a restrictionless mutant and was therefore expected to be ideal as an all-round plasmid recipient. Since WA802 is able to modify DNA, the further transfer of plasmids from this strain to other K-12 laboratory strains should still be unrestricted. As seen in Table 6, two-step matings using the recipient strain also as the intermediate host were more effective than one-step matings, at least for the Tc plasmids carried by the wild E. coli strains. The use of strain WA802 as recipient offered no advantage
over the standard K-12 strain Dl. However, an opposite situation was found for Ap plasmids (Table 7). One-step experiments were, in most cases, equal to two-step matings, but the restrictionless recipient WA802 enabled the detection of many additional cases of Ap transfer. Finally, fecal specimens of 100 healthy individuals were studied with regard to bacterial resistance to ampicillin, chloramphenicol, streptomycin, and tetracycline. The drugresistant clones of aerobic gram-negative bacteria selected were subject to two-step MIP conjugation experiments employing strain Dlnal, rif, as the recipient and intermediate host. The transconjugants obtained were tested in each case for resistance against the three drugs not selected for by using
TABLE
7
DETECTIONOF TRANSFERABLERESISTANCETO AMPICILLIN AMONGGRAM-NEGATIVE URINARY BACTERIA Number of donors of ampicillin resistancec Expt 1 2 3 4
Type of MIP mating One Two One Two
Intermediate host=
step step step step
a R, recipient. b See Table 6, footnote b. c See Table 6, footnote c.
R R
Recipien@ E. coli K-12, hsr+, E. coli K-12, hsr+, E. coli K-12, hsr-, E. coli K-12, hsr-,
hsm+ hsm+ hsm+ hsm+
E. coli
Proteus
(35)
(10)
Total cases of transfer
20 20 28 29
3 5 4 6
23 25 32 35
354
BURMAN AND OSTENSSON TABLE 8
mids also could be performed in parallel using the same equipment. It was of particular DRUG RESISTANCE IN GRAM-NEGATIVE FECAL BACTERIA OF 100 HEALTHY SUBJECTS interest to compare the efficiency of MIP matings with that of established conjugation Number of subjects” with methods. Employing a set of 45 R plasmids (Table 2), this issue was therefore studied Drug-resistant Transferable in greater detail. Resistance to bacteria resistance Whereas transfer between two almost-isoAmpicillin 45 21 genie strains ofE. coli K-12 was easily demChloramphenicol 22 16 onstrated by the MIP method with all R plasStreptomycin 30 8 mids studied (Table 3) this process was less Tetracycline 45 20 efficient when other pairs of parental strains One or more drugs 58 23 were used (Tables 3,4, and 5). Nevertheless, a Of 100 fecal specimens studied, 2 did not yield the sensitivity of the MIP conjugation method growth of aerobic gram-negative bacteria. was found to be greater than that of conventional matings in all situations tested, the multipoint inoculator. Gram-negative fecal despite the 50- to loo-fold difference in the bacteria resistant to one or several of the amount of mating mixture plated. Without drugs tested were found in 58 subjects, 23 of much extra effort, the efficiency of the MIP whom carried strains with conjugative R plas- transfer of many plasmids could be increased mids (Table 8). Similar fecal R plasmid car- by performing the matings as a two-step prorier rates were observed in Sweden by other cedure. Thereby the MIP method apparently workers using conventional conjugation became superior to the optimized but laborious two-step mating method (HFRT) of methods (12). As seen in Table 8, resistances to ampicil- Watanabe [see above and Ref. (14)]. One possible explanation for the supelin and tetracycline were the most common; riority of the MIP conjugation method is that they were found in 45 subjects and were transferable in 21 and 20 subjects, respec- small inocula of donor and recipient bacteria tively. Although chloramphenicol is very are used; hence growth takes place in the mating mixtures. Many wild-type R plasmids rarely used in human medicine today, transferable resistance to this drug was surprisingly common (in 16 subjects). Among the TABLE 9 23 R plasmid-carrying subjects, 40 separate SEPARATE PATTERNS OF TRANSFERABLE DRUG transferable drug-resistance patterns were RESISTANCE IN GRAM-NEGATIVE FECAL observed (Table 9). Ap was mediated by 31 BACTERIA OF 100 SUBJECTS plasmids, whereas Tc was carried by 27, Number of separate Cm by 25, and Sm by 8 plasmids. Patterna
DISCUSSION
Ap, Cm, Tc
patterns observed 10
6 Ap, Tc The chief aim of the present investiga6 Tc tion was to evaluate a simple manually op4 Ap, Cm, Sm, Tc erated multipoint inoculation apparatus for 4 Ap, Cm, Sm the performance of R plasmid transfer ex4 AP, Cm 3 periments. Since this equipment is very pracAP 2 Cm tical, e.g., for the identification of gram-nega1 Cm, Tc tive bacteria (7) and for large-scale antibiotic susceptibility testing of bacteria using the a Abbreviations: Ap, ampicillin; Cm, chlorampheniagar dilution method, it would be extra con- col; Sm, streptomycin; Tc, tetracycline. No other antivenient if screening of the strains for R plas- biotics were studied.
