Vol. 131, No. 1 Printed in U.S.A.
JOURNAL OF BACTERIOLOGY, JUlY 1977, p. 356-362 Copyright © 1977 American Society for Microbiology
Relationships Among Some R Plasmids Found in Haemophilus influenzae LYNN P. ELWELL,* J. R. SAUNDERS, M. H. RICHMOND, AND STANLEY FALKOW Department of Microbiology and Immunology, School of Medicine, University of Washington, Seattle, Washington 98195*; Department of Microbiology, University of Liverpool, Liverpool L69 3 Bx, United Kingdom; and Department of Bacteriology, Medical School, University of Bristol, Bristol B58 ITD, United Kingdom
Received for publication 21 March 1977
Tetracycline resistance in a strain of Haemophilus influenzae isolated in the United Kingdom was found to be determined by an apparently non-selftransmissible plasmid of 31 x 106 daltons (31 MDal), designated pUB701. Deoxyribonucleic acid hybridization studies indicated that pUB701 shares about 70% base sequence homology with the 30-MDal ampicillin resistance R plasmid RSFOO7, isolated in the United States from H. influenzae, and 64% sequence homology with the 38-MDal tetracycline and chloramphenicol resistance R plasmid pRI234, isolated in the Netherlands. Heteroduplex studies between RSFOO7 and pUB701 confirmed the fact that these plasmids were largely homologous, except that pUB701 contained the tetracycline resistance transposon TnD, whereas RSFOO7 contained the ampicillin resistance transposon TnA. A strain of H. parainfluenzae resistant to both chloramphenicol and tetracycline carried two species of plasmid deoxyribonucleic acid of 2.7 and 0.75 MDal. We were unable to prove that either resistance was plasmid-borne in this strain. Hybridization studies with a [3H]thymine-labeled tetracycline resistance enteric plasmid suggested that the tetracycline transposon was integrated into the chromosome of H. parainfluenzae UJB2832. We conclude either that the strains we studied received R factors ofthe same incompatibility group bearing different resistance genes, or that different resistance genes were translocated to a common resident plasmid of H. influenzae.
Haemophilus influenzae type b causes a number of serious infections of children, notably meningitis and epiglottitis. The emergence of ampicillin resistance in an H. influenzae population previously regarded as uniformly susceptible to this antibiotic is, therefore, of particularly great concern. The relatively recent and rapid acquisition of resistance to ampicillin and other antibiotics by H. influenzae suggests that R factors are involved. Indeed, plasmidlinked resistance to a number of antibiotics, including ampicillin, kanamycin (9), and tetracycline (8), as well as a combination of tetracycline and chloramphenicol (32), has now been demonstrated. Furthermore, ampicillin resistance (27, 31), kanamycin resistance (9), and linked resistance to tetracycline and chloramphenicol (32) are transferable by conjugation to other bacteria. The resistance mechanisms found in H. influenzae are very similar to those mediated by R factors in the Enterobacteriaceae. Thus, ampicillin resistance in strains of H. influenzae that have been studied is due exclusively to the 356
possession of the TEM or type IIIa p-lactamase (13, 22, 30, 33). This enzyme, which is determined by the translocatable genetic element transposon A (TnA) (17, 18), is found on a variety of phylogenetically distinct plasmids resident in gram-negative bacteria (19). Similarly, other resistance genes, i.e., those coding for tetracycline resistance (14, 21) and kanamycin resistance (3), are also borne on transposons and are consequently widespread on different bacterial replicons. The question therefore arises as to the origin of the resistance genes recently detected in Haemophilus species. This paper describes relationships between three Haemophilus R plasmids of similar molecular weights but of diverse geographical origins and specifying different drug resistances, and proposes mechanisms for the accretion of drug resistance by this group of organisms. MATERIALS AND METHODS Bacterial strains and plasmids. The bacterial strains and plasmids used in this study are shown in Table 1.
