2003, 47(7):2236. DOI:
Antimicrob. Agents Chemother.
Pietro E. Varaldo
Maria P. Montanari, Ileana Cochetti, Marina Mingoia and
Erythromycin-Resistant Strains of
of Tetracycline- and
Phenotypic and Molecular Characterization
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ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, July 2003, p. 2236–2241
0066-4804/03/$08.00?0 DOI: 10.1128/AAC.47.7.2236–2241.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.
Vol. 47, No. 7
Phenotypic and Molecular Characterization of Tetracycline- and
Erythromycin-Resistant Strains of Streptococcus pneumoniae
Maria P. Montanari, Ileana Cochetti, Marina Mingoia, and Pietro E. Varaldo*
Department of Microbiology and Biomedical Sciences, University of Ancona
Medical School, 60131 Ancona, Italy
Received 17 December 2002/Returned for modification 17 March 2003/Accepted 15 April 2003
Sixty-five clinical isolates of Streptococcus pneumoniae, all collected in Italy between 1999 and 2002 and re-
sistant to both tetracycline (MIC, >8 ?g/ml) and erythromycin (MIC, >1 ?g/ml), were investigated. Of these
strains, 11% were penicillin resistant and 23% were penicillin intermediate. With the use of the erythromycin-
clindamycin-rokitamycin triple-disk test, 14 strains were assigned to the constitutive (cMLS) phenotype of
macrolide resistance, 44 were assigned to the partially inducible (iMcLS) phenotype, 1 was assigned to the
inducible (iMLS) phenotype, and 6 were assigned to the efflux-mediated (M) phenotype. In PCR assays, 64 of
the 65 strains were positive for the tetracycline resistance gene tet(M), the exception being the one M isolate
susceptible to kanamycin, whereas tet(K), tet(L), and tet(O) were never found. All cMLS, iMcLS, and iMLS
isolates had the erythromycin resistance gene erm(B), and all M phenotype isolates had the mef(A) or mef(E)
gene. No isolate had the erm(A) gene. The int-Tn gene, encoding the integrase of the Tn916-Tn1545 family of
conjugative transposons, was detected in 62 of the 65 test strains. Typing assays showed the strains to be to a
great extent unrelated. Of 16 different serotypes detected, the most numerous were 23F (n ? 13), 19A (n ? 10),
19F (n ? 9), 6B (n ? 8), and 14 (n ? 6). Of 49 different pulsed-field gel electrophoresis types identified, the
majority (n ? 39) were represented by a single isolate, while the most numerous type included five isolates. By
high-resolution restriction analysis of PCR amplicons with four endonucleases, the tet(M) loci from the 64
tet(M)-positive pneumococci were classified into seven distinct restriction types. Overall, a Tn1545-like trans-
poson could reasonably account for tetracycline and erythromycin resistance in the vast majority of the
pneumococci of cMLS, iMcLS, and iMLS phenotypes, whereas a Tn916-like transposon could account for
tetracycline resistance in most M phenotype strains.
In Streptococcus pneumoniae, tetracycline resistance is pre-
dominantly due to ribosomal protection, i.e., the production of
cytoplasmic proteins—-encoded by tet(M) or, less often, other
tet genes—capable of interacting with the ribosome and mak-
ing it insensitive to tetracycline inhibition (3, 38). An efflux-
mediated mechanism reducing the intracellular tetracycline
concentration to subtoxic levels is less common in streptococci,
where this mechanism is due to membrane proteins encoded
by the gene tet(K) or tet(L) (3).
Pneumococcal resistance to macrolides is due to either tar-
get site modification or active efflux (40). The former, preva-
lent mechanism usually depends on a posttranscriptional,
methylase-mediated modification of 23S rRNA encoded by the
erm(B) gene (19, 40). erm(B)-associated coresistance to mac-
rolide, lincosamide, and streptogramin B (MLS) antibiotics
can be expressed either constitutively, with high-level resis-
tance to all MLS antibiotics (cMLS phenotype), or inducibly
(iMLS phenotype). More often, pneumococcal strains appear
inducibly resistant to only 16-membered macrolides and con-
stitutively resistant to lincosamides (iMcLS phenotype) (22).
