Vol. 133, No. 2
JOURNAL OF BACTERIOLOGY, Feb. 1978, p. 852859
0021-9193/78/0133-0852$02.00/0 Copyright i 1978 American Society for Microbiology
Prined in U.S.A.
Genetic Analysis of Antibiotic Resistance in Streptococcus pyogenes JAMES G. STUARTt AND JOSEPH J. FERRETTI* Department of Microbiology and Immunology, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma 73190 Received for publication 12 April 1977
The genetics of antibiotic resistance in mutant strains of Streptococcus pyogenes was studied. Utilizing a type 6 strain (9440) primarily resistant to streptomycin (Strr), classes of mutant strains were isolated that were resistant to one of the following antibiotics: rifampin (RiP), erythromycin (Eryr), thiostrepton (Tstr), spiramycin (Spr), fusidic acid (Fusr), gramicidin (Grcr), ethidium bromide (Ebrr), kanamycin (Kanr), neomycin (Neor), oleandomycin (Oler), gentamicin (Genr), and novobiocin (Novr). Transduction experiments separated antibiotic resistance markers into two distinct groups: transducible markers, including Fusr, Bacr, Ksg, SpCr, EYr, Sprr, Rift, Stlr, and Tstr (Bacr, Ksgr, Spcr, and Stlr refer to resistance to bacitracin, kasugamycin, spectinomycin, and streptolydigan, respectively), and nontransducible markers, including Grcr, Ebrr, Kanr, Neor, Oler, Genr, and Novr. By means of two- and three-point crosses, transducible markers (excluding tst) were located in three separate linkage groups. spr was found to be linked with ery and spc in the order spc-ery-spr, whereas in a separate linkage group the order was determined to be str-fus-bac-ksg. The third linkage group contained the rif and stl markers.
Studies on the genetics of antibiotic resistance in Streptococcuspyogenes were started by Leonard et al. (12), who described transduction of streptomycin resistance. Subsequently, Malke (13, 14) refined the transduction technique and described two groups of markers that were cotransduced into sensitive recipients. In one linkage group, resistance to streptomycin (str) was cotransduced at high frequency with resistance to kanamycin (kan), and in another group resistance to spectinomycin (spc), erythromycin (ery), and lincomycin (lin) were found to be tightly linked in the order described. Another antibiotic resistance marker, rifampin (rit), was shown to be separate from the streptomycin linkage group (24). All of these studies utilized strains possessing laboratory-induced antibiotic resistance markers, presumably chromosomal in
origin. Antibiotic resistance markers found in clinical isolates, which are extrachromosomal in origin, have also been transduced to sensitive recipient strains. Ubakata et al. (26) have shown that transfer of resistance occurs in two different patterns; i.e., transfer of tetracycline resistance alone or joint transfer of chloramphenicol, macrolide antibiotics, lincomycin, and clindamycin resistance. Malke (15) has shown that antibiotic t Present address: Department of Biological Sciences, Murray State University, Murray, KY 42071.
852
resistance to erythromycin, lincomycin, and staphylomycin S, known to be plasmid directed (16), is transferred by transduction with high efficiency. The present study of antibiotic resistance markers of chromosomal origin substantiates linkage data previously reported by Malke (14), and, further, orders four new antibiotic resistance markers within two linkage groups. Additional data establish the presence of a third linkage group including resistance to rifampin and streptolydigan (stl).
MATERIALS AND METHODS Strains. Parent strains of streptococci and streptococcal phages used in this research were obtained from the laboratory of Lewis Wannamaker at the University of Minnesota. Bacterial strains include two group A strains of S. pyogenes, a type 12 strain designated K56 and a type 6, streptomycin-resistant (Strr) strain designated 9440 str. Mutant strains were derived from 9440 str and are listed in Table 1. Antibioticresistant strains of K56 were constructed by transduction and are also listed in Table 1. The genetic symbols and nomenclature used are described by Demerec et al. (5). One strain of bacteriophage A25 was utilized in this study in transduction experiments. This strain is a double temperature-sensitive mutant designated A25 tsl-2 and was originally isolated by Malke (13). Media. Proteose peptone broth has been described
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ANTIBIOTIC RESISTANCE IN S. PYOGENES
TABLE 1. Bacterial strains Strain K56 derivativesb AS1 AS2
AS16-AS19 AS21 AS29 AS37 AS47-AS49 AS55 AS61 AS68 AS74
Markerse str-1O
rif-l 8pC
ery-l spr-4 stl-1
ksg
fus-l fus-1 bac-6 bac-6 tst
9440 derivativesc
AS7-AS10 rif AS11-AS15 spc AS20 ery-1 AS22-AS24 ery spr AS25-AS28 spr AS30-AS31 ery spr spc AS32-AS36 stl AS38-AS41 sti rif AS42-AS46 ksg AS50-AS54 fus AS56-AS58 ksg fus AS59 ksg-2 fus-1 bac-6 AS60 fus-I bac-6 AS62-AS67 bac AS69-AS73 tst AS76-AS80 grc AS81-AS85 ebr AS86-AS90 kan AS91-AS95 neo AS96-AS100 gen AS101-AS102 ole AS103 nov a Lack of an allele number following a gene designation indicates a group of independently isolated markers. b All K56 derivatives were constructed by transduction. c All 9440 derivatives contained str-10. Additional markers were obtained by selection of spontaneous mutants or by transduction into each subline.
previously by Wannamaker et al. (28) as no. 1 broth. Three types of solid media were employed. Serum Todd-Hewitt (STH) agar was prepared by adding 0.38% Na2HPO4 and 1.5% bacteriological agar to ToddHewitt broth (Difco). This solution was autoclaved, and sterile solutions of 0.02% CaCl2 and 5% normal horse serum were added. Blood agar was prepared by substituting 5% sheep blood or human blood for normal horse serum in STH agar plates. Serum soft agar was prepared by substituting 3% proteose peptone 2 (Difco) and 0.6% NaCl in proteose peptone broth with the addition of 0.7% bacteriological agar. Mutant Isolation. Spontaneous antibiotic-resistant mutants were isolated by the following procedure. Bacteria (9440 str) were grown to log phase at 30°C in proteose peptone broth, and 0.1-ml samples of this culture were added to 3 ml of serum soft agar, which
853
was then overlayed on STH agar plates. After incubation for 2 h at 37°C, a second 3-ml overlay of serum soft agar containing an appropriate level of antibiotic was added to the seeded STH agar plate. After overnight incubation at 37°C, resistant colonies were selected and tested for antibiotic susceptibility by a modification of the liquid microdilution method described by Baker and Thornsberry (1). Antibiotics. The antibiotics used in this study, the nominal concentration in agar overlays, and their commercial sources were as follows: streptomycin (2 mg/ml), Nutritional Biochemicals; rifampin (1 mg/ml), Becton, Dickinson and Co.; spectinomycin (266
,ug/ml), Upjohn; erythromycin (1.66 ,g/ml), Sigma; spiramycin (33 ,g/ml), Phone-Paulenc, Paris; streptolydigan (133 ,ug/ml), Upjohn; kasugamycin (13.3 mg/ml), Bristol Laboratories; fusidic acid (833,ug/ml), Leo Pharmaceutical; bacitracin (10 pug/ml), Sigma; thiostrepton (3.33 pg/ml), Squibb and Sons; gentamicin (1 mg/ml), Schering Corp.; gramicidin (100 pg/ml), Sigma; kanamycin (6.66 mg/ml), Sigma; neomycin (6.66 mg/ml), Sigma; oleandomycin (50 ug/ml), Sigma; and novobiocin (250 pug/ml), Sigma. Transduction. The procedure employed for transduction was a modification of techniques described by Colon et al. (4) and Malke (13). Bacteriophage for transduction experiments were propagated by adding 1 drop of phage (with titer exceeding 107 plaque-forming units per ml) plus 1 drop of donor bacteria from an overnight culture to 10 ml of proteose peptone broth containing hyaluronidase (0.1 mg/ml). After incubation overnight at 30°C, bacteriophage were separated from bacteria by membrane (Millipore Corp.) filtration and stored at 4°C. Transduction was performed according to the following procedure: 1 ml of bacteriophage was added to a plastic petri dish (20 by 120 mm) and subjected to irradiation with a model GT15T8 UV lamp for 1 min. The intensity of the lamp was 12,500 erg/s per cm2, as measured by a model J-225 Black-Ray UV light intensity meter. After irradiation, bacteriophage were added to stationaryphase K56 recipients at a multiplicity of infection of 0.1 to 0.5 phage particles per recipient cell. This mixture was incubated for 20 min at 370C to allow for adsorption. After incubation, 0.1-ml samples of the adsorption mixture were added to 3.0 ml of serum soft agar, mixed, and gently overlayed on STH agar plates. The STH agar plates were incubated for 2 h at 37°C to allow for recombination and expression of the donor phenotype. A second overlay containing appropriate concentrations of antibiotic was next added to the STH agar plates, and the plates were allowed to incubate for 24 to 48 h at 37°C. The net number of transductants per plate was calculated by subtracting the number of colonies arising on control plates without phage (0 to 15 colonies per plate, depending upon the antibiotic used) from the number of colonies on experimental plates.