R PLASMIDS OF H. INFLUENZAE
VOL. 131, 1977
Strain/plasmid Strain G32 G32(RSF007)
TABLE 1. Bacterial strains and plasmids used Characteristics" Species
CL17 UB2811(pUB701) UB2820
H. influenzae H. influenzae H. influenzae
HC234(pR1234)
H. influenzae
Drug sensitive Ap; transformant with RSFOO7 Drug sensitive Tc Cured, drug-sensitive derivative of UB2811(pUB701) Cm Tc
UB2832
H. parainfluenzae
Cm Tc
Plasmid RSFOO7
H. influenzae H. influenzae
357
Source
L. P. Elwell et al. (12) L. P. Elwell et al. (12)
M. Coyle P. Cavanagh This paper
B. van Klingeren et al. (32) P. Cavanagh (4)
U.S.A.; L. P. Elwell et al. (12) U.K.; this paper Tc H. influenzae pUB701 Netherlands; B. van KlinTc Cm H. influenzae pRI1234 geren et al. (32) K. Timmis Tc Km E. coli KTO07 a Ap, Resistance to ampicillin; Tc, resistance to tetracycline; Cm, resistance to chloramphenicol; Km, resistance to kanamycin. H. influenzae
Media and other materials. In the United Kingdom, Haemophilus species were grown in brain heart infusion (Difco Laboratories) containing 1% (vol/vol) Fildes peptic digest (BHF medium). Where required, this was solidified by the incorporation of 1 to 2% (wt/vol) agar (Difco). Haemophilus strains were generally grown in an atmosphere supplemented with 5 to 10% CO2. Other media and materials used in both Seattle and the United Kingdom were described previously (11, 12, 16, 27). Labeling of DNA and preparation of plasmid DNA. The labeling and preparation of plasmid deoxyribonucleic acid (DNA) were described previously (11, 12, 16, 28). Preparation of unlabeled whole-cell DNA for hybridization studies. Unlabeled whole-cell DNA was prepared by the method of So et al. (28). Preparation and detection of DNA-DNA duplexes. DNA-DNA duplex formation between labeled plasmid DNA and whole cellular DNA of relevant strains was carried out by the S1 endonuclease method of Crosa et al. (7). Determination of G+C content DNA. The guanine-plus-cytosine (G+C) content of DNA was determined as described by de Graaff et al. (11). Preparation of heteroduplexes and their analysis by electron microscopy. The method used to analyze heteroduplexes was described previously (11, 18). Open circles were generated by X-ray nicking covalently closed circular molecules of pUB701 and RSFOO7. In addition, it was found necessary to treat Haemophilus plasmid DNA with Pronase (10 ,ug/ml) to obtain good-quality electron microscope grids. Determination of contour length and molecular weight of plasmid DNA. Molecular weights of plasmid DNA molecules were determined from contour lengths as previously described (12). Elimination of drug resistance. Organisms were
Ap
grown in BHF medium containing ethidium bromide (2 jig/ml) for 18 h at 37°C. Dilutions of the culture were then plated on drug-free BHF agar. After incubation for 18 h at 37°C, suitable plates were replicated onto similar medium containing tetracycline (10 ,ug/ml) or chloramphenicol (10 ,ug/ml). After a further incubation for 18 h at 37°C, clones that were drug sensitive were selected. Transformation. Transformation was performed by the method of Cohen et al. (6).
RESULTS Origin and properties of resistant Haemophilus strains. The origin and properties of plasmid RSFOO7 were described previously (11, 12). We compared strains of H. influenzae carrying this plasmid with several other strains exhibiting resistance to antibiotics commonly used to treat Haemophilus infections. H. influenzae UB2811(pUB701) is a tetracycline-resistant strain (minimal inhibitory concentration [MIC], 16 ,ug/ml) isolated from a patient with bronchitis, at Stoke-on-Trent, U.K. H. influenzae HC234(pRI234) is a tetracycline-resistant (MIC, 32 Mg/ml) and chloramphenicol-resistant (MIC, 16 ,ug/ml) strain isolated in the Netherlands from the pharynx of a patient with lymphatic leukemia (32). Both resistance markers were found to be transferable as a single unit to other H. influenzae strains as well as to Escherichia coli, presumably by conjugation (32). H. parainfluenzae UB2832 is a strain also isolated from a bronchitis case in England (4). It is resistant to both tetracycline (MIC, 16 pg/ml) and chloramphenicol (MIC, 20 ug/ml) (4). Nei-
358
J. BACTERIOL.
ELWELL ET AL.
ther resistant strain H. influenzae UB2811 nor H. parainfluenzae UB2832 could act as a donor of resistance by contact with either other Haemophilus species or E. coli. Molecular properties of plasmids found in resistant Haemophilus strains. All resistant strains of Haemophilus were found to contain covalently closed circular DNA when either crude Sarkosyl or cleared lysates were analyzed by ethidium bromide-cesium chloride gradient centrifugation (data not shown). The contour lengths of circular DNA species in each strain were estimated by electron microscopy. Table 2 shows the molecular weights of the plasmids present. Strain UB2811(pUB701) contains a plasmid of 31 x 106 daltons (31 MDal) believed to encode tetracycline resistance (see below), and we named it pUB701. Strain CL17 is 1 of 60 H. influenzae isolates that were drug sensitive. It contains a 26-Mdal plasmid of unknown function. H. parainfluenzae UB2832 contains two plasmids of 2.7 and 0.75 MDal. The function of either of these plasmids is as yet unclear (see below). Assuming that the molecular weight of the Haemophilus chromosome is 1.66 x 109 (15) and knowing the amount of covalently closed circular DNA per chromosome equivalent, it is possible to calculate the minimum copy number of each plasmid in its host (Table 2). Thus, the larger plasmids, RSF007 and pUB701, and that from strain CL17 are present at about two copies per chromosome in H. influenzae. On the other hand, both the 2.7- and 0.75-MDal plasmids from H. parainfluenzae UB2832 are present as multiple copies. This is typical of the copy number/size relationship observed with plasmids in other bacteria (5). TABLE 2. Characteristics ofplasmids from resistant Haemophilus strains MiniStrain
Sizea (MDal)
mum
G+Cc
copy
(mol%)
no.b
H. influenzae
G32(RSF007) UB2811(pUB701) CL17(RSF017)
H. parainfluenzae UB2832
30.2 + 0.59 (58) 31 ± 0.86 (11) 26 + 1.12 (6)
2.7 ± 0.06 (35) 0.75 ± 0.02 (30)
2.3 1.0 1.5
39 39 36
20 NDd 25 ND a Number in parentheses indicates the number of molecules measured. bThe minimum copy size per chromosome equivalent assuming a molecular weight of the Haemophilus chromosome of 1.66 x 109 (15). c The G+C of the chromosome of H. influenzae G32 was 38 mol% in these experiments. d ND, Not determined.