Another methylase first described (31) and then found to be
extensively present (15, 17) in Streptococcus pyogenes, medi-
ated by a gene originally called erm(TR) and now designated
erm(A) according to current nomenclature (30), has only oc-
casionally been reported in S. pneumoniae (2, 35). Recently,
mutations in 23S rRNA or ribosomal proteins leading to mac-
rolide resistance in clinical isolates of S. pneumoniae have also
been described (10, 37). A macrolide efflux mechanism, first
demonstrated with S. pneumoniae and S. pyogenes and then
with other streptococci, is associated with a resistance pattern
(M phenotype) characterized by low-level resistance to only
14- and 15-membered macrolides among MLS antibiotics (34).
M phenotype resistance is mediated by mef genes, two variants
of which, mef(A) and mef(E)—originally discovered in S. pyo-
genes (4) and S. pneumoniae (36), respectively—have 90%
identity (36) and have been regarded as a single gene class
designated mef(A) (30). However, the two variants have re-
cently been shown to be carried by different genetic elements
in S. pneumoniae (9), and due to a number of important dif-
ferences in the properties of mef(A)- and mef(E)-carrying
pneumococci (9, 22), it has been recommended that the dis-
tinction between the two genes be maintained. A novel eryth-
romycin efflux system, not associated with mef(A) or with other
known macrolide efflux genes, has lately been described in
erm(A)-positive strains of S. pyogenes with inducible, high-level
resistance to erythromycin (14). However, its presence in S.
pneumoniae and other streptococci has not yet been addressed.
In streptococci, drug resistance determinants occur more
frequently on conjugative transposons than on plasmids. In
particular, in S. pneumoniae the association of erythromycin
resistance and tetracycline resistance may be due to Tn1545
and related conjugative transposons, which encode erythromy-
cin resistance via erm(B) and tetracycline resistance via tet(M)
* Corresponding author. Mailing address: Department of Microbi-
ology and Biomedical Sciences, University of Ancona Medical School,
Via Ranieri, Monte d’Ago, 60131 Ancona, Italy. Phone: 39 071
2204694. Fax: 39 071 2204693. E-mail: firstname.lastname@example.org.
on May 29, 2013 by guest
and also kanamycin resistance via aphA3 (8). These transpo-
sons belong to a larger class of conjugative transposons, typi-
cally represented by Tn916, which encode tet(M)-mediated
resistance to tetracycline but not resistance to erythromycin or
kanamycin (6). An integrase gene usually called int-Tn, related
to the second of the 24 open reading frames of Tn916, is
characteristic of the Tn916-Tn1545 family of conjugative trans-
In this study, a collection of clinical S. pneumoniae isolates
resistant to both tetracycline and erythromycin were typed and
investigated for a number of phenotypic and genotypic char-
acteristics inherent to either resistance.
MATERIALS AND METHODS
Bacterial strains. Sixty-five clinical isolates of S. pneumoniae, all resistant to
both tetracycline (MIC, ?8 ?g/ml) and erythromycin (MIC, ?1 ?g/ml), were
tested. All strains, collected from several Italian laboratories between 1999 and
2002, were isolated from upper respiratory tract specimens (the vast majority),
sputum, blood, or cerebrospinal fluid. Strain identification was confirmed in our
laboratory by conventional tests, such as susceptibility to optochin and solubility
in bile, and by the API system (Biome ´rieux, Marcy-l’Etoile, France).
