Macrolide resistance mechanisms and expression of phenotypes among Streptococcus pneumoniae circulating in Italy
A. Marchese,
E. Tonoli,
E. A. Debbia and
G. C. Schito*
Institute of Microbiology, University of Genoa, Largo R. Benzi 10, 16132 Genoa, Italy
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Abstract
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In Italy, macrolide-resistant pneumococci have been isolated at a rate
increasing
from 6% in
1993 to 31.7% in 1998. A collection of 161 erythromycin-resistant Streptococcus
pneumoniae recovered between 1993 and 1997 has now been phenotypically and
genotypically characterized. Approximately 90% of these microorganisms possessed a
constitutive MLSB mechanism of resistance. PCR detected ermB and mefE genes in strains showing MLSB and M phenotypes, respectively. Using
pulsed-field gel electrophoresis of chromosomal DNA, one dominant restriction profile and its
variations were detected in 51 S. pneumoniae isolates collected from different locations,
indicating the circulation of a clone characterized by the possession of a great ability to spread.
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Introduction
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Erythromycin resistance in Streptococcus pneumoniae has increased over recent
years in several geographical areas, Italy included.1,2 Currently, 31.7% of all pneumococci circulating in Italy
are resistant to macrolides,2 compared with 6% in 1993.3
In Canada4 and the USA,5 the M phenotype specified by the mefE gene6 represents the prevailing mechanism, while in Spain7 an MLSB phenotype is observed almost exclusively. Since M phenotype
strains display low-level macrolide resistance, their local preponderance might have an impact on
antibiotic choice for non-meningeal infections. Their incidence has therefore been evaluated
using a large collection of S. pneumoniae isolated in Italy by phenotypic determination
and PCR analysis. In addition, to ascertain whether spreading of resistance is due to clonal
expansion, the nature of all strains has been analysed by pulsed-field gel electrophoresis (PFGE).
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Materials and methods
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Erythromycin-resistant S. pneumoniae (161) selected from among 1046 respiratory
strains collected between 1993 and 1997, and originating from widely dispersed laboratories in
Italy (Ancona, Bologna, Genoa, Florence, Milan, Naples, Parma, Turin and Vercelli), were
studied. Susceptibility to antimicrobial drugs was assessed by microdilution assay as detailed in
NCCLS guidelines (1997).8,9 Erythromycin was supplied by Abbott S.p.a. (Campoverde, Italy), clavulanic acid by
SmithKline Beecham Pharmaceuticals (Milan, Italy), imipenem by the hospital pharmacy, and
clindamycin, penicillin G, amoxycillin, cefotaxime, ceftriaxone, co-trimoxazole, tetracycline,
chloramphenicol, rifampicin and vancomycin were purchased commercially (Sigma-Aldrich,
Milan, Italy).
Resistance phenotypes were classified using a double-disc test with erythromycin and
clindamycin discs. After 1842 h of incubation at 35°C, a blunting of the
clindamycin zone of inhibition proximal to the erythromycin disc was taken to indicate inducible
resistance (I), while resistance to clindamycin with no blunting indicated constitutive resistance
(C). The M phenotype was characterized by susceptibility to clindamycin with no blunting of the
zone of inhibition around the clindamycin disc.
ermB and mefE genes were amplified by PCR as described previously,10 and PFGE of chromosomal DNA and pattern analysis were
performed as reported elsewhere.10
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Results and discussion
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Table I shows the susceptibility patterns of erythromycin-resistant S. pneumoniae.
Rifampicin and vancomycin emerged as the most potent drugs tested (100% susceptible strains)
followed by third-generation cephalosporins (>90%), amoxycillin, co-amoxiclav and
imipenem (84%), penicillin (79%), chloramphenicol (60%), co-trimoxazole (40%), tetracycline
(13%) and clindamycin (10%).