The G+C content of the R plasmids RSFOO7 and pUB701 and the indigenous, phenotypically cryptic plasmid harbored by the drugsensitive strain CL17 ranged from 36 to 39 mol%. In terms of their G+C content, therefore, they appear to be unlike any other known R plasmid with the possible exception of certain plasmids isolated from Proteus species. The fact that the G+C content of the H. influenzae chromosome is 38 mol% supports the hypothesis that these R plasmids are indigenous to Haemophilus. However, they could have originated from a donor with a similar DNA base composition. Elimination of drug resistance from Haemophilus. Attempts were made to cure H. influenzae UB2811(pUB701) and H. parainfluenzae UB2832 of their resistance to determine whether such resistance was plasmid determined. It proved possible to eliminate tetracycline resistance, in 11 of 541 colonies tested, from H. influenzae UB2811(pUB701) by growth of the strain in a medium containing ethidium bromide. In contrast, it proved impossible to eliminate either tetracycline or chloramphenicol resistance from H. parainfluenzae UB2832 by this method (0 of 3,500 colonies tested). Strains of H. influenzae UB2811(pUB701) that had been cured of tetracycline resistance contained no detectable extrachromosomal DNA (data not shown). It, therefore, seemed likely that the tetracycline resistance determinant was linked to the plasmid resident in this strain. Transformation with plasmid DNA from Haemophilus. Preparations of covalently closed circular DNA from H. influenzae UB2811(pUB701) would not transform strains of E. coli or H. influenzae. However, it has already been shown that RSFOO7, which is of a size similar to that of pUB701, will not transform E. coli to ampicillin resistance (11). On the other hand, the smaller (about 4 MDal) ampicillin resistance plasmids, RSF0885 (11, 12) and pUB702 (27), will transform H. influenzae and E. coli. This may indicate that RSFOO7 and pUB701 are particularly fragile or that these plasmids are too large to be transformed successfully. Attempts to transforn either H. influenzae, H. parainfluenzae, or E. coli to either chloramphenicol or tetracycline resistance with plasmid DNA from H. parainfluenzae UB2832 proved uniformly unsuccessful. Relationships among plasmids from resistant Haemophilus strains. The tetracycline-resistant strain H. influenzae UTB2811(pUB701) and the tetracycline- and chloramphenicol-resistant strain H. influenzae HC234(pRI234)
VOL. 131, 1977
contained plasmids having molecular properties similar to those of the well-characterized ampicillin resistance plasmid RSFOO7. This led us to examine the relationship among these three plasmids to determine whether they were derived from a common source and differed only in their resistance genes. We, therefore, hybridized [3H]thymidine-labeled RSF0O7 DNA and [3H]thymidine-labeled pUB701 DNA with unlabeled whole-cell DNA from each host strain and appropriate control strains. RSF007 shared 65% of its polynucleotide sequence with pUB701, and the reciprocal hybridization value of 69% was in close agreement (Table 3). Both RSF0O7 and pUB701 were approximately 65% related to the 38-MDal tetracycline and chloramphenicol resistance R plasmid, pRI234, harbored by H. influenzae HC234. In contrast, the cryptic plasmid from drug-sensitive H. influenzae CL17 showed minimal homology with either plasmid. Plasmid DNA from H. parainfluenzae UB2832 shared very few nucleotide sequences with the ampicillin resistance plasmid RSF0O7, but there was significant homology, approximately 10%, with the tetracycline resistance plasmid pUB701. The probable reason for this will be discussed in more detail below. To visualize the exact extent of homology between RSF007 and pUB701, we prepared heteroduplex molecules for examination in an electron microscope. Heteroduplex molecules observed were indeed largely double stranded (Fig. 1). RSFOO7 has the characteristic singlestrand loop of about 3.2 MDal which composes
R PLASMIDS OF H. INFLUENZAE
359
FIG. 1. (A) HIeteroduplex of RSFOO7 and pUB701 plasmid DNA. Approximately 0.2 Mg of X-ray-nicked plasmid DNAs of RSFOO7 and pUB701 was mixed with 0.25 ml of a solution containing 0.1 N NaOH and 0.01 M ethylenediaminetetraacetate. The pH was then lowered to 8.5 with 2 M tris(hydroxymethyl)aminomethane (pH 7.1) and 0.25 ml offormamide (Mallinckrodt). The DNA was allowed to reanneal for 5 h under these conditions at room temperature. Cytochrome c was added to a final concentration of 0.1 Mg/ml, and the reannealed DNA was spread at the air-water interface on a glass slide TABLE 3. Hybridization between 3H-labeled onto a hypophase of a solution containing 60% formHaemophilus plasmid DNA and whole-cell DNA amide, 0.01 M tris(hydroxymethyl)aminomethane, and 0.001 M ethylenediaminetetraacetate (pH 8.5). Relative DNA seAfter 2 min the DNA was picked up from the surface quence homology of the hypophase on a Parlodion-coated electron Source of unlabeled DNA DNAa microscope grid. The grid was stained with 5 x 10-5 M uranyl acetate in 90% ethanol for 30 s and fixed RSFOO7 pUB701 in isopentane for 10 s. Grids were shadowed with platinum palladium and examined in a Jeol model H. influenzae G32 1 1 100B electron microscope. (B) Diagram of RSFOO71 H. influenzae G32(RSF007) 69 100 pUB701 heteroduplex. Thick line, Double-stranded H. influenzae UB2811(pUB701) 65 100 DNA; thin line, single-stranded DNA; Ap, ampi64 66 H. influenzae HC234(pRI234) cillin transposon; Tc, tetracycline transposon; IR, H. influenzae CL17 2.9 1.6 inverted repeat sequence. H. parainfluenzae UB2832 10.5 1 a The degree of DNA-DNA duplex formation was TnA (18). DNA from pUB701 lacks this strucassayed by the Si endonuclease method (7). In each ture and, in turn, contains the prominent incase, the degree of duplex formation was calculated verted repeat sequence, approximately 1,400 relative to the homologous reaction. The actual ex- base pairs in length, characteristic of the tetratent of binding of 3H-labeled RSFOO7 and pUB701 cycline determinant TnD (20, 21, 25), and a purified plasmid DNA with whole-cell DNA of their respective parent strains was approximately 80%. single-strand loop of about 4 MDal (Fig. 1). It is All other reactions were normalized to these values also clear from the heteroduplex molecule that taken as 100%. Each value shown is the average of polynucleotide sequence differences between RSFOO7 and pUB701 are not limited to the drug three reactions.