RESULTS Transduction. Each mutant strain was tested for its ability to donate antibiotic resistance via transduction by using mutants of strain 9440 str as donors and K56 as the recipient.
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J. BACTERIOL.
The streptomycin marker served as a convenient reference for comparison of transduction frequencies of new markers with the transduction frequency of streptomycin resistance. A summary of data obtained in transduction experiments of antibiotic-resistant markers obtained in this study is listed in Table 2. In each case the streptomycin marker was transferred with high frequency. The collective data indicated that there were two groups of antibiotic resistance markers; i.e., transducible, including resistance to bacitracin, erythromycin, fusidic acid, kasugamycin, rifampin, spectinomycin, spiramycin, and streptolydigan (bac, ery, fus, ksg, rif, spc, spr, and sti, respectively), and nontransducible (data not shown, frequency less than 10-10), including resistance to ethidium bromide, gen-
tamicin, gramicidin, kanamycin, neomycin, no-
vobiocin, and oleandomycin (ebr, gen, grc, kan, neo, nov, and ole, respectively). TABLE 2. Summary of transduction of antibiotic resistance markers Selected recombit class
Antibiotic-
resistant donor
pr Ua per PFa
Efficiency (%)b
AS62-67 BaCr 3.3 x 10-7 24 AS20 3.1 x 10-6 35 Eryr FUsr AS50-53 4.7 x 10-6 208 AS42-46 4.5 x 10-6 Ksgr 20 AS7-10 4.6 x 10-6 Rif 184 AS11-15 4.2 x 10-6 32 Spcr AS25-28 4.2 x 10-6 Sprr 85 AS32-36 3.7 x 10-6 Stlr 136 AS69-73 Tstr 1.5 x 10-6 20 a PFU, Plaque-forming units. b Efficiency was determined as frequency of transduction versus frequency of str transduction in the same cross.
Duplicate sets of transduction experiments were also performed with sensitive 9440 str recipients. These strains served as competent recipients for all transducible markers, but not for the nontransducible markers. Generally, transduction frequencies were lower with 9440 str recipients than with K56 recipients, so transduction experiments were routinely performed with K56 recipients (24). Fortunately, the competency of 9440 str as a recipient permitted the construction of multiresistant strains, which were subsequently used to perform genetic analyses with two- and three-point reciprocal crosses. Two-point crosses. Each marker was first tested for linkage with the str marker by transduction into K56 recipient cells. One hundred transductants from each cross were selected and tested for streptomycin resistance by streaking on STH agar plates containing streptomycin (150 jig/ml). Reciprocal crosses were performed by adding phage lysates derived from 9440 str donors to K56 recipients carrying a single antibiotic resistance marker. One hundred Strr recombinants were selected and tested for the presence of the original marker by streaking onto STH agar plates containing the appropriate antibiotic. The data in Table 3 indicate that the streptomycin marker is linked to fus, bac, and ksg at average cotransduction frequencies of
85%, 24%, and 20%, respectively. Streptomycin resistance was not cotransducible with resistance to erythromycin, spectinomycin, spiramycin, streptolydigan, or tetracycline. It should be noted that the cross 9440 str-10 x K56 tst-1 was performed, but the recipient would not accept the streptomycin marker. These results were consistent with the behavior exhibited by thio-
TABLE 3. Two-point crosses involving streptomycin resistance Donor
AS43- 45 str ksg 9440 str
AS50-53 str fus 9440 str
Recipient
K56 AS47 ksg K56
AS55 fus
Selected phenotype Strr
Strr Fusr strr
Unselected phenotype
Ksgr
33 373
8
Ksg" KSgr
KsW
35 75
32 77
strr
391
Stre
122
Fuse
460 40
92
30 439
6
Stre Bac8 Bacr
226 331
41
Fusr
AS67 str bac 9440 str
K56 AS68 bac
Bacr
Strr
No. of recom- % Cotrnaducbinants tion
strr
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ANTIBIOTIC RESISTANCE IN S. PYOGENES
VOL. 133, 1978
strepton-resistant (Tstr) strains throughout the study. Generally, thiostrepton-resistant strains, in addition to being slow growers, served as poor recipients. Consequently, frequencies of transduction with Tst' recipients were at least one or two logs lower than with Tst8 recipients. iJnkage relationships among other antibiotic resistance markers were determined by twopoint reciprocal crosses as above. Data from additional two-point reciprocal crosses established the presence of three separate linkage groups (Table 4). sti and rif comprised one linkage group, and were cotransduced at an average frequency of 55%. Resistance to erythromycin, spiramycin, and spectinomycin were also linked by cotransduction experiments. Marker pairs were cotransduced with the following average frequencies: 94% for ery-spc, 79% for ery-spr, and 84% for spc-spr. Since ery and spr were
partially cross-resistant, it was at first difficult to conclude that they were separate loci. This problem was overcome by constructing Eryr and Sprr strains and testing for separation of these markers by transduction. Results from the cross AS22 and AS24 (ery spr) x K56 indicated that 8% of 159 Sprr colonies selected were sensitive to erythromycin. These data showed that Eryr and Sprr could be separated by transduction and therefore were specified by separate loci. The third linkage group was comprised of str, bac, fus, and ksg. Marker pairs in this linkage group were cotransduced with the following frequencies: 32% for fus-ksg, 28% for fus-bac, and 4% for bac-ksg. Some reciprocal crosses with kasugamycin-resistant (Ksgr) donors were not performed, due to a limited supply of kasugamycin. Crosses involving Sprr, Eryr, Spcr, or Ksgr donors and Tstr recipients were performed, but appar-
TABLE 4. Two-point crosses Phenotype Recipient
Donor
Selected
No. of recombiUnselected nants
Cotrans-
duction
AS38-40 stl rif
K56
Rift
Stlr
269 249
52
Stll
AS32-36 sti
AS2 rif
Stlr
Rif Rif
292 208
58
AS22 and AS23 ery
AS12, AS14, and AS15 spc
Eryr
Spc'
375 6
98
AS12, AS14, and AS15 spc
AS22 and AS23 ery
Spcr
379 45
89
Eryr
Ery8
319 166
66
Eryr
AS25-28 spr
AS21 ery
Sprr
Spr EryW
AS22 and AS24 ery spr
K56
Sprr
Eryr Ery-
146 13
92
AS12, AS14, and AS15 spc
AS22 and AS23 spr
Spcr
Spr8 Sprr
320 104
76
AS22 and AS23 spr
AS17-19 spc
Sprr
Spc'
365 26
93
Spcr
AS67 bac
AS47 ksg
Ksgm
7 156
4
Ksggr
AS50-52 fus
AS47-49 ksg
Fusr
KsgT Ksgr
123 249
33
AS43-45
AS55 fus
Ksgr
Fuse
54 129
30
48 323
13
FUSr
Bac' Bacr
238 309
44
ksg
Bacr
Fus r
AS67 bac
AS55 fus
A850 fis
AS68 bac
BaCr Fusr
Fus8
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J. BACTERIOL.
