Evolution of erythromycin resistance in Streptococcus pneumoniae in Italy

Monica Monaco, Romina Camilli, Fabio D'Ambrosio, Maria Del Grosso and Annalisa Pantosti*

Department of Infectious, Parasitic and Immunomediated Diseases, Istituto Superiore di Sanità, Viale Regina Elena 299, 00161 Rome, Italy


* Corresponding author. Tel: +39-0649902852; Fax: +39-0649387112; Email: pantosti{at}iss.it

Received 21 September 2004; returned 7 October 2004; revised 23 November 2004; accepted 24 November 2004


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Objectives: To evaluate erythromycin resistance in recent invasive isolates of Streptococcus pneumoniae in Italy, to study the phenotypic and genotypic characteristics of the isolates, and to compare data with those obtained in a previous survey.

Methods: Invasive pneumococcal isolates were obtained from 56 laboratories throughout the country, in 2001–2003. Isolates were serotyped and antimicrobial susceptibilities determined by Sensititre panels and Etest. A new PCR was performed to detect erythromycin resistance genes. Typing methods for selected erythromycin-resistant isolates included PFGE and multilocus sequence typing (MLST).

Results: One hundred and fifty-five isolates out of 444 (34.9%) were resistant to erythromycin: 95 isolates (21.4%) carried erm(B), 56 (12.6%) carried mef(A) and three carried both genes. One isolate, carrying neither erm(B) nor mef(A), showed a point mutation in domain V of the 23S rRNA genes. The mef(A)-positive isolates carried subtype mef(A) (47 isolates), subtype mef(E) (nine isolates), and both subtype mef(E) and erm(B) (three isolates). All subtype mef(A) strains, except two, belonged to serotype 14, appeared to be clonally related by PFGE and related to the England14-9 clone by MLST. The two isolates belonging to other serotypes showed different genetic backgrounds.

Conclusions: Erythromycin resistance in S. pneumoniae has increased in the last few years in Italy. erm(B) is still the predominant resistance determinant; however, the increase in erythromycin resistance (34.9% versus 28.8% of the previous years) is mainly due to an increase in the proportion of isolates carrying the efflux pump mef(A), whereas the proportion of isolates carrying erm(B) has not changed.

Keywords: macrolides , molecular typing , erm(B) , mef(A)


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
In recent years, resistance to macrolides in Streptococcus pneumoniae has increased in many parts of the world, including Italy.1 Besides the well-known resistance determinants—erm(B), conferring the MLSB phenotype and mef(A), conferring the M phenotype—new mechanisms involving mutations of ribosomal proteins or 23S rRNA genes have been described.2 The mef(A) gene comprises two variants, mef(A) and mef(E), which are 90% identical at the nucleotide level, and are carried by different genetic elements, Tn1207.1 and mega, respectively.3,4 We and others have chosen to examine the two variant genes separately, due to differences in the properties of the genetic elements, and therefore we will refer to them as subtype mef(A) and subtype mef(E), respectively.5,6

In a previous study, on a large collection of S. pneumoniae strains from invasive pneumococcal disease (IPD) isolated in Italy in the years 1997–2000, the resistance rate to erythromycin was found to be 28.8%,7 one of the highest in Europe.1 The majority of the erythromycin-resistant isolates showed an MLSB phenotype and carried erm(B), whereas one quarter of the resistant isolates carried mef(A), commonly subtype mef(A). The aim of the present study was to evaluate the evolution of erythromycin resistance in S. pneumoniae in Italy in terms of resistance phenotypes and genetic determinants.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Bacterial isolates

S. pneumoniae isolates from blood or CSF were obtained from 56 Italian hospital laboratories as part of the National Surveillance of Bacterial Meningitis (http://www.simi.iss.it/meningite_batterica.htm) and the AR-ISS Surveillance (http://www.simi.iss.it/antibiotico_resistenza.htm). Identification of the pneumococcal isolates was confirmed by conventional techniques and serotyping was performed using the antisera produced by the Staten Serum Institut (Copenhagen, Denmark). The control strains previously characterized and used for the multiplex PCR were: PN99 [erm(B)],8 PN137 [subtype mef(A)],5 PN150 [subtype mef(E)],5 PGX1416 [both erm(B) and subtype mef(E)], obtained from Glaxo, Verona, Italy, and ATCC 49619 (erythromycin susceptible, negative control).