360
ELWELL ET AL.
resistance determinants. For example, a small substitution loop near the TnA sequence and three additional, closely grouped substitution loops are clearly visible in the heteroduplex molecule. Therefore, although the "cores" of RSFOO7 and pUB701 are highly related, in terms of their polynucleotide sequences they are not identical. Demonstration of the tetracycline transposon (TnD) in resistant Haemophilus. As a probe for the tetracycline transposon, we used plasmid KTOO7, kindly given to us by K. Timmis. KTO07 is a 9.9-MDal, tetracycline resistance, kanamycin resistance plasmid that was generated by the digestion of the enteric plasmids R6 and PML21. The size of the tetracycline determinant in KTO07 was estimated to be approximately 2.8 MDal by agarose gel electrophoresis after EcoRI enzyme digestion. The results of hybridizing [3Hlthymine-labeled KTO07 with unlabeled whole-cell DNAs are shown in Table 4. KTO07 shared 23 to 32% of its polynucleotide sequences with Haemophilus strains UB2811 (Tcr), HC234 (Tcr Cmr), and UB2832 (Tcr, Cmr) and less than 1% with the ampicillinresistant strain G32(RSF007). Since it was demonstrated by heteroduplexing that the R plasmids RSFOO7 and pUB701 are closely related, differing mainly in their respective resistance markers, the homology detected between KTO07 and pUB701 is most likely confined to the tetracycline resistance determinant. Also notable is the significant homology between KTO07 and H. parainfluenzae UB2832. Since it is unlikely that the 2.7-MDal or the 0.75-MDal
J. BACTERIOL.
plasmid harbored by UB2832 contributes to the tetracycline resistance of this strain (discussed above), these data suggest that the tetracycline determinant is integrated into the chromosome of this strain. This also explains the 10% homology noted when [3H]thymidine-labeled pUB701 plasmid DNA was hybridized with UB2832 whole-cell DNA (Table 3). Presumably, the sequences in common are limited to the tetracycline determinant of pUB701 since no homology was seen when [3H]thymidine-labeled RSFOO7 (Apr) plasmid DNA was used as a probe (Table 3). DISCUSSION The tetracycline-resistant strain of H. influenzae UB2811(pUB701) contains a 31-MDal plasmid species. It was not possible to transfer resistance to other bacteria either by cell-to-cell contact or by transformation with purified plasmid DNA. It is possible that the inability to transfer pUB701 into other strains is a result of restriction modification; however, the highly related plasmid RSFOO7 was not so excluded. Nonetheless, cured strains of this organism showed concomitant loss of tetracycline resist-
ance and extrachromosomal DNA. Further-
more, DNA-DNA hybridization and heteroduplex analyses revealed that the 31-MDal plasmid, which we designated pUB701, contains the sequence of DNA characteristic of the tetracycline resistance transposon TnD (20, 25). It thus seems certain that pUB701 determines tetracycline resistance in this strain. In contrast, we were unable to prove that either the tetracycline resistance or the chloramphenicol resistance found in H. parainfluTABLE 4. Hybridization between the 3H-labeled enzae UB2832 was specified by plasmids. This tetracycline resistance enteric plasmid KTO07 and strain contained at least two plasmid species: whole-cell DNA one of 2.7 MDal and the other of 0.75 MDal. The former is certainly of a size similar to that of Relative DNA the ampicillin resistance plasmids RSF0885 (11, sequence homolSource of unlabeled DNA ogy (%) with 12) and pUB702 (27), which are 4.0 and 2.5 KT007 plasmid MDal, respectively, and which are found in H. DNAa The 0.75-MDal plasmid from H. influenzae. H. influenzae G32 ...................... 0 parainfluenzae is extremely small and contains E. coli C600(KTO07) .................... 100 only enough genetic information to code for one H. influenzae G32(RSFOO7) .............