STUART AND FERRETTI
ently these marker combinations were not compatible, and no transductants were observed from these crosses or reciprocal crosses with these combinations. Three-point cross analyses. Four groups of three-point crosses within the spc-ery-spr
linkage group were performed (Table 5). The first group of three-point crosses involved transfer of spc into recipients resistant to erythromycin and/or spiramycin. Of 417 recombinants analyzed, 76% were Spcr Ery Spre, 15% were SpCr Erys Sprr, 8% were Spcr, Eryr Sprr, and
TABLE 5. Three-point crosses Phenotype
Selected
Unselected
No. of recombinants
AS12, AS14, and AS15 spc
AS22 and AS23 ery spr
Sprr
Erym Spe Ery" Sprr Eryr Sprr Eryr Spes
316 63 34 4
AS22 and AS23 ery spr
AS12, AS14, and AS15 spc
Eryr
Sprr Spre Sprr Spre
Spcr
243 131 6
Spcr
1
Spce Eryr
365 26 0 0
Donor
AS22 and AS23 ery spr
Recipient
AS17-19 spc
Sprr
Spe
Spce
Spcr Eryr
Spc" EryW Spcr Ery8 Eryr
Spr' Spcr
AS30 and AS31 ery spr spc
K56
AS50 str fus
AS68 bac
Fusr
Strr Strr Stre Stre
AS50 str fus
AS68 bac
AS67 bac str
Ery"
129 0
Bac' Bace Bac' Bace
224 203 85 35
Strr
Fusr Bacr Fusr Bace Fuse Bacr Fus" Bace
199 173 91 5
AS55 [us
Bacr
Str Fusr Stre Fus" Strr Fus" Strr Fusr
323 30 18 0
AS60 str fus bac
K56
Strr Bacr
Fusr Fus"
196 15
AS43-45 str ksg
AS55 fus
Ksgr
Stre Fusr Stre Fuse Strr Fuse
129 34
Strr FUsr
AS43-45 str ksg
AS50-52 fus str
AS57 and AS58 str fus ksg
AS55 fus
AS47-49 ksg
K56
Strr
Fusr
Stef Ksge
0
0
Ksg" Fus" Ksgr Fus" Ksg" Fusr Ksgr Fusr
248 33 25
Str' Ksg' Strr Ksg" Str Ksgr Str" Ksg"
163 116 86 7
Fusr Fus"
23
0
1
ANTIBIOTIC RESISTANCE IN S. PYOGENES
VOL. 133, 1978
only 1% were Spcr Eryr SprO. Since the number of recombinants in the last phenotypic class was so low, it was assumed to result from four crossing-over events. These data place ery between spc and spr (Fig. 1). The second group of crosses was performed with Eryr Sprr donors and Spcr recipients. Erythromycin-resistant recombinants were selected and tested for the presence of Spcr and Sprr by streaking on STH agar plates containing spectinomycin and/or spiramycin. Of 381 recombinants analyzed, 64% were Eryr Sprr Spcr, 34% were Ery' Sprs Spe, 2% were Eryr Sprr Spcr, and one recombinant was Eryr Spre Spcr. The last phenotype mentioned was assumed to arise from quadruple crossovers. The third group of crosses was performed with EryW Spr' donors and Spcr recipients. Sprr recombinants were selected and tested for the presence of Ery' and Sprr by streaking on STH agar plates containing appropriate levels of these antibiotics. The data from these crosses indicated that spr is closer to ery than to spc, since the spr ery pair was cotransduced at a frequency of 93%. Since spr is an outside marker, it is suggested on the basis of cotransduction frequencies that the gene order is spc-ery-spr. To confirm the above gene order, a fourth group of crosses was performed using donors triply resistant to erythromycin, spiramycin, and spectinomycin, and sensitive K56 recipients. Spcr Sprr transductants were selected and tested for the presence of Eryr by streaking recombinants on STH agar plates contining erythromycin. As shown in Table 5, 100% of 129 recombinants selected were Ery'. These data confirmed the gene order spc-ery-spr. Distances between these genes were considered a function of average cotransduction frequencies of twoand three-point crosses and are presented in Fig. 1.
With the same methodological approach,
qPC
94%
my
857
three-point crosses were performed within the following triplet sets of markers: str-fus-ksg and str-fus-bac. Data from these crosses are presented in Table 5, and established definitively the gene orders str-fus-ksg and str-fus-bac. Distances between these genes were considered a function of average cotransduction frequencies. On the basis of contransduction frequencies obtained from two- and three-point crosses, it is logical to infer the gene order str-fus-bac-ksg. Cotransduction frequencies among these genes are presented in Fig. 2, which indicates the relative distances between these genes. DISCUSSION Transduction experiments separated antibiotic resistant markers into two distinct groups, transducible and nontransducible markers. It is not known why certain mutant classes failed to be transduced; however, there are several possible explanations that might be offered. Nontransducible phenotypes could be a result of multiple mutations in genes that are not closely linked. Alternatively, genes of nontransducible markers may originate from extrachromosomal genetic elements, which in this transduction system are nontransferable. Among transducible markers, transfer of rif, str, ery, spc, and bac has been reported earlier (13, 24, 28) in S. pyogenes, and data from this study confirmed these results. On the other hand, this study presented the first descriptions of transduction ofstl, fus, ksg, spr, and tst markers in S.pyogenes. Additionally, bac was transferred within group A streptococci for the first time since the report by Wannamaker et al. (28) describing intergroup transfer of bac from a group C strain into a group A recipient. Two- and three-point cross analyses established the presence of three separate linkage groups, rif-stl, str-fus-bac-ksg, and spc-ery-spr. It is interesting to note that all transducible 81%
spr
85% FIG. 1. Distances between spc, ery, and spr expressed as a function of average cotransduction frequencies. 80%
fus
28%
bac
4%
ksg
31% 22% 12%
FIG. 2. Distances between str, fus, bac, and ksg expressed
frequencies.