Antibiotics and susceptibility tests. Tetracycline, erythromycin, minocycline,
and penicillin were purchased from Sigma Chemical Co., St. Louis, Mo. Broth
microdilution MICs were determined and MIC breakpoints of tetracycline and
erythromycin were interpreted as recommended by the National Committee for
Clinical Laboratory Standards (24). Mueller-Hinton II broth (BBL Microbiology
Systems, Cockeysville, Md.) supplemented with 3% lysed horse blood was used
as the test medium. The inoculum was 5 ? 105CFU/ml, and S. pneumoniae
ATCC 49619 was used for quality control. Kanamycin susceptibility was deter-
mined by a standard agar diffusion test (25) using 30-?g commercial disks (Oxoid
Ltd., Basingstoke, United Kingdom) with the following zone diameter break-
points: susceptible, ?18 mm; intermediate, 14 to 17 mm; resistant, ?13 mm.
Determination of the macrolide resistance phenotype. Test strains were as-
signed to the constitutive (cMLS), the partially inducible (iMcLS), the inducible
(iMLS), or the efflux-mediated (M) macrolide resistance phenotype on the basis
of the triple-disk (erythromycin plus clindamycin and rokitamycin) test, as de-
scribed previously (23).
Gene detection by PCR. Tetracycline resistance genes tet(K) and tet(L) were
detected by using the primer pairs described by Trzcinski et al. (39), and tet(M)
and tet(O) were detected by using those described by Corso et al. (7) and Olsvik
et al. (26), respectively. Erythromycin resistance genes erm(A) and erm(B) were
detected by using the oligonucleotide primers designated III8and III10by Sep-
pa ¨la ¨ et al. (31) and the primer pair developed by Sutcliffe et al. (33), respectively.
The mef gene was detected by using the primer pair described by Sutcliffe et al.
(33); mef(A) and mef(E) were then distinguished by PCR restriction fragment
length polymorphism analysis of the 348-bp amplicon with BamHI (New En-
gland Biolabs, Beverly, Mass.), which has no restriction site in mef(E) and one in
mef(A), generating two fragments of 284 and 64 bp (22). The integrase gene
int-Tn, associated with the Tn916-Tn1545 family of conjugative transposons, was
detected by using the primer pair described by Poyart-Salmeron et al. (27). DNA
preparation and amplification and electrophoresis of PCR products were carried
out by adapting established methods (16) to the procedures described for the
individual primer pairs.
Serotyping. All isolates were serotyped by the capsular swelling test using
specific antisera (Statens Seruminstitut, Copenhagen, Denmark). Serotypes were
indicated with conventional capsular type designations.
PFGE. SmaI macrorestriction fragment patterns were analyzed by pulsed-field
gel electrophoresis (PFGE); macrorestriction and PFGE were performed, and
the relevant patterns were analyzed and compared as recently described else-
where (29). For clusters with at least two isolates, types were designated with
lowercase letters in order of size.
HRRA. High-resolution restriction analysis (HRRA) of the tet(M) gene was
carried out essentially as described by Doherty et al. (12). Briefly, a 10-?l aliquot
of the PCR product obtained by using the primer pair described by Corso et al.
(7) from each tet(M)-positive isolate was digested with the following restriction
endonucleases: AciI, MseI, RsaI, and TaqI (New England Biolabs). Restriction
fragments were separated by agarose (4%) gel electrophoresis and visualized by
staining with ethidium bromide. The molecular size marker (100-bp DNA lad-
der) was from M-Medical Genenco, Florence, Italy. Each restriction pattern
yielded by each endonuclease was assigned a number; restriction genotypes were
determined on the basis of the combined restriction patterns of all four enzymes
and lettered with capitals in order of size.
Antibiotic susceptibility. The distribution of MICs of tetra-
cycline, erythromycin, and penicillin for the 65 tetracycline-
and erythromycin-resistant isolates of S. pneumoniae is sum-
marized in Table 1. Forty-three strains were susceptible to
penicillin (MIC, ?0.12 ?g/ml), 15 were intermediate, and sev-
en were resistant (the MIC for five of these strains was 2 ?g/ml
and that for the other two was 4 ?g/ml). All the strains but one
were resistant to kanamycin according to the results of the disk
Macrolide resistance phenotypes. On the basis of the eryth-
romycin-clindamycin-rokitamycin triple-disk test, 14 of the 65
test strains were assigned to the cMLS phenotype, 44 were
assigned to the iMcLS phenotype, 1 was assigned to the iMLS
phenotype, and 6 were assigned to the M phenotype. These
findings, together with the ranges of MICs of erythromycin, are
summarized in Table 2.