Table II summarizes the antibiotype, phenotype and genotype
distribution in this collection
of strains. The majority (65%) of microorganisms (105/161) carried resistance to two or more
additional drugs with the pattern: erythromycin,
-co-trimoxazoletetracyclinechloramphenicol being the most represented (46
strains out of 161; 28.6%). In total, 87% of the 161 S. pneumoniae strains tested were
resistant to tetracycline, 60% to co-trimoxazole, 40% to chloramphenicol and 21% to penicillin
(12.4% and 8.6% low- and high-level resistance, respectively). As expected, penicillin resistance
in erythromycin-resistant S. pneumoniae (21%) was higher than that observed in the
general S. pneumoniae population circulating in Italy (12.7%).2 The high incidence of associated erythromycin and tetracycline
resistance is not surprising since ermB and TetM determinants appear to be carried by
transposon Tn1545. Erythromycin resistance alone was rare (14 strains, 8.6%), the
majority (11/14) of these showing the M phenotype (Table II).
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Table II. Distribution of antibiotic resistance patterns, macrolide phenotypes and genotypes among
the 161 S. pneumoniae studied
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Most S. pneumoniae (145 out of 161; 90%) expressed a constitutive phenotype in
keeping with MIC results, while 15 displayed the M phenotype. Only one isolate possessed an
inducible mechanism of resistance (Table II). All strains belonging to the
MLSB
phenotype were PCR-positive for the ermB gene and 15 strains categorized as M carried
the mefE gene (Table II). M phenotype S. pneumoniae
showed erythromycin
MIC90 not exceeding 8 mg/L, as expected,6
while erythromycin and clindamycin MIC90 values for those with a constitutive
mechanism were 128 mg/L (Table III).
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Table III. In-vitro activities of erythromycin and clindamycin against macrolide constitutive (C),
inducible (I) and M-type resistant (M) S. pneumoniae
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Overall, 38 different clones could be distinguished by PFGE, indicating a substantial
heterogeneity among the erythromycin-resistant isolates. The most prevalent are illustrated in the
Figure. The most represented PFGE profile, designated L, was found in
17 strains, and its
variations in 34 other microorganisms. S. pneumoniae belonging to this clone circulate
in all Italian centres. The majority (32/51) of strains characterized by profile L or by its variations
were also resistant to co-trimoxazole, tetracycline and chloramphenicol. The second most
represented profile (11 strains) corresponded to the Italian autochthonous clone described
previously.10 These microorganisms were isolated in
northern and central Italy exclusively, and were resistant to erythromycin, penicillin,
co-trimoxazole and tetracycline. The third profile, found in four isolates and in eight other
variants, is identical to that displayed by the Spanish/USA clone whose presence in Italy has been
reported before.10 Among the 15 M phenotypes of S.
pneumoniae, six different profiles emerged. Only one strain was characterized by restriction
pattern L, dominating among constitutive macrolide-resistant microorganisms.

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Figure. Most prevalent PFGE patterns abserved
among erythromycin resistant pneumococcal isolates. Lanes 1 and 13: molecular weight marker
ladder; lanes 212: profiles A, A', B, G, L, L', L'', LV, LVI, O, P.
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In 1993, percentages of penicillin and erythromycin resistance were very similar (5.5 and 6%,
respectively) while erythromycin resistance (31.7%) currently exceeds the rate of penicillin
resistance (12.7%):2 it therefore seems clear that, in Italy,
the evolution of erythromycin resistance is driven by independent forces, possibly including the
spread of clone L (penicillin-susceptible). The molecular mechanisms underlying macrolide
resistance in S. pneumoniae appear to be similar in Spain7 and Italy, where ermB genes confer the MLSB phenotype
(mostly in the constitutive variant) to the vast majority of strains. The situation is reversed in
Canada4 and the USA,5 where the mefE gene predominates. Our observation that the ermB gene is carried by S. pneumoniae clones that are only rarely colonized by mefE genes may implicate a different host range of the two genetic determinants and/or a
geographic segregation of S. pneumoniae subtypes. Widely divergent prescription
habits, rates of overall resistance, amounts of antibiotic consumption and gene pool sizes may
also be responsible for the differences observed.
Resistance to antibiotics is a complex phenomenon and actual levels of expression markedly
influence the clinical significance of this trait. There is now unanimous agreement on the
contention that ß-lactam resistance in S. pneumoniae does not necessarily equate
with treatment failures in non-meningeal infections.1
Moreno et al.11 observed surprising cure rates
among a small number of patients with pneumonia caused by erythromycin-resistant S.
pneumoniae treated with erythromycin. In addition, response to the drug was found to be
independent of both MIC values for the isolated strains and presence of bacteraemia. These
findings, while limited and requiring confirmation, are extremely interesting and point to the
possibility that the clinical response described for ß-lactam-resistant S. pneumoniae
may also hold true for other classes of drug.