I as a
function of
average cotransduction
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STUART AND FERRETTI
markers except tst were mapped within one of these linkage groups. It seems possible that tst is linked to one of the ribosomal gene clusters in S. pyogenes because of its binding action with bacterial 50S ribosomal subunits (29). Data in this study showed that tst is not linked to str, fus, or bac, which contrasts with the genetic structure of the Bacillus subtilis chromosome, where tst is linked to str and fus (6, 7, 20). ksg or the spc-ery-spr linkage group are therefore the most promising possibilities for linkage with tst. Surprisingly, no recombinants could be obtained from reciprocal crosses involving Tstr and any of these four remaining loci. The reason for the lack of recombinants is not known, but it is plausible that recipient cells were not viable under the influence of the paired phenotypes Tstr and Ksgr, and Spcr, Eryr, or Sprr. Linkage of rif and stl was not surprising, since these markers were also linked in Escherichia coli (21) and B. subtilis (9). Neither rif nor sti was linked by cotransduction to other antibioticresistant markers in S. pyogenes. This linkage relationship is similar to that of E. coli (25) and Salmonella typhimurium (18), but dissimilar to B. subtilis (7) and Neisseria gonorrhoeae (19), where rif is linked to other genes conferring antibiotic resistance. Linkage of ery and spc confirmed similar observations by Malke (14) in S. pyogenes and corresponds with the linkage of these genes in E. coli (25) and B. subtilis (7). Linkage of ery and spr was not surprising, since they are both macrolide antibiotics and have similar modes of action (17). Presumably lincomycin resistance (lin) is located within this linkage group, since Malke (14) reported linkage of spc-ery-lin, in that order. Unfortunately, Linr mutants were not isolated in this study and could not be ordered within the linkage group spc-ery-spr. The str-fus-ksg linkage group appeared to be near the size limit of the transduced segment of DNA, since the donor phenotype was transduced only about 8% of the time when Strr was selected, and 0% when Ksgr was selected (Table 5). The observation of close linkage between str and fus was not surprising, because these markers are tightly linked in E. coli (25) and B. subtilis (6). However, it was interesting to discover the presence of ksg in the Strr linkage group, since none of the three loci that specify Ksgr in E. coli is linked to the ribosome gene cluster containing strA (22, 30). The position of bac within the str-fus-ksg linkage group was inferred on the basis of cotransduction frequencies presented in Fig. 2. Although bacr has been transduced in S. pyogenes, this is the first report to map the bac locus in any bacterial species. Presumably, resistance to
bacitracin involves some change in the cell wall or the cell membrane (23), whereas resistance to streptomycin, fusidic acid, and kasugamycin probably involves a mutational change in the protein-synthesizing system of the cell (3, 8, 27). The relationship among the genes in this linkage group might involve the production of products found at the cell wall of bacteria. For example, Ilida and Koike (10) found cell alterations in gram-negative bacteria induced by aminoglycoside antibiotics, and Klainer (11) reported an abnormal surface morphology of S. aureus after exposure to aminoglycosides. ACKNOWLEDGMENT This work was supported by Oklahoma Heart Asociation grant G74-118.
LITERATURE CITED 1. Baker, C. N., and C. Thornaberry. 1974. Antimicrobial susceptibility of Streptococcus mutans isolated from patients with endocarditis. Antimicrob. Agents Chemother. 5:268-271. 2. Bolien, A., J. Savies, M. Ozaki, and S. M ui 1969. Ribosomal protein confering sensitivity to the antibiotic spectinomycin in Escherichia coli. Science
165:85-86. 3. Carrasco, L, and D. Vasquez. 1973. Ribosomal sites involved in binding of aminoacyl-tRNA and EF2. Mode of action of fusidic acid. FEBS Lett 32:152-156. 4. Colon, A. E., R. M. Cole, and C. G. Leonard. 1970. Transduction in group A streptococci by ultraviolet irradiated phages. Can. J. Microbiol. 16:201-202. 5. Demerec, AL, E. A. Adleberg, A. J. Clark, and P. E. Hartman- 1965. A proposal for a uniform nomenclature in bacterial genetics Genetics 54:61-76. 6. Goldthwaite, C., and L Smith. 1972. Genetic mapping of aminoglycoside and fusidic acid resistant mutations in BaciUus subtilis. MoL Gen. Genet. 114:181-189. 7. Hartford, N., and N. Sueoka. 1970. Chromosomal location of antibiotic resistance markers in Bacillus subtilis. J. Mol. Biol. 51:267-286. 8. Hash, J. H. 1972. Antibiotic mechanism. Annu. Rev.
PharmacoL 12:35-56. 9. Haworth, S. R., and L R. Brown. 1973. Genetic analysis of ribonucleic acid polymerase mutants of Bacillus subtilis. J. Bacteriol. 114:103-113. 10. lida, K., and M. Koike. 1974. Cell wall alterations of gram-negative bacteria by aminoglycoside antibiotics.
Antimicrob. Agents Chemother. 5:95-97. 11. Klainer, A. S. 1974. The normal and abnormal surface morphology of staphylococci. Ann. N.Y. Acad. Sci.
236:63-75. 12. Leonard, C. G., A. E. Colon, and R. M. Cole. 1968. Transduction in group A streptococcus. Biochem. Biophys. Res. Commun. 30:130-135. 13. Malke, H. 1969. Transduction of Streptococcus pyogenes K56 by temperature sensitive mutants of transducing phage A25. Z. Naturforsch. 246:1556-1561. 14. Malke, H. 1972. Linkage relationships of mutations endowing Streptococcus pyogenes with resistance to antibiotics that affect the ribosome. Mol. Gen. Genet. 116:299-308. 15. Malke, H. 1975. Transfer of a plasmid mediating antibiotic resistance between strains of Streptococcus pyogenes in mixed cultures. Z. AUg. Mikrobiol. 15:645-649. 16. Malke, H., H. E. Jacob, and K. Storl. 1976. Characterization of the antibiotic resistance plasmid ERLI from Streptococcuspyogenes. Mol. Gen. Genet. 144:333-338. 17. Pestka, S. 1971. Inhibitors of ribosome function. Annu.
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Rev. Microbiol. 26:487-562. 18. Sanderson, K. E. 1972. Linkage map of Sabnonella typhimurium, edition IV. Bacteriol. Rev. 36:558-586. 19. Sarubbi, F. A., Jr., E. Blackman, and P. F. Sparling. 1974. Genetic mapping of linked antibiotic resistance loci in Neisseria gonorrhoeae. J. Bacteriol. 120:1284-1292. 20. Smith, I., and H. Smith. 1973. Location of the SP02 attachment site and the bryamycin resistance marker on the Bacillus subtilis chromosome. J. Bacteriol. 114:1138-1142. 21. Sokolova, E. V., M. I. Ovadis, Z. M. Gorlenko, and R. B. Khesdn. 1970. Localization of streptolydigin resistant mutation in E. coli and effect of streptolydigin on T2 phage development in stl-r and stl-s of E. coli. Biochem. Biophys. Res. Commun. 41:8704876. 22. Sparling, F. P., Y. Ikeya, and D. Elliot. 1973. Two genetic loci for resistance to kasugamycin in Escherichia coli. J. Bacteriol. 113:704-710. 23. Stone, K. J., and J. L Strominger. 1971. Mechanism of action of bacitracin: complexation with metal ion and Cj6.isoprenyl pyrophosphate. Proc. Natl. Acad. Sci. U.S.A. 68:3223-3227. 24. Stuart, J. G., and J. J. Ferretti. 1973. Transduction of
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rifampin resistance in group A streptococci. J. Bacteriol. 115:709-710. 25. Taylor, A. L, and C. D. Trotter. 1972. Linkage map of Escherichia coli strain K- 12. Bacteriol. Rev. 36:504-524. 26. Ubakata, K., M. Konno, and R. Fujii. 1975. Transduction of drug resistance to tetracycline, chloramphenicol, macrolides, lincomycin, and clindamycin with phages induced from Streptococcus pyogenes. J. Antibiot. 28:681-688. 27. Umezawa, H., H. Hamada, Y. Suhara, T. Hashimoto, T. Ekekawa, N. Tanaka, K. Maeda, Y. Okami, and T. Takeuchi. 1966. Kasugamycin, a new antibiotic, p. 753-757. Antimicrob. Agents Chemother. 1965. 28. Wannamaker, L W., S. Almquist, and S. Skjold. 1973. Intergroup phage reactions and transduction between group C and group A streptococci. J. Exp. Med. 137:1338-1353. 29. Weisblum, B., and V. Demohn. 1970. Thiostrepton, an inhibitor of 50S ribosome subunit function. J. Bacteriol. 101:1073-1075. 30. Yoshikawa, M., A. Okuyama, and N. Tanaka. 1975. A third kasugamycin resistance locus, ksgC, affecting ribosomal protein S2 in Escherichia coli K-12. J. Bacteriol. 122:796-797.