JOURNAL OF BACTERIOLOGY, JUlY 1977, p. 356-362 Copyright © 1977 American Society for Microbiology
Relationships Among Some R Plasmids Found in Haemophilus influenzae LYNN P. ELWELL,* J. R. SAUNDERS, M. H. RICHMOND, AND STANLEY FALKOW Department of Microbiology and Immunology, School of Medicine, University of Washington, Seattle, Washington 98195*; Department of Microbiology, University of Liverpool, Liverpool L69 3 Bx, United Kingdom; and Department of Bacteriology, Medical School, University of Bristol, Bristol B58 ITD, United Kingdom
Received for publication 21 March 1977
Tetracycline resistance in a strain of Haemophilus influenzae isolated in the United Kingdom was found to be determined by an apparently non-selftransmissible plasmid of 31 x 106 daltons (31 MDal), designated pUB701. Deoxyribonucleic acid hybridization studies indicated that pUB701 shares about 70% base sequence homology with the 30-MDal ampicillin resistance R plasmid RSFOO7, isolated in the United States from H. influenzae, and 64% sequence homology with the 38-MDal tetracycline and chloramphenicol resistance R plasmid pRI234, isolated in the Netherlands. Heteroduplex studies between RSFOO7 and pUB701 confirmed the fact that these plasmids were largely homologous, except that pUB701 contained the tetracycline resistance transposon TnD, whereas RSFOO7 contained the ampicillin resistance transposon TnA. A strain of H. parainfluenzae resistant to both chloramphenicol and tetracycline carried two species of plasmid deoxyribonucleic acid of 2.7 and 0.75 MDal. We were unable to prove that either resistance was plasmid-borne in this strain. Hybridization studies with a [3H]thymine-labeled tetracycline resistance enteric plasmid suggested that the tetracycline transposon was integrated into the chromosome of H. parainfluenzae UJB2832. We conclude either that the strains we studied received R factors ofthe same incompatibility group bearing different resistance genes, or that different resistance genes were translocated to a common resident plasmid of H. influenzae.
Haemophilus influenzae type b causes a number of serious infections of children, notably meningitis and epiglottitis. The emergence of ampicillin resistance in an H. influenzae population previously regarded as uniformly susceptible to this antibiotic is, therefore, of particularly great concern. The relatively recent and rapid acquisition of resistance to ampicillin and other antibiotics by H. influenzae suggests that R factors are involved. Indeed, plasmidlinked resistance to a number of antibiotics, including ampicillin, kanamycin (9), and tetracycline (8), as well as a combination of tetracycline and chloramphenicol (32), has now been demonstrated. Furthermore, ampicillin resistance (27, 31), kanamycin resistance (9), and linked resistance to tetracycline and chloramphenicol (32) are transferable by conjugation to other bacteria. The resistance mechanisms found in H. influenzae are very similar to those mediated by R factors in the Enterobacteriaceae. Thus, ampicillin resistance in strains of H. influenzae that have been studied is due exclusively to the 356
possession of the TEM or type IIIa p-lactamase (13, 22, 30, 33). This enzyme, which is determined by the translocatable genetic element transposon A (TnA) (17, 18), is found on a variety of phylogenetically distinct plasmids resident in gram-negative bacteria (19). Similarly, other resistance genes, i.e., those coding for tetracycline resistance (14, 21) and kanamycin resistance (3), are also borne on transposons and are consequently widespread on different bacterial replicons. The question therefore arises as to the origin of the resistance genes recently detected in Haemophilus species. This paper describes relationships between three Haemophilus R plasmids of similar molecular weights but of diverse geographical origins and specifying different drug resistances, and proposes mechanisms for the accretion of drug resistance by this group of organisms. MATERIALS AND METHODS Bacterial strains and plasmids. The bacterial strains and plasmids used in this study are shown in Table 1.