Genotypic strain characterization. As shown in Table 3, the
tetracycline resistance genes tet(K), tet(L), and tet(O) were not
found. In contrast, 64 of the 65 test strains were positive for
tet(M), the exception being one M phenotype isolate (MIC of
penicillin, 0.25 ?g/ml; MIC of tetracycline, 16 ?g/ml; MIC of
minocycline, 0.25 ?g/ml; MIC of erythromycin, 16 ?g/ml), the
only isolate susceptible to kanamycin, which was negative for
all four tetracycline resistance genes tested. All cMLS and
iMcLS isolates as well as the iMLS isolate had the erm(B)
gene, and all six M phenotype isolates had the mef gene [four
with mef(E) and two with mef(A)]. The mef gene was also
found in six isolates with the erm(B) gene: five [four with
mef(E) and one with mef(A)] of the iMcLS phenotype and one
[with mef(A)] of the cMLS phenotype. No isolate had the
erm(A) gene. Of the 59 strains carrying both tet(M) and
erm(B), i.e., all the strains of the cMLS, iMcLS, and iMLS
phenotypes, 57 were also positive for the int-Tn gene (all ex-
TABLE 1. Distribution of MICs of tetracycline, erythromycin, and
penicillin for 65 tetracycline- and erythromycin-resistant
isolates of S. pneumoniae
No. of strains inhibited by MIC (?g/ml) ofa:
?0.12 0.12 0.250.5248 16 3264 128
5 13 15 13
1 3 1
aNo strain was inhibited by a MIC of 1 ?g/ml.
TABLE 2. Macrolide resistance phenotypes of 65 tetracycline- and
erythromycin-resistant isolates of S. pneumoniae
No. (%) of
Range of MICs (?g/ml)
VOL. 47, 2003 TETRACYCLINE- AND ERYTHROMYCIN-RESISTANT S. PNEUMONIAE2237
on May 29, 2013 by guest
cept a cMLS isolate and an iMcLS isolate). Of the six M
phenotype strains, which carried mef(A) or mef(E) as the only
erythromycin resistance determinant, the five with the tet(M)
gene were also int-Tn positive, whereas the isolate lacking
tet(M) as well as the other tetracycline resistance genes tested
was also int-Tn negative.
Typing. Sixteen different serotypes, of which 11 were repre-
sented by at least two isolates, were detected. The most nu-
merous was 23F (n ? 13), followed by 19A (n ? 10), 19F (n ?
9), 6B (n ? 8), and 14 (n ? 6).
Forty-nine PFGE types, of which 10 were represented by at
least two isolates, were detected. Type a, the most numerous,
included five isolates.
The distribution of serotypes and PFGE types among the 65
erythromycin- and tetracycline-resistant S. pneumoniae isolates
and their associations with the characteristics described above
(macrolide resistance phenotypes, tetracycline and erythromy-
cin resistance genes, and the int-Tn gene) are summarized in
Restriction types of the tet(M) gene. With HRRA of allelic
variation within tet(M), endonucleases MseI and TaqI each
yielded consistently the same fingerprinting profiles from the
PCR amplicons of all strains, whereas AciI and RsaI produced
five and two distinct profiles, respectively (Fig. 1). Altogether,
the tet(M) loci from the 64 tet(M)-positive pneumococci ana-
lyzed fell into seven restriction types (A to G). Their distribu-
tion is reported in Table 3. Over half (31 of 59) of the strains
with the erm(B) gene (cMLS, iMcLS, and iMLS phenotypes)
had restriction type A, whereas four of the five strains with the
mef gene (M phenotype) exhibited restriction type B. Restric-
tion types A and B are shown in Fig. 2.