JOURNAL OF BACTERIOLOGY, Feb. 1978, p. 852859
0021-9193/78/0133-0852$02.00/0 Copyright i 1978 American Society for Microbiology
Prined in U.S.A.
Genetic Analysis of Antibiotic Resistance in Streptococcus pyogenes JAMES G. STUARTt AND JOSEPH J. FERRETTI* Department of Microbiology and Immunology, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma 73190 Received for publication 12 April 1977
The genetics of antibiotic resistance in mutant strains of Streptococcus pyogenes was studied. Utilizing a type 6 strain (9440) primarily resistant to streptomycin (Strr), classes of mutant strains were isolated that were resistant to one of the following antibiotics: rifampin (RiP), erythromycin (Eryr), thiostrepton (Tstr), spiramycin (Spr), fusidic acid (Fusr), gramicidin (Grcr), ethidium bromide (Ebrr), kanamycin (Kanr), neomycin (Neor), oleandomycin (Oler), gentamicin (Genr), and novobiocin (Novr). Transduction experiments separated antibiotic resistance markers into two distinct groups: transducible markers, including Fusr, Bacr, Ksg, SpCr, EYr, Sprr, Rift, Stlr, and Tstr (Bacr, Ksgr, Spcr, and Stlr refer to resistance to bacitracin, kasugamycin, spectinomycin, and streptolydigan, respectively), and nontransducible markers, including Grcr, Ebrr, Kanr, Neor, Oler, Genr, and Novr. By means of two- and three-point crosses, transducible markers (excluding tst) were located in three separate linkage groups. spr was found to be linked with ery and spc in the order spc-ery-spr, whereas in a separate linkage group the order was determined to be str-fus-bac-ksg. The third linkage group contained the rif and stl markers.
Studies on the genetics of antibiotic resistance in Streptococcuspyogenes were started by Leonard et al. (12), who described transduction of streptomycin resistance. Subsequently, Malke (13, 14) refined the transduction technique and described two groups of markers that were cotransduced into sensitive recipients. In one linkage group, resistance to streptomycin (str) was cotransduced at high frequency with resistance to kanamycin (kan), and in another group resistance to spectinomycin (spc), erythromycin (ery), and lincomycin (lin) were found to be tightly linked in the order described. Another antibiotic resistance marker, rifampin (rit), was shown to be separate from the streptomycin linkage group (24). All of these studies utilized strains possessing laboratory-induced antibiotic resistance markers, presumably chromosomal in
origin. Antibiotic resistance markers found in clinical isolates, which are extrachromosomal in origin, have also been transduced to sensitive recipient strains. Ubakata et al. (26) have shown that transfer of resistance occurs in two different patterns; i.e., transfer of tetracycline resistance alone or joint transfer of chloramphenicol, macrolide antibiotics, lincomycin, and clindamycin resistance. Malke (15) has shown that antibiotic t Present address: Department of Biological Sciences, Murray State University, Murray, KY 42071.
852
resistance to erythromycin, lincomycin, and staphylomycin S, known to be plasmid directed (16), is transferred by transduction with high efficiency. The present study of antibiotic resistance markers of chromosomal origin substantiates linkage data previously reported by Malke (14), and, further, orders four new antibiotic resistance markers within two linkage groups. Additional data establish the presence of a third linkage group including resistance to rifampin and streptolydigan (stl).
MATERIALS AND METHODS Strains. Parent strains of streptococci and streptococcal phages used in this research were obtained from the laboratory of Lewis Wannamaker at the University of Minnesota. Bacterial strains include two group A strains of S. pyogenes, a type 12 strain designated K56 and a type 6, streptomycin-resistant (Strr) strain designated 9440 str. Mutant strains were derived from 9440 str and are listed in Table 1. Antibioticresistant strains of K56 were constructed by transduction and are also listed in Table 1. The genetic symbols and nomenclature used are described by Demerec et al. (5). One strain of bacteriophage A25 was utilized in this study in transduction experiments. This strain is a double temperature-sensitive mutant designated A25 tsl-2 and was originally isolated by Malke (13). Media. Proteose peptone broth has been described
VOL. 133, 1978
ANTIBIOTIC RESISTANCE IN S. PYOGENES
TABLE 1. Bacterial strains Strain K56 derivativesb AS1 AS2
AS16-AS19 AS21 AS29 AS37 AS47-AS49 AS55 AS61 AS68 AS74
Markerse str-1O
rif-l 8pC
ery-l spr-4 stl-1
ksg
fus-l fus-1 bac-6 bac-6 tst
9440 derivativesc
AS7-AS10 rif AS11-AS15 spc AS20 ery-1 AS22-AS24 ery spr AS25-AS28 spr AS30-AS31 ery spr spc AS32-AS36 stl AS38-AS41 sti rif AS42-AS46 ksg AS50-AS54 fus AS56-AS58 ksg fus AS59 ksg-2 fus-1 bac-6 AS60 fus-I bac-6 AS62-AS67 bac AS69-AS73 tst AS76-AS80 grc AS81-AS85 ebr AS86-AS90 kan AS91-AS95 neo AS96-AS100 gen AS101-AS102 ole AS103 nov a Lack of an allele number following a gene designation indicates a group of independently isolated markers. b All K56 derivatives were constructed by transduction. c All 9440 derivatives contained str-10. Additional markers were obtained by selection of spontaneous mutants or by transduction into each subline.
previously by Wannamaker et al. (28) as no. 1 broth. Three types of solid media were employed. Serum Todd-Hewitt (STH) agar was prepared by adding 0.38% Na2HPO4 and 1.5% bacteriological agar to ToddHewitt broth (Difco). This solution was autoclaved, and sterile solutions of 0.02% CaCl2 and 5% normal horse serum were added. Blood agar was prepared by substituting 5% sheep blood or human blood for normal horse serum in STH agar plates. Serum soft agar was prepared by substituting 3% proteose peptone 2 (Difco) and 0.6% NaCl in proteose peptone broth with the addition of 0.7% bacteriological agar. Mutant Isolation. Spontaneous antibiotic-resistant mutants were isolated by the following procedure. Bacteria (9440 str) were grown to log phase at 30°C in proteose peptone broth, and 0.1-ml samples of this culture were added to 3 ml of serum soft agar, which
853
was then overlayed on STH agar plates. After incubation for 2 h at 37°C, a second 3-ml overlay of serum soft agar containing an appropriate level of antibiotic was added to the seeded STH agar plate. After overnight incubation at 37°C, resistant colonies were selected and tested for antibiotic susceptibility by a modification of the liquid microdilution method described by Baker and Thornsberry (1). Antibiotics. The antibiotics used in this study, the nominal concentration in agar overlays, and their commercial sources were as follows: streptomycin (2 mg/ml), Nutritional Biochemicals; rifampin (1 mg/ml), Becton, Dickinson and Co.; spectinomycin (266
,ug/ml), Upjohn; erythromycin (1.66 ,g/ml), Sigma; spiramycin (33 ,g/ml), Phone-Paulenc, Paris; streptolydigan (133 ,ug/ml), Upjohn; kasugamycin (13.3 mg/ml), Bristol Laboratories; fusidic acid (833,ug/ml), Leo Pharmaceutical; bacitracin (10 pug/ml), Sigma; thiostrepton (3.33 pg/ml), Squibb and Sons; gentamicin (1 mg/ml), Schering Corp.; gramicidin (100 pg/ml), Sigma; kanamycin (6.66 mg/ml), Sigma; neomycin (6.66 mg/ml), Sigma; oleandomycin (50 ug/ml), Sigma; and novobiocin (250 pug/ml), Sigma. Transduction. The procedure employed for transduction was a modification of techniques described by Colon et al. (4) and Malke (13). Bacteriophage for transduction experiments were propagated by adding 1 drop of phage (with titer exceeding 107 plaque-forming units per ml) plus 1 drop of donor bacteria from an overnight culture to 10 ml of proteose peptone broth containing hyaluronidase (0.1 mg/ml). After incubation overnight at 30°C, bacteriophage were separated from bacteria by membrane (Millipore Corp.) filtration and stored at 4°C. Transduction was performed according to the following procedure: 1 ml of bacteriophage was added to a plastic petri dish (20 by 120 mm) and subjected to irradiation with a model GT15T8 UV lamp for 1 min. The intensity of the lamp was 12,500 erg/s per cm2, as measured by a model J-225 Black-Ray UV light intensity meter. After irradiation, bacteriophage were added to stationaryphase K56 recipients at a multiplicity of infection of 0.1 to 0.5 phage particles per recipient cell. This mixture was incubated for 20 min at 370C to allow for adsorption. After incubation, 0.1-ml samples of the adsorption mixture were added to 3.0 ml of serum soft agar, mixed, and gently overlayed on STH agar plates. The STH agar plates were incubated for 2 h at 37°C to allow for recombination and expression of the donor phenotype. A second overlay containing appropriate concentrations of antibiotic was next added to the STH agar plates, and the plates were allowed to incubate for 24 to 48 h at 37°C. The net number of transductants per plate was calculated by subtracting the number of colonies arising on control plates without phage (0 to 15 colonies per plate, depending upon the antibiotic used) from the number of colonies on experimental plates.