R PLASMIDS OF H. INFLUENZAE
VOL. 131, 1977
Strain/plasmid Strain G32 G32(RSF007)
TABLE 1. Bacterial strains and plasmids used Characteristics" Species
CL17 UB2811(pUB701) UB2820
H. influenzae H. influenzae H. influenzae
HC234(pR1234)
H. influenzae
Drug sensitive Ap; transformant with RSFOO7 Drug sensitive Tc Cured, drug-sensitive derivative of UB2811(pUB701) Cm Tc
UB2832
H. parainfluenzae
Cm Tc
Plasmid RSFOO7
H. influenzae H. influenzae
357
Source
L. P. Elwell et al. (12) L. P. Elwell et al. (12)
M. Coyle P. Cavanagh This paper
B. van Klingeren et al. (32) P. Cavanagh (4)
U.S.A.; L. P. Elwell et al. (12) U.K.; this paper Tc H. influenzae pUB701 Netherlands; B. van KlinTc Cm H. influenzae pRI1234 geren et al. (32) K. Timmis Tc Km E. coli KTO07 a Ap, Resistance to ampicillin; Tc, resistance to tetracycline; Cm, resistance to chloramphenicol; Km, resistance to kanamycin. H. influenzae
Media and other materials. In the United Kingdom, Haemophilus species were grown in brain heart infusion (Difco Laboratories) containing 1% (vol/vol) Fildes peptic digest (BHF medium). Where required, this was solidified by the incorporation of 1 to 2% (wt/vol) agar (Difco). Haemophilus strains were generally grown in an atmosphere supplemented with 5 to 10% CO2. Other media and materials used in both Seattle and the United Kingdom were described previously (11, 12, 16, 27). Labeling of DNA and preparation of plasmid DNA. The labeling and preparation of plasmid deoxyribonucleic acid (DNA) were described previously (11, 12, 16, 28). Preparation of unlabeled whole-cell DNA for hybridization studies. Unlabeled whole-cell DNA was prepared by the method of So et al. (28). Preparation and detection of DNA-DNA duplexes. DNA-DNA duplex formation between labeled plasmid DNA and whole cellular DNA of relevant strains was carried out by the S1 endonuclease method of Crosa et al. (7). Determination of G+C content DNA. The guanine-plus-cytosine (G+C) content of DNA was determined as described by de Graaff et al. (11). Preparation of heteroduplexes and their analysis by electron microscopy. The method used to analyze heteroduplexes was described previously (11, 18). Open circles were generated by X-ray nicking covalently closed circular molecules of pUB701 and RSFOO7. In addition, it was found necessary to treat Haemophilus plasmid DNA with Pronase (10 ,ug/ml) to obtain good-quality electron microscope grids. Determination of contour length and molecular weight of plasmid DNA. Molecular weights of plasmid DNA molecules were determined from contour lengths as previously described (12). Elimination of drug resistance. Organisms were
Ap
grown in BHF medium containing ethidium bromide (2 jig/ml) for 18 h at 37°C. Dilutions of the culture were then plated on drug-free BHF agar. After incubation for 18 h at 37°C, suitable plates were replicated onto similar medium containing tetracycline (10 ,ug/ml) or chloramphenicol (10 ,ug/ml). After a further incubation for 18 h at 37°C, clones that were drug sensitive were selected. Transformation. Transformation was performed by the method of Cohen et al. (6).
RESULTS Origin and properties of resistant Haemophilus strains. The origin and properties of plasmid RSFOO7 were described previously (11, 12). We compared strains of H. influenzae carrying this plasmid with several other strains exhibiting resistance to antibiotics commonly used to treat Haemophilus infections. H. influenzae UB2811(pUB701) is a tetracycline-resistant strain (minimal inhibitory concentration [MIC], 16 ,ug/ml) isolated from a patient with bronchitis, at Stoke-on-Trent, U.K. H. influenzae HC234(pRI234) is a tetracycline-resistant (MIC, 32 Mg/ml) and chloramphenicol-resistant (MIC, 16 ,ug/ml) strain isolated in the Netherlands from the pharynx of a patient with lymphatic leukemia (32). Both resistance markers were found to be transferable as a single unit to other H. influenzae strains as well as to Escherichia coli, presumably by conjugation (32). H. parainfluenzae UB2832 is a strain also isolated from a bronchitis case in England (4). It is resistant to both tetracycline (MIC, 16 pg/ml) and chloramphenicol (MIC, 20 ug/ml) (4). Nei-
358
J. BACTERIOL.
ELWELL ET AL.
ther resistant strain H. influenzae UB2811 nor H. parainfluenzae UB2832 could act as a donor of resistance by contact with either other Haemophilus species or E. coli. Molecular properties of plasmids found in resistant Haemophilus strains. All resistant strains of Haemophilus were found to contain covalently closed circular DNA when either crude Sarkosyl or cleared lysates were analyzed by ethidium bromide-cesium chloride gradient centrifugation (data not shown). The contour lengths of circular DNA species in each strain were estimated by electron microscopy. Table 2 shows the molecular weights of the plasmids present. Strain UB2811(pUB701) contains a plasmid of 31 x 106 daltons (31 MDal) believed to encode tetracycline resistance (see below), and we named it pUB701. Strain CL17 is 1 of 60 H. influenzae isolates that were drug sensitive. It contains a 26-Mdal plasmid of unknown function. H. parainfluenzae UB2832 contains two plasmids of 2.7 and 0.75 MDal. The function of either of these plasmids is as yet unclear (see below). Assuming that the molecular weight of the Haemophilus chromosome is 1.66 x 109 (15) and knowing the amount of covalently closed circular DNA per chromosome equivalent, it is possible to calculate the minimum copy number of each plasmid in its host (Table 2). Thus, the larger plasmids, RSF007 and pUB701, and that from strain CL17 are present at about two copies per chromosome in H. influenzae. On the other hand, both the 2.7- and 0.75-MDal plasmids from H. parainfluenzae UB2832 are present as multiple copies. This is typical of the copy number/size relationship observed with plasmids in other bacteria (5). TABLE 2. Characteristics ofplasmids from resistant Haemophilus strains MiniStrain
Sizea (MDal)
mum
G+Cc
copy
(mol%)
no.b
H. influenzae
G32(RSF007) UB2811(pUB701) CL17(RSF017)
H. parainfluenzae UB2832
30.2 + 0.59 (58) 31 ± 0.86 (11) 26 + 1.12 (6)
2.7 ± 0.06 (35) 0.75 ± 0.02 (30)
2.3 1.0 1.5
39 39 36
20 NDd 25 ND a Number in parentheses indicates the number of molecules measured. bThe minimum copy size per chromosome equivalent assuming a molecular weight of the Haemophilus chromosome of 1.66 x 109 (15). c The G+C of the chromosome of H. influenzae G32 was 38 mol% in these experiments. d ND, Not determined.