In clinical isolates of S. pneumoniae, tetracycline resistance
is frequently associated with erythromycin resistance. In one
large U.S. survey carried out from 1999 to 2000, ?60% of
multiresistant pneumococci exhibiting erythromycin resistance
were also resistant to tetracycline (11); in Europe, associations
of ?80% in erythromycin-resistant pneumococci isolated in
Spain (32) and Italy (21) have recently been reported. This
association may reflect the widespread presence in pneumo-
coccal populations of transposons, typified by Tn1545, thought
to result from the insertion over time of resistance deter-
minants, such as erm(B) for erythromycin and aphA3 for
kanamycin, into primitive gram-positive conjugative trans-
posons carrying tet(M) and the integrase gene int-Tn, typified
by Tn916 (3, 6).
Among the 65 tetracycline- and erythromycin-resistant clin-
ical strains of S. pneumoniae investigated in this study, the
presence of Tn1545-like transposons could reasonably account
for tetracycline and erythromycin resistance in the vast major-
ity (at least 57 of 59) of the strains of the cMLS, iMcLS, and
iMLS phenotypes, i.e., those sharing tet(M), erm(B), kanamy-
cin resistance, and the int-Tn gene. The remaining two isolates
shared tet(M), erm(B), and kanamycin resistance but were
int-Tn negative. However, tet(M) was also associated with
int-Tn in five of the six strains of the M phenotype, i.e., strains
lacking erm genes and expressing erythromycin resistance due
to mef-mediated active efflux. Unlike erm(B) in pneumococci
with constitutive or inducible MLS resistance, the mef gene in
M phenotype pneumococci is not known to be linked to tetra-
cycline resistance, which in these strains could be due to a
Tn916-like transposon. Similar findings—i.e., M phenotype
TABLE 3. Macrolide resistance phenotypes and their association with tetracycline and erythromycin resistance genes, the int-Tn gene,
serotypes, PFGE types, and restriction types of the tet(M) gene in 65 tetracycline- and erythromycin-resistant isolates of S. pneumoniae
(no. of strains)b
(no. of strains)b,c
Restriction type of tet(M)
loci (no. of strains)b,d
12 23F (3), 19F (3), 14 (2), 3,
6B, 9A, 15A
b (2), d (2), c, Ost (7) A (4), E (3), G (3), B (2)
38 19A (8), 23F (7), 19F (5),
6B (3), 3 (2), 6A (2),
10A (2), 10F (2), 14 (2),
15A, 33F, 36
6B, 7F, 19F, 23F
a (4), c (2), e (2), f (2),
i (2), b, h, j, Ost (23)
A (22), C (5), D (4), B
(3), F (3), E
a, b, g, h
A, B, C, D
1 23F OstA
6B, 11A, 19A, 23F
j, Ost (3)
B (3), D
atet(K), tet(L), tet(O), and erm(A) were not detected in any test strain. ?, present; ?, absent.
bThe number of strains is indicated for types represented by more than one strain.
cOst, one-strain type.
dRestriction profiles were as follows (different profiles yielded by the same endonucleases are distinguished by subscript numerals): A, RsaI1, MseI, TaqI, AciI1; B,
RsaI2, MseI, TaqI, AciI2; C, RsaI2, MseI, TaqI, AciI3; D, RsaI1, MseI, TaqI, AciI3; E, RsaI1, MseI, TaqI, AciI4; F, RsaI2, MseI, TaqI, AciI1; G, RsaI1, MseI, TaqI, AciI5.
eThis strain was the only kanamycin-susceptible isolate and was also found to be susceptible to minocycline.
2238 MONTANARI ET AL.ANTIMICROB. AGENTS CHEMOTHER.
on May 29, 2013 by guest
pneumococci carrying mef(A) or mef(E), tet(M), and int-Tn—
have recently been reported in Spain (32) and Scotland (1).