RESULTS Transduction. Each mutant strain was tested for its ability to donate antibiotic resistance via transduction by using mutants of strain 9440 str as donors and K56 as the recipient.
854
STUART AND FERRETTI
J. BACTERIOL.
The streptomycin marker served as a convenient reference for comparison of transduction frequencies of new markers with the transduction frequency of streptomycin resistance. A summary of data obtained in transduction experiments of antibiotic-resistant markers obtained in this study is listed in Table 2. In each case the streptomycin marker was transferred with high frequency. The collective data indicated that there were two groups of antibiotic resistance markers; i.e., transducible, including resistance to bacitracin, erythromycin, fusidic acid, kasugamycin, rifampin, spectinomycin, spiramycin, and streptolydigan (bac, ery, fus, ksg, rif, spc, spr, and sti, respectively), and nontransducible (data not shown, frequency less than 10-10), including resistance to ethidium bromide, gen-
tamicin, gramicidin, kanamycin, neomycin, no-
vobiocin, and oleandomycin (ebr, gen, grc, kan, neo, nov, and ole, respectively). TABLE 2. Summary of transduction of antibiotic resistance markers Selected recombit class
Antibiotic-
resistant donor
pr Ua per PFa
Efficiency (%)b
AS62-67 BaCr 3.3 x 10-7 24 AS20 3.1 x 10-6 35 Eryr FUsr AS50-53 4.7 x 10-6 208 AS42-46 4.5 x 10-6 Ksgr 20 AS7-10 4.6 x 10-6 Rif 184 AS11-15 4.2 x 10-6 32 Spcr AS25-28 4.2 x 10-6 Sprr 85 AS32-36 3.7 x 10-6 Stlr 136 AS69-73 Tstr 1.5 x 10-6 20 a PFU, Plaque-forming units. b Efficiency was determined as frequency of transduction versus frequency of str transduction in the same cross.
Duplicate sets of transduction experiments were also performed with sensitive 9440 str recipients. These strains served as competent recipients for all transducible markers, but not for the nontransducible markers. Generally, transduction frequencies were lower with 9440 str recipients than with K56 recipients, so transduction experiments were routinely performed with K56 recipients (24). Fortunately, the competency of 9440 str as a recipient permitted the construction of multiresistant strains, which were subsequently used to perform genetic analyses with two- and three-point reciprocal crosses. Two-point crosses. Each marker was first tested for linkage with the str marker by transduction into K56 recipient cells. One hundred transductants from each cross were selected and tested for streptomycin resistance by streaking on STH agar plates containing streptomycin (150 jig/ml). Reciprocal crosses were performed by adding phage lysates derived from 9440 str donors to K56 recipients carrying a single antibiotic resistance marker. One hundred Strr recombinants were selected and tested for the presence of the original marker by streaking onto STH agar plates containing the appropriate antibiotic. The data in Table 3 indicate that the streptomycin marker is linked to fus, bac, and ksg at average cotransduction frequencies of
85%, 24%, and 20%, respectively. Streptomycin resistance was not cotransducible with resistance to erythromycin, spectinomycin, spiramycin, streptolydigan, or tetracycline. It should be noted that the cross 9440 str-10 x K56 tst-1 was performed, but the recipient would not accept the streptomycin marker. These results were consistent with the behavior exhibited by thio-
TABLE 3. Two-point crosses involving streptomycin resistance Donor
AS43- 45 str ksg 9440 str
AS50-53 str fus 9440 str
Recipient
K56 AS47 ksg K56
AS55 fus
Selected phenotype Strr
Strr Fusr strr
Unselected phenotype
Ksgr
33 373
8
Ksg" KSgr
KsW
35 75
32 77
strr
391
Stre
122
Fuse
460 40
92
30 439
6
Stre Bac8 Bacr
226 331
41
Fusr
AS67 str bac 9440 str
K56 AS68 bac
Bacr
Strr
No. of recom- % Cotrnaducbinants tion
strr
855
ANTIBIOTIC RESISTANCE IN S. PYOGENES
VOL. 133, 1978
strepton-resistant (Tstr) strains throughout the study. Generally, thiostrepton-resistant strains, in addition to being slow growers, served as poor recipients. Consequently, frequencies of transduction with Tst' recipients were at least one or two logs lower than with Tst8 recipients. iJnkage relationships among other antibiotic resistance markers were determined by twopoint reciprocal crosses as above. Data from additional two-point reciprocal crosses established the presence of three separate linkage groups (Table 4). sti and rif comprised one linkage group, and were cotransduced at an average frequency of 55%. Resistance to erythromycin, spiramycin, and spectinomycin were also linked by cotransduction experiments. Marker pairs were cotransduced with the following average frequencies: 94% for ery-spc, 79% for ery-spr, and 84% for spc-spr. Since ery and spr were
partially cross-resistant, it was at first difficult to conclude that they were separate loci. This problem was overcome by constructing Eryr and Sprr strains and testing for separation of these markers by transduction. Results from the cross AS22 and AS24 (ery spr) x K56 indicated that 8% of 159 Sprr colonies selected were sensitive to erythromycin. These data showed that Eryr and Sprr could be separated by transduction and therefore were specified by separate loci. The third linkage group was comprised of str, bac, fus, and ksg. Marker pairs in this linkage group were cotransduced with the following frequencies: 32% for fus-ksg, 28% for fus-bac, and 4% for bac-ksg. Some reciprocal crosses with kasugamycin-resistant (Ksgr) donors were not performed, due to a limited supply of kasugamycin. Crosses involving Sprr, Eryr, Spcr, or Ksgr donors and Tstr recipients were performed, but appar-
TABLE 4. Two-point crosses Phenotype Recipient
Donor
Selected
No. of recombiUnselected nants
Cotrans-
duction
AS38-40 stl rif
K56
Rift
Stlr
269 249
52
Stll
AS32-36 sti
AS2 rif
Stlr
Rif Rif
292 208
58
AS22 and AS23 ery
AS12, AS14, and AS15 spc
Eryr
Spc'
375 6
98
AS12, AS14, and AS15 spc
AS22 and AS23 ery
Spcr
379 45
89
Eryr
Ery8
319 166
66
Eryr
AS25-28 spr
AS21 ery
Sprr
Spr EryW
AS22 and AS24 ery spr
K56
Sprr
Eryr Ery-
146 13
92
AS12, AS14, and AS15 spc
AS22 and AS23 spr
Spcr
Spr8 Sprr
320 104
76
AS22 and AS23 spr
AS17-19 spc
Sprr
Spc'