The G+C content of the R plasmids RSFOO7 and pUB701 and the indigenous, phenotypically cryptic plasmid harbored by the drugsensitive strain CL17 ranged from 36 to 39 mol%. In terms of their G+C content, therefore, they appear to be unlike any other known R plasmid with the possible exception of certain plasmids isolated from Proteus species. The fact that the G+C content of the H. influenzae chromosome is 38 mol% supports the hypothesis that these R plasmids are indigenous to Haemophilus. However, they could have originated from a donor with a similar DNA base composition. Elimination of drug resistance from Haemophilus. Attempts were made to cure H. influenzae UB2811(pUB701) and H. parainfluenzae UB2832 of their resistance to determine whether such resistance was plasmid determined. It proved possible to eliminate tetracycline resistance, in 11 of 541 colonies tested, from H. influenzae UB2811(pUB701) by growth of the strain in a medium containing ethidium bromide. In contrast, it proved impossible to eliminate either tetracycline or chloramphenicol resistance from H. parainfluenzae UB2832 by this method (0 of 3,500 colonies tested). Strains of H. influenzae UB2811(pUB701) that had been cured of tetracycline resistance contained no detectable extrachromosomal DNA (data not shown). It, therefore, seemed likely that the tetracycline resistance determinant was linked to the plasmid resident in this strain. Transformation with plasmid DNA from Haemophilus. Preparations of covalently closed circular DNA from H. influenzae UB2811(pUB701) would not transform strains of E. coli or H. influenzae. However, it has already been shown that RSFOO7, which is of a size similar to that of pUB701, will not transform E. coli to ampicillin resistance (11). On the other hand, the smaller (about 4 MDal) ampicillin resistance plasmids, RSF0885 (11, 12) and pUB702 (27), will transform H. influenzae and E. coli. This may indicate that RSFOO7 and pUB701 are particularly fragile or that these plasmids are too large to be transformed successfully. Attempts to transforn either H. influenzae, H. parainfluenzae, or E. coli to either chloramphenicol or tetracycline resistance with plasmid DNA from H. parainfluenzae UB2832 proved uniformly unsuccessful. Relationships among plasmids from resistant Haemophilus strains. The tetracycline-resistant strain H. influenzae UTB2811(pUB701) and the tetracycline- and chloramphenicol-resistant strain H. influenzae HC234(pRI234)
VOL. 131, 1977
contained plasmids having molecular properties similar to those of the well-characterized ampicillin resistance plasmid RSFOO7. This led us to examine the relationship among these three plasmids to determine whether they were derived from a common source and differed only in their resistance genes. We, therefore, hybridized [3H]thymidine-labeled RSF0O7 DNA and [3H]thymidine-labeled pUB701 DNA with unlabeled whole-cell DNA from each host strain and appropriate control strains. RSF007 shared 65% of its polynucleotide sequence with pUB701, and the reciprocal hybridization value of 69% was in close agreement (Table 3). Both RSF0O7 and pUB701 were approximately 65% related to the 38-MDal tetracycline and chloramphenicol resistance R plasmid, pRI234, harbored by H. influenzae HC234. In contrast, the cryptic plasmid from drug-sensitive H. influenzae CL17 showed minimal homology with either plasmid. Plasmid DNA from H. parainfluenzae UB2832 shared very few nucleotide sequences with the ampicillin resistance plasmid RSF0O7, but there was significant homology, approximately 10%, with the tetracycline resistance plasmid pUB701. The probable reason for this will be discussed in more detail below. To visualize the exact extent of homology between RSF007 and pUB701, we prepared heteroduplex molecules for examination in an electron microscope. Heteroduplex molecules observed were indeed largely double stranded (Fig. 1). RSFOO7 has the characteristic singlestrand loop of about 3.2 MDal which composes
R PLASMIDS OF H. INFLUENZAE
359
FIG. 1. (A) HIeteroduplex of RSFOO7 and pUB701 plasmid DNA. Approximately 0.2 Mg of X-ray-nicked plasmid DNAs of RSFOO7 and pUB701 was mixed with 0.25 ml of a solution containing 0.1 N NaOH and 0.01 M ethylenediaminetetraacetate. The pH was then lowered to 8.5 with 2 M tris(hydroxymethyl)aminomethane (pH 7.1) and 0.25 ml offormamide (Mallinckrodt). The DNA was allowed to reanneal for 5 h under these conditions at room temperature. Cytochrome c was added to a final concentration of 0.1 Mg/ml, and the reannealed DNA was spread at the air-water interface on a glass slide TABLE 3. Hybridization between 3H-labeled onto a hypophase of a solution containing 60% formHaemophilus plasmid DNA and whole-cell DNA amide, 0.01 M tris(hydroxymethyl)aminomethane, and 0.001 M ethylenediaminetetraacetate (pH 8.5). Relative DNA seAfter 2 min the DNA was picked up from the surface quence homology of the hypophase on a Parlodion-coated electron Source of unlabeled DNA DNAa microscope grid. The grid was stained with 5 x 10-5 M uranyl acetate in 90% ethanol for 30 s and fixed RSFOO7 pUB701 in isopentane for 10 s. Grids were shadowed with platinum palladium and examined in a Jeol model H. influenzae G32 1 1 100B electron microscope. (B) Diagram of RSFOO71 H. influenzae G32(RSF007) 69 100 pUB701 heteroduplex. Thick line, Double-stranded H. influenzae UB2811(pUB701) 65 100 DNA; thin line, single-stranded DNA; Ap, ampi64 66 H. influenzae HC234(pRI234) cillin transposon; Tc, tetracycline transposon; IR, H. influenzae CL17 2.9 1.6 inverted repeat sequence. H. parainfluenzae UB2832 10.5 1 a The degree of DNA-DNA duplex formation was TnA (18). DNA from pUB701 lacks this strucassayed by the Si endonuclease method (7). In each ture and, in turn, contains the prominent incase, the degree of duplex formation was calculated verted repeat sequence, approximately 1,400 relative to the homologous reaction. The actual ex- base pairs in length, characteristic of the tetratent of binding of 3H-labeled RSFOO7 and pUB701 cycline determinant TnD (20, 21, 25), and a purified plasmid DNA with whole-cell DNA of their respective parent strains was approximately 80%. single-strand loop of about 4 MDal (Fig. 1). It is All other reactions were normalized to these values also clear from the heteroduplex molecule that taken as 100%. Each value shown is the average of polynucleotide sequence differences between RSFOO7 and pUB701 are not limited to the drug three reactions.