R PLASMID
TRANSFER
BY MULTIPOINT
are repressed with regard to fertility; consequently, their transfer to other host cells might lead to a temporary depression of their fertility functions and thus a more efficient propagation of the plasmid among the recipient bacteria. This provision for amplification is employed in the lirst step of the HFRT method and may be active also in MIP matings. The fact that the two-step MIP matings often were even more efficient than the one-step experiments supports this hypothesis. However, for reasons we do not know, the degree of increased fertility thus obtained was dependent not only on the plasmid in question but also on the plasmid host (strong in E. cofi K-12, moderate in S. murcescens, and poor in E. coli C). The choice of parental strains greatly influenced the transfer of our laboratory plasmids. E. coli K-12 was a more efficient donor than S. marcescens and also seemed to be the most suitable recipient for the set of plasmids studied, followed by S. typhimurium, S. sonnei, E. coli C, and S. marcescens. The apparent superiority of E. co/i K-12 in these respects and the surprisingly inefficient matings with E. coli C could be related to the degree of improved transfer observed in two-step experiments (often marked in K-12, and poor or absent in C). Furthermore, the envelope lipopolysaccharide (LPS) of E. cofi K- 12 contains very little O-antigen side chains (13). Since Salmonella mutants, having lost the polysaccharide portion of their LPS, show improved R plasmid recipient ability (II ,15), an analogous situation in E. cofi could contribute to the unusual recipient ability of the K-12 strains. Finally, the laboratory plasmids studied were originally recovered in E. co/i K-12. Thus, it is possible that they are selected for or adapted to K-12 as host. S. marcescens appeared to be a particularly unsuitable host for most of the R plasmids studied. Several plasmids were not received or retained at all by S. marcescens, and all but two of the plasmids caused greatly reduced host growth rates on the selective plates where loss of the plasmids was prevented. The recipient ability of S.
INOCULATION
355
marcescens was not improved by using an almost-isogenic Serratia strain as plasmid donor. Nevertheless, passage through Serratia as the intermediate recipient improved the fertility of matings involving Serratiu, as well as other genera, as the donors and final recipients. Although the transfer efficiency and host range of various plasmids were not the primary questions at issue in this investigation, we obtained results suggesting that the expression of fertility in parental bacteria as well as other factors (vegetative replication?) in the new host may be of greater importance than the host specificity of DNA to the outcome of R plasmid matings. The MIP method of plasmid transfer and testing of transconjugants was useful not only in the experiments with certain plasmids present in laboratory strains but also in the detection and studies of conjugative R plasmids in wild strains of bacteria. In this situation, we expected a restrictionless K-12 mutant to be particularly useful as recipient, since degradation of foreign DNA may lead to a lOO-fold reduction in transfer frequency with a restricting K-12 recipient (I). The use of such a mutant, however, increased the yield of Ap transconjugants but did not increase the yield of Tc transconjugants. Thus, plasmid-specific modification/restriction systems may be as important as host-specific systems in this context. On the other hand, two-step mating seemed to offer less advantage with Ap plasmids. It should be emphasized finally that MIP matings also represent a minimum estimate of plasmid transfer. Mating mixtures containing about lo2 transconjugants per milliliter may give either positive or negative MIP results, and lower yields of transconjugants lead to a falsely negative outcome unless larger volumes of the undiluted mixtures are plated. Thus, occasional MIP matings may alternate between fertile and negative upon repetition. In conclusion, the labor and media-saving MIP conjugation method described and evaluated here appears to be more efficient than conventional methods. The MIP method
356
BURMANANDOSTENSSON
is particularly useful in large-scale R plasmid screening programs, in investigations of host range and mobilization of plasmids, and in studies of other aspects of plasmid biology where large numbers of plasmid transfer experiments are necessary (e.g., in compatibility grouping of plasmids). ACKNOWLEDGMENTS We thank Naomi Datta for gifts of plasmids. This work was supported by Swedish Medical Research Council Grants 4508 and 5217.
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