365 26
93
Spcr
AS67 bac
AS47 ksg
Ksgm
7 156
4
Ksggr
AS50-52 fus
AS47-49 ksg
Fusr
KsgT Ksgr
123 249
33
AS43-45
AS55 fus
Ksgr
Fuse
54 129
30
48 323
13
FUSr
Bac' Bacr
238 309
44
ksg
Bacr
Fus r
AS67 bac
AS55 fus
A850 fis
AS68 bac
BaCr Fusr
Fus8
856
J. BACTERIOL.
STUART AND FERRETTI
ently these marker combinations were not compatible, and no transductants were observed from these crosses or reciprocal crosses with these combinations. Three-point cross analyses. Four groups of three-point crosses within the spc-ery-spr
linkage group were performed (Table 5). The first group of three-point crosses involved transfer of spc into recipients resistant to erythromycin and/or spiramycin. Of 417 recombinants analyzed, 76% were Spcr Ery Spre, 15% were SpCr Erys Sprr, 8% were Spcr, Eryr Sprr, and
TABLE 5. Three-point crosses Phenotype
Selected
Unselected
No. of recombinants
AS12, AS14, and AS15 spc
AS22 and AS23 ery spr
Sprr
Erym Spe Ery" Sprr Eryr Sprr Eryr Spes
316 63 34 4
AS22 and AS23 ery spr
AS12, AS14, and AS15 spc
Eryr
Sprr Spre Sprr Spre
Spcr
243 131 6
Spcr
1
Spce Eryr
365 26 0 0
Donor
AS22 and AS23 ery spr
Recipient
AS17-19 spc
Sprr
Spe
Spce
Spcr Eryr
Spc" EryW Spcr Ery8 Eryr
Spr' Spcr
AS30 and AS31 ery spr spc
K56
AS50 str fus
AS68 bac
Fusr
Strr Strr Stre Stre
AS50 str fus
AS68 bac
AS67 bac str
Ery"
129 0
Bac' Bace Bac' Bace
224 203 85 35
Strr
Fusr Bacr Fusr Bace Fuse Bacr Fus" Bace
199 173 91 5
AS55 [us
Bacr
Str Fusr Stre Fus" Strr Fus" Strr Fusr
323 30 18 0
AS60 str fus bac
K56
Strr Bacr
Fusr Fus"
196 15
AS43-45 str ksg
AS55 fus
Ksgr
Stre Fusr Stre Fuse Strr Fuse
129 34
Strr FUsr
AS43-45 str ksg
AS50-52 fus str
AS57 and AS58 str fus ksg
AS55 fus
AS47-49 ksg
K56
Strr
Fusr
Stef Ksge
0
0
Ksg" Fus" Ksgr Fus" Ksg" Fusr Ksgr Fusr
248 33 25
Str' Ksg' Strr Ksg" Str Ksgr Str" Ksg"
163 116 86 7
Fusr Fus"
23
0
1
ANTIBIOTIC RESISTANCE IN S. PYOGENES
VOL. 133, 1978
only 1% were Spcr Eryr SprO. Since the number of recombinants in the last phenotypic class was so low, it was assumed to result from four crossing-over events. These data place ery between spc and spr (Fig. 1). The second group of crosses was performed with Eryr Sprr donors and Spcr recipients. Erythromycin-resistant recombinants were selected and tested for the presence of Spcr and Sprr by streaking on STH agar plates containing spectinomycin and/or spiramycin. Of 381 recombinants analyzed, 64% were Eryr Sprr Spcr, 34% were Ery' Sprs Spe, 2% were Eryr Sprr Spcr, and one recombinant was Eryr Spre Spcr. The last phenotype mentioned was assumed to arise from quadruple crossovers. The third group of crosses was performed with EryW Spr' donors and Spcr recipients. Sprr recombinants were selected and tested for the presence of Ery' and Sprr by streaking on STH agar plates containing appropriate levels of these antibiotics. The data from these crosses indicated that spr is closer to ery than to spc, since the spr ery pair was cotransduced at a frequency of 93%. Since spr is an outside marker, it is suggested on the basis of cotransduction frequencies that the gene order is spc-ery-spr. To confirm the above gene order, a fourth group of crosses was performed using donors triply resistant to erythromycin, spiramycin, and spectinomycin, and sensitive K56 recipients. Spcr Sprr transductants were selected and tested for the presence of Eryr by streaking recombinants on STH agar plates contining erythromycin. As shown in Table 5, 100% of 129 recombinants selected were Ery'. These data confirmed the gene order spc-ery-spr. Distances between these genes were considered a function of average cotransduction frequencies of twoand three-point crosses and are presented in Fig. 1.
With the same methodological approach,
qPC
94%
my
857
three-point crosses were performed within the following triplet sets of markers: str-fus-ksg and str-fus-bac. Data from these crosses are presented in Table 5, and established definitively the gene orders str-fus-ksg and str-fus-bac. Distances between these genes were considered a function of average cotransduction frequencies. On the basis of contransduction frequencies obtained from two- and three-point crosses, it is logical to infer the gene order str-fus-bac-ksg. Cotransduction frequencies among these genes are presented in Fig. 2, which indicates the relative distances between these genes. DISCUSSION Transduction experiments separated antibiotic resistant markers into two distinct groups, transducible and nontransducible markers. It is not known why certain mutant classes failed to be transduced; however, there are several possible explanations that might be offered. Nontransducible phenotypes could be a result of multiple mutations in genes that are not closely linked. Alternatively, genes of nontransducible markers may originate from extrachromosomal genetic elements, which in this transduction system are nontransferable. Among transducible markers, transfer of rif, str, ery, spc, and bac has been reported earlier (13, 24, 28) in S. pyogenes, and data from this study confirmed these results. On the other hand, this study presented the first descriptions of transduction ofstl, fus, ksg, spr, and tst markers in S.pyogenes. Additionally, bac was transferred within group A streptococci for the first time since the report by Wannamaker et al. (28) describing intergroup transfer of bac from a group C strain into a group A recipient. Two- and three-point cross analyses established the presence of three separate linkage groups, rif-stl, str-fus-bac-ksg, and spc-ery-spr. It is interesting to note that all transducible 81%
spr
85% FIG. 1. Distances between spc, ery, and spr expressed as a function of average cotransduction frequencies. 80%
fus
28%
bac
4%
ksg
31% 22% 12%
FIG. 2. Distances between str, fus, bac, and ksg expressed
frequencies.