Interestingly, the same tet(M) allele (type II) was identified by
HRRA in four of our five M phenotype strains carrying mef(A)
or mef(E), tet(M), and int-Tn. In contrast, the prevalent tet(M)
allele (type I) of the seven detected in the 64 tet(M)-positive
pneumococci was identified in isolates of all phenotypes
(cMLS, iMcLS, and iMLS) with erm(B)-mediated erythromy-
cin resistance but in no strain with mef(A) or mef(E)-mediated
resistance (M phenotype). The sixth M phenotype pneumo-
coccus was the only test strain susceptible to kanamycin and
the only one lacking tet(M). Since this strain was also negative
for tet(O), tet(K), and tet(L), it is possible that some less com-
mon tet gene capable of conferring tetracycline resistance on
streptococci (3, 5) was involved; however, its susceptibility to
minocycline suggests an efflux mechanism (3).
It is worth noting that in none of our tetracycline- and
erythromycin-resistant S. pneumoniae isolates did we detect
the tet(O) gene, whose presence in this species has occasionally
been reported in limited numbers of South African (41), North
FIG. 1. Different fingerprinting profiles obtained by digesting the tet(M) amplicons from 64 tet(M)-positive pneumococci with four endonucle-
ases. Lane M, molecular size marker (100-bp ladder). Lane A, undigested tet(M) amplicon. Lanes 1a to 1e, different AciI profiles (AciI1to AciI5).
Lanes 2a and 2b, different RsaI profiles (RsaI1and RsaI2). Lane 3, MseI profile. Lane 4, TaqI profile.
FIG. 2. HRRA patterns of two tet(M)-positive pneumococci with restriction types A and B. Lane M, molecular size marker (100-bp ladder).
Lane A, undigested tet(M) amplicon of the strain exhibiting restriction type A; lanes A1 to A4, restriction profiles yielded by endonucleases AciI,
RsaI, MseI, and TaqI, respectively. Lane B, undigested tet(M) amplicon of the strain exhibiting restriction type B; lanes B1 to B4, restriction profiles
yielded by endonucleases AciI, RsaI, MseI, and TaqI, respectively.
VOL. 47, 2003 TETRACYCLINE- AND ERYTHROMYCIN-RESISTANT S. PNEUMONIAE2239
on May 29, 2013 by guest
American (20), and German (28) isolates. Conversely, it has
very recently been shown that tet(O) largely accounts for tet-
racycline resistance in tetracycline- and erythromycin-resistant
isolates of S. pyogenes, in which it is typically associated with
erm(A) or mef(A) (E. Giovanetti, A. Brenciani, R. Lupidi,
M. C. Roberts, and P. E. Varaldo, Program Abstr. 12th Eur.
Congr. Clin. Microbiol. Infect. Dis., abstr. P472, p. 80, 2002).
There are, however, few reports of erm(A) in S. pneumoniae
isolates (2, 35); in particular, we have never found this gene
in Italian pneumococci. As regards mef(A), while only true
mef(A) is found in S. pyogenes isolates (E. Giovanetti and P. E.
Varaldo, unpublished results), both mef(A) and the mef(E)
variant can be found in M phenotype erythromycin-resistant
pneumococci (9, 22). However, in the case of associated tetra-
cycline resistance, whereas in S. pyogenes isolates the mef gene
is associated with tet(O) (Giovanetti et al., 12th, ECCMID),
this study shows that in pneumococci it is associated with
The 65 tetracycline- and erythromycin-resistant S. pneumo-
niae strains tested were distributed over 16 serotypes and 49
PFGE types. These findings, in addition to the other pheno-
typic and genotypic differences, indicate that the test strains were
substantially unrelated. This suggests a spread of resistance
due to horizontal transfer of transposons or resistance deter-
minants rather than to clonal dissemination. The most numer-
ous serotypes (23F, 19A, 19F, 6B, 14, etc.) are essentially those
that have more frequently been seen to combine macrolide and
erythromycin resistance in other studies in Italy (18, 21) and in
other European countries (1, 28). The rates of both penicillin-
resistant (11%) and penicillin-intermediate (23%) isolates in
our collection of tetracycline- and erythromycin-resistant pneu-
mococci were higher than those recently reported among clin-
ical S. pneumoniae isolates in Italy (13, 21).
This work was supported in part by a grant from the Italian Ministry
of Education, University and Research.
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