Antimicrobial susceptibility tests

Determination of MICs to penicillin, erythromycin, clindamycin, tetracycline and chloramphenicol was performed by a microdilution method (Sensititre panel, Biomedical s.r.l., Scorzé, Venice, Italy) and when necessary was confirmed by Etest. The NCCLS breakpoints were applied for the interpretation of the results.9

Detection of erythromycin resistance genes

A multiplex PCR was developed for the simultaneous detection of erm(B) and of subtype mef(A) and subtype mef(E) in one reaction. Five oligonucleotide primers were used: EB1 and EB2 to detect erm(B),10 OM10 (5'- AGCATTGGAACAGCTTTTCA-3'), a forward primer designed in a consensus sequence of the mef(A), and two reverse primers, MEFA (5'-ATTTTGCCGTAGTACAGCC-3') and MEFE (5'-TACATGCTTTTCGAAGCC-3') to detect subtype mef(A) and subtype mef(E), respectively. The assay was based on a previously described duplex PCR10 with the following modifications: concentration of MgCl2 was 4.5 mM, concentration of EB1 and EB2, 1 µM each, concentration of OM10, MEFA and MEFE, 0.5 µM each. The expected sizes of the PCR products were 639, 519 and 318 bp for erm(B), subtype mef(E) and subtype mef(A), respectively. Positive and negative controls were run concurrently with the isolates under study.

To detect mutations in the ribosomal proteins L4 and L22 or in the domain V of each of the four 23S rRNA alleles, amplicons were obtained and sequenced according to published methods.2,11

Molecular typing of the isolates

Relatedness among selected strains carrying mef(A) was studied by PFGE, as described previously,5 comparing visually the macrorestriction profiles obtained following SmaI digestion.

MLST was performed on representative isolates following the recommended procedure.12


    Results and discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
In the years 2001–2003, 444 S. pneumoniae isolates (excluding duplicate isolates from the same patient) were obtained from 56 hospital laboratories around Italy. Of these isolates, 164 (37%) were from CSF and 280 (63%) from blood.

Forty-five (10.1%) strains were non-susceptible to penicillin; of these, 22 (5%) isolates were fully resistant (MIC range 2–4 mg/L) and 23 (5.1%) intermediately resistant to penicillin. This rate is similar to the rate obtained in isolates from the years 1997–2000 (9.9%).7

One-hundred and fifty-five isolates (34.9%) were resistant to erythromycin: 99 isolates (22.3%) showed high-level resistance (MIC ≥ 256 mg/L) to erythromycin and resistance to clindamycin (MLSB phenotype), and 56 strains (12.6%) showed varying levels of erythromycin resistance (MIC range 4–128 mg/L) and susceptibility to clindamycin (M phenotype). Among the MLSB phenotype isolates, penicillin, tetracycline and chloramphenicol resistance rates were 30%, 89.4% and 23.5%, respectively. The M phenotype isolates, with one exception (a single tetracycline resistant isolate), exhibited susceptibility to penicillin, tetracycline and chloramphenicol.

By the multiplex PCR, products of the expected size for each of the five positive controls were obtained, whereas no amplicon was yielded by the negative control. After examining a sample of isolates by both the previous method5 and the multiplex PCR, this assay was applied to all the isolates under study. Among the 99 strains showing the MLSB phenotype, 95 isolates carried erm(B), three isolates erm(B) in association with subtype mef(E), whereas in one isolate neither erm(B) nor mef(A) was detected. All the 56 isolates showing an M phenotype carried mef(A): in particular 47 isolates (84%) carried subtype mef(A) and nine (16%) subtype mef(E).

Table 1 shows the comparison between the rate of erythromycin resistance and the frequency of different resistance genotypes in the isolates examined in this study and in a previous study in the years 1997–2000, as part of similar surveillance systems.7 Interestingly, the increase in erythromycin resistance in 2001–2003 appears to be due to an increase in the percentage of isolates carrying mef(A), whereas the percentage of isolates carrying erm(B) appears unchanged over the years. Within mef(A), the relative percentages of the two subtypes, mef(A) and mef(E), have remained unchanged (not shown). Moreover, an increase in the MIC conferred by the efflux mechanism has been noted, up to 128 mg/L, a level we had not observed before using the same method (Etest).


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Table 1. Erythromycin resistance rates and resistance determinants in pneumococcal invasive isolates in Italy

 
There was also a new finding in that three isolates carried both erm(B) and subtype mef(E), a genotype not found previously in Italian invasive isolates. This is in accordance with Farrell et al.,6 who found that subtype mef(E) but not subtype mef(A), was associated with erm(B) in a large series of double-gene isolates from different geographical areas.