I as a
function of
average cotransduction
858
J. BACTERIOL.
STUART AND FERRETTI
markers except tst were mapped within one of these linkage groups. It seems possible that tst is linked to one of the ribosomal gene clusters in S. pyogenes because of its binding action with bacterial 50S ribosomal subunits (29). Data in this study showed that tst is not linked to str, fus, or bac, which contrasts with the genetic structure of the Bacillus subtilis chromosome, where tst is linked to str and fus (6, 7, 20). ksg or the spc-ery-spr linkage group are therefore the most promising possibilities for linkage with tst. Surprisingly, no recombinants could be obtained from reciprocal crosses involving Tstr and any of these four remaining loci. The reason for the lack of recombinants is not known, but it is plausible that recipient cells were not viable under the influence of the paired phenotypes Tstr and Ksgr, and Spcr, Eryr, or Sprr. Linkage of rif and stl was not surprising, since these markers were also linked in Escherichia coli (21) and B. subtilis (9). Neither rif nor sti was linked by cotransduction to other antibioticresistant markers in S. pyogenes. This linkage relationship is similar to that of E. coli (25) and Salmonella typhimurium (18), but dissimilar to B. subtilis (7) and Neisseria gonorrhoeae (19), where rif is linked to other genes conferring antibiotic resistance. Linkage of ery and spc confirmed similar observations by Malke (14) in S. pyogenes and corresponds with the linkage of these genes in E. coli (25) and B. subtilis (7). Linkage of ery and spr was not surprising, since they are both macrolide antibiotics and have similar modes of action (17). Presumably lincomycin resistance (lin) is located within this linkage group, since Malke (14) reported linkage of spc-ery-lin, in that order. Unfortunately, Linr mutants were not isolated in this study and could not be ordered within the linkage group spc-ery-spr. The str-fus-ksg linkage group appeared to be near the size limit of the transduced segment of DNA, since the donor phenotype was transduced only about 8% of the time when Strr was selected, and 0% when Ksgr was selected (Table 5). The observation of close linkage between str and fus was not surprising, because these markers are tightly linked in E. coli (25) and B. subtilis (6). However, it was interesting to discover the presence of ksg in the Strr linkage group, since none of the three loci that specify Ksgr in E. coli is linked to the ribosome gene cluster containing strA (22, 30). The position of bac within the str-fus-ksg linkage group was inferred on the basis of cotransduction frequencies presented in Fig. 2. Although bacr has been transduced in S. pyogenes, this is the first report to map the bac locus in any bacterial species. Presumably, resistance to
bacitracin involves some change in the cell wall or the cell membrane (23), whereas resistance to streptomycin, fusidic acid, and kasugamycin probably involves a mutational change in the protein-synthesizing system of the cell (3, 8, 27). The relationship among the genes in this linkage group might involve the production of products found at the cell wall of bacteria. For example, Ilida and Koike (10) found cell alterations in gram-negative bacteria induced by aminoglycoside antibiotics, and Klainer (11) reported an abnormal surface morphology of S. aureus after exposure to aminoglycosides. ACKNOWLEDGMENT This work was supported by Oklahoma Heart Asociation grant G74-118.
LITERATURE CITED 1. Baker, C. N., and C. Thornaberry. 1974. Antimicrobial susceptibility of Streptococcus mutans isolated from patients with endocarditis. Antimicrob. Agents Chemother. 5:268-271. 2. Bolien, A., J. Savies, M. Ozaki, and S. M ui 1969. Ribosomal protein confering sensitivity to the antibiotic spectinomycin in Escherichia coli. Science
165:85-86. 3. Carrasco, L, and D. Vasquez. 1973. Ribosomal sites involved in binding of aminoacyl-tRNA and EF2. Mode of action of fusidic acid. FEBS Lett 32:152-156. 4. Colon, A. E., R. M. Cole, and C. G. Leonard. 1970. Transduction in group A streptococci by ultraviolet irradiated phages. Can. J. Microbiol. 16:201-202. 5. Demerec, AL, E. A. Adleberg, A. J. Clark, and P. E. Hartman- 1965. A proposal for a uniform nomenclature in bacterial genetics Genetics 54:61-76. 6. Goldthwaite, C., and L Smith. 1972. Genetic mapping of aminoglycoside and fusidic acid resistant mutations in BaciUus subtilis. MoL Gen. Genet. 114:181-189. 7. Hartford, N., and N. Sueoka. 1970. Chromosomal location of antibiotic resistance markers in Bacillus subtilis. J. Mol. Biol. 51:267-286. 8. Hash, J. H. 1972. Antibiotic mechanism. Annu. Rev.
PharmacoL 12:35-56. 9. Haworth, S. R., and L R. Brown. 1973. Genetic analysis of ribonucleic acid polymerase mutants of Bacillus subtilis. J. Bacteriol. 114:103-113. 10. lida, K., and M. Koike. 1974. Cell wall alterations of gram-negative bacteria by aminoglycoside antibiotics.
Antimicrob. Agents Chemother. 5:95-97. 11. Klainer, A. S. 1974. The normal and abnormal surface morphology of staphylococci. Ann. N.Y. Acad. Sci.
236:63-75. 12. Leonard, C. G., A. E. Colon, and R. M. Cole. 1968. Transduction in group A streptococcus. Biochem. Biophys. Res. Commun. 30:130-135. 13. Malke, H. 1969. Transduction of Streptococcus pyogenes K56 by temperature sensitive mutants of transducing phage A25. Z. Naturforsch. 246:1556-1561. 14. Malke, H. 1972. Linkage relationships of mutations endowing Streptococcus pyogenes with resistance to antibiotics that affect the ribosome. Mol. Gen. Genet. 116:299-308. 15. Malke, H. 1975. Transfer of a plasmid mediating antibiotic resistance between strains of Streptococcus pyogenes in mixed cultures. Z. AUg. Mikrobiol. 15:645-649. 16. Malke, H., H. E. Jacob, and K. Storl. 1976. Characterization of the antibiotic resistance plasmid ERLI from Streptococcuspyogenes. Mol. Gen. Genet. 144:333-338. 17. Pestka, S. 1971. Inhibitors of ribosome function. Annu.
VOL. 133, 1978
ANTIBIOTIC RESISTANCE IN S. PYOGENES
Rev. Microbiol. 26:487-562. 18. Sanderson, K. E. 1972. Linkage map of Sabnonella typhimurium, edition IV. Bacteriol. Rev. 36:558-586. 19. Sarubbi, F. A., Jr., E. Blackman, and P. F. Sparling. 1974. Genetic mapping of linked antibiotic resistance loci in Neisseria gonorrhoeae. J. Bacteriol. 120:1284-1292. 20. Smith, I., and H. Smith. 1973. Location of the SP02 attachment site and the bryamycin resistance marker on the Bacillus subtilis chromosome. J. Bacteriol. 114:1138-1142. 21. Sokolova, E. V., M. I. Ovadis, Z. M. Gorlenko, and R. B. Khesdn. 1970. Localization of streptolydigin resistant mutation in E. coli and effect of streptolydigin on T2 phage development in stl-r and stl-s of E. coli. Biochem. Biophys. Res. Commun. 41:8704876. 22. Sparling, F. P., Y. Ikeya, and D. Elliot. 1973. Two genetic loci for resistance to kasugamycin in Escherichia coli. J. Bacteriol. 113:704-710. 23. Stone, K. J., and J. L Strominger. 1971. Mechanism of action of bacitracin: complexation with metal ion and Cj6.isoprenyl pyrophosphate. Proc. Natl. Acad. Sci. U.S.A. 68:3223-3227. 24. Stuart, J. G., and J. J. Ferretti. 1973. Transduction of
859
rifampin resistance in group A streptococci. J. Bacteriol. 115:709-710. 25. Taylor, A. L, and C. D. Trotter. 1972. Linkage map of Escherichia coli strain K- 12. Bacteriol. Rev. 36:504-524. 26. Ubakata, K., M. Konno, and R. Fujii. 1975. Transduction of drug resistance to tetracycline, chloramphenicol, macrolides, lincomycin, and clindamycin with phages induced from Streptococcus pyogenes. J. Antibiot. 28:681-688. 27. Umezawa, H., H. Hamada, Y. Suhara, T. Hashimoto, T. Ekekawa, N. Tanaka, K. Maeda, Y. Okami, and T. Takeuchi. 1966. Kasugamycin, a new antibiotic, p. 753-757. Antimicrob. Agents Chemother. 1965. 28. Wannamaker, L W., S. Almquist, and S. Skjold. 1973. Intergroup phage reactions and transduction between group C and group A streptococci. J. Exp. Med. 137:1338-1353. 29. Weisblum, B., and V. Demohn. 1970. Thiostrepton, an inhibitor of 50S ribosome subunit function. J. Bacteriol. 101:1073-1075. 30. Yoshikawa, M., A. Okuyama, and N. Tanaka. 1975. A third kasugamycin resistance locus, ksgC, affecting ribosomal protein S2 in Escherichia coli K-12. J. Bacteriol. 122:796-797.