Given these possible therapeutic implications and in analogy with what is currently done
with ß-lactams, assessment of macrolide resistance phenotypes should routinely be
performed in clinical microbiology laboratories.
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Acknowledgments
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This study was supported in part by Abbott S.p.a., Campoverde, Italy.
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Notes
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* Corresponding author. Tel:
+39-10-353-7655; Fax: +39-10-504-837; E-mail: schitogc{at}aleph.it 
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References
|
---|
1
.
Kaplan, S. L. & Mason, E. O. (1998).
Management of infections due to antibiotic-resistant Streptococcus pneumoniae. Clinical Microbiology Reviews 11, 62844.[Abstract/Free Full Text]
2
.
Schito, G. C., Mannelli, S., Cibrario-Sent, M., Pesce, A.
& Marchese, A. (1999). Evoluzione delle resistenze ai farmaci antimicrobici
in Streptococcus pneumoniae circolante in Italia. Analisi dei dati
dell'Osservatiorio
Epidemiologico Italiano. Giornale Italiano di Microbiologia Medica Odontoiatrica e
Clinica 3, 4357.
3
.
Marchese, A., Debbia, E. A., Arvigo, A., Pesce, A.
&
Schito, G. C. (1995). Susceptibility of Streptococcus pneumoniae strains
isolated in Italy to penicillin and ten other antibiotics. Journal of Antimicrobial
Chemotherapy 36, 8337.[Abstract]
4
.
Johnston, N. J., De Azavedo, J., Kellner, J. D. &
Low, D.
E. (1998). Prevalence and characterization of the mechanisms of macrolide,
lincosamide and streptogramin resistance in Streptococcus pneumoniae. Antimicrobial
Agents and Chemotherapy 42, 24256.[Abstract/Free Full Text]
5
.
McDougal, L. K. & Tenover, F. C. (1997).
Characterisation of macrolide resistance phenotypes in Streptococcus pneumoniae. In Program and Abstracts of the Thirty-Seventh Interscience Conference on Antimicrobial
Agents and Chemotherapy, Toronto, Canada, 1997. Abstract C-77b, p. 59, American
Society for Microbiology, Washington, DC.
6
.
Sutcliffe, J., Grebe, T., Tait-Kamradt, A. &
Wondrack, L.
(1996). Detection of erythromycin-resistant determinants by PCR. Antimicrobial Agents and Chemotherapy 40, 2562
6.[Abstract]
7
.
Lantero, M., Portillo, A., Gastanares, M. J., Ruiz-Larrea,
F., Zarazaga, M., Olarte, I. et al. (1998). MLS resistance phenotypes and
mechanisms in S. pneumoniae. In Program and Abstracts of the Fourth International
Conference on the Macrolides, Azalides, Streptogramins and Ketolides. Barcelona,
Spain, 1998. Abstract 3.10, p. 34.
8
.
National Committee for Clinical Laboratory Standards.
(1997). Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria
that Grow AerobicallyFourth Edition: Approved Standard M7-A4. NCCLS,
Wayne, PA.
9
.
National Committee for Clinical Laboratory Standards.
(1998). Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria
that Grow AerobicallyFourth Edition: Approved Standard M100-S8. NCCLS,
Wayne, PA.
10
.
Marchese, A., Ramirez, M., Schito, G. C. &
Tomasz, A.
(1998). Molecular epidemiology of penicillin-resistant Streptococcus
pneumoniae isolates recovered in Italy from 1993 to 1996. Journal of Clinical
Microbiology 36, 29449.[Abstract/Free Full Text]
11
.
Moreno, S., Garcià-Leoni, M. E., Cercenado,
E., Diaz, M. D., Bernaldo de Quiros, J. C. L. & Bouza, E. (1995). Infections
caused by erythromycin-resistant Streptococcus pneumoniae: incidence, risk factors and
response to therapy in a prospective study. Clinical Infectious Diseases 20, 1195200.[ISI][Medline]
Received 11 December 1998;
returned 11 March 1998; revised 19 April 1999;
accepted 12 May 1999