In one erythromycin-resistant isolate showing an erythromycin MIC ≥ 256 mg/L and a clindamycin MIC=4 mg/L, neither erm(B) nor mef(A) were detected. In an experimental microarray designed to detect macrolide resistance genes, this isolate did not appear to contain any of the known erythromycin resistance determinants (M. Cassone, Università degli Studi di Siena, personal communication). No mutations were detected in the relevant regions of the ribosomal proteins L4 and L22; however, the mutation A2059G was present in three 23S rRNA alleles. This is the most common mutation responsible for erythromycin resistance in S. pneumoniae.2,11 Although we were unable to amplify the fourth 23S rRNA allele using the published methods, the high level of erythromycin resistance of the isolate indicates that the mutation should involve all the 23S rRNA alleles, since the level seems to be dependent on the number of mutated alleles.11

The erythromycin-resistant isolates belonged to a variety of serotypes. The 95 erm(B)-positive isolates belonged to 15 different serotypes, the most frequent being 19F (22.1%), 14 (15.7%), 6B (13.7%), 23F (12.6%) and 15B (8.4%), whereas 45 of 47 isolates carrying subtype mef(A) belonged to serotype 14. By PFGE, these isolates appeared to be clonally related, as their profiles differed by three bands or fewer (not shown). However, no epidemiological connection was apparent as the strains were isolated in nine different Italian regions over 3 years. The two isolates carrying subtype mef(A) and belonging to serotypes 6A and 11A, respectively, showed very different PFGE profiles (not shown), indicating different genetic backgrounds.

The nine isolates carrying subtype mef(E) belonged to six different serotypes. The three isolates carrying both erm(B) and subtype mef(E) belonged to serotype 14 and displayed identical or similar profiles by PFGE, related to those of the serotype 14 isolates previously described (not shown).

The MLST allelic profiles obtained for some representative isolates are shown in Table 2. Two isolates carrying subtype mef(A) had identical sequence type (ST9), already observed in a similar isolate from 1999 and corresponding to the England14-9 clone.5 This indicates that in Italy the efflux mechanism is carried predominantly by a well-established penicillin-susceptible serotype 14 clone carrying subtype mef(A). The two isolates belonging to serotypes 6A and 11A show different STs, ruling out the possibility of a capsular switch. The serotype 14 isolate carrying both erm(B) and subtype mef(E) belonged to ST15, a single-locus variant of ST9, already described in an Italian serotype 14 strain from 1998 carrying erm(B).8


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Table 2. Characteristics and allelic profiles (MLST) of selected erythromycin-resistant S. pneumoniae isolates

 
In conclusion, an increase in erythromycin resistance in pneumococcal isolates in Italy is mainly due to an increase in the proportion of isolates carrying the efflux mechanisms. Evolution includes the appearance of new genotypes, namely isolates carrying two erythromycin resistance genes or mutations in 23S rRNA, and M phenotypes with higher levels of resistance. Clonal expansion seems to account for the spreading of serotype 14 isolates carrying subtype mef(A). However, horizontal transfer of resistance determinants appears to play a role in the spreading of both erm(B) and subtype mef(E), since isolates with different serotypes are recruited among the resistant isolates, and genetic determinants of resistance tend to accumulate in the same strains.


    Acknowledgements
 
We are indebted to Stefania Salmaso, Delia Boccia and Paolo Fortunato D'Ancona for scientific organization and data management of the AR-ISS project, and to the colleagues who provided isolates.

This study was supported in part by grants from the Italian Ministero della Salute (Progetto Sorveglianza della Resistanza agli Agenti Antimicrobici 2002 and Progetti finalizzati 2003).


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
1 . EARSS Annual Report 2002. (2003). [Online.] http://www.earss.rivm.nl (20 September 2004, date last accessed).

2 . Tait-Kamradt, A., Davies, T., Cronan, M. et al. (2000). Mutations in 23S rRNA and ribosomal protein L4 account for resistance in pneumococcal strains selected in vitro by macrolide passage. Antimicrobial Agents and Chemotherapy 44, 2118–25.[Abstract/Free Full Text]

3 . Santagati, M., Iannelli, F., Oggioni, M. et al. (2000). Characterization of a genetic element carrying the macrolide efflux gene mef(A) in Streptococcus pneumoniae. Antimicrobial Agents and Chemotherapy 44, 2585–7.[Abstract/Free Full Text]

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5 . Del Grosso, M., Iannelli, F., Messina, C. et al. (2002). Macrolide efflux genes mef(A) and mef(E) are carried by different genetic elements in Streptococcus pneumoniae. Journal of Clinical