360
ELWELL ET AL.
resistance determinants. For example, a small substitution loop near the TnA sequence and three additional, closely grouped substitution loops are clearly visible in the heteroduplex molecule. Therefore, although the "cores" of RSFOO7 and pUB701 are highly related, in terms of their polynucleotide sequences they are not identical. Demonstration of the tetracycline transposon (TnD) in resistant Haemophilus. As a probe for the tetracycline transposon, we used plasmid KTOO7, kindly given to us by K. Timmis. KTO07 is a 9.9-MDal, tetracycline resistance, kanamycin resistance plasmid that was generated by the digestion of the enteric plasmids R6 and PML21. The size of the tetracycline determinant in KTO07 was estimated to be approximately 2.8 MDal by agarose gel electrophoresis after EcoRI enzyme digestion. The results of hybridizing [3Hlthymine-labeled KTO07 with unlabeled whole-cell DNAs are shown in Table 4. KTO07 shared 23 to 32% of its polynucleotide sequences with Haemophilus strains UB2811 (Tcr), HC234 (Tcr Cmr), and UB2832 (Tcr, Cmr) and less than 1% with the ampicillinresistant strain G32(RSF007). Since it was demonstrated by heteroduplexing that the R plasmids RSFOO7 and pUB701 are closely related, differing mainly in their respective resistance markers, the homology detected between KTO07 and pUB701 is most likely confined to the tetracycline resistance determinant. Also notable is the significant homology between KTO07 and H. parainfluenzae UB2832. Since it is unlikely that the 2.7-MDal or the 0.75-MDal
J. BACTERIOL.
plasmid harbored by UB2832 contributes to the tetracycline resistance of this strain (discussed above), these data suggest that the tetracycline determinant is integrated into the chromosome of this strain. This also explains the 10% homology noted when [3H]thymidine-labeled pUB701 plasmid DNA was hybridized with UB2832 whole-cell DNA (Table 3). Presumably, the sequences in common are limited to the tetracycline determinant of pUB701 since no homology was seen when [3H]thymidine-labeled RSFOO7 (Apr) plasmid DNA was used as a probe (Table 3). DISCUSSION The tetracycline-resistant strain of H. influenzae UB2811(pUB701) contains a 31-MDal plasmid species. It was not possible to transfer resistance to other bacteria either by cell-to-cell contact or by transformation with purified plasmid DNA. It is possible that the inability to transfer pUB701 into other strains is a result of restriction modification; however, the highly related plasmid RSFOO7 was not so excluded. Nonetheless, cured strains of this organism showed concomitant loss of tetracycline resist-
ance and extrachromosomal DNA. Further-
more, DNA-DNA hybridization and heteroduplex analyses revealed that the 31-MDal plasmid, which we designated pUB701, contains the sequence of DNA characteristic of the tetracycline resistance transposon TnD (20, 25). It thus seems certain that pUB701 determines tetracycline resistance in this strain. In contrast, we were unable to prove that either the tetracycline resistance or the chloramphenicol resistance found in H. parainfluTABLE 4. Hybridization between the 3H-labeled enzae UB2832 was specified by plasmids. This tetracycline resistance enteric plasmid KTO07 and strain contained at least two plasmid species: whole-cell DNA one of 2.7 MDal and the other of 0.75 MDal. The former is certainly of a size similar to that of Relative DNA the ampicillin resistance plasmids RSF0885 (11, sequence homolSource of unlabeled DNA ogy (%) with 12) and pUB702 (27), which are 4.0 and 2.5 KT007 plasmid MDal, respectively, and which are found in H. DNAa The 0.75-MDal plasmid from H. influenzae. H. influenzae G32 ...................... 0 parainfluenzae is extremely small and contains E. coli C600(KTO07) .................... 100 only enough genetic information to code for one H. influenzae G32(RSFOO7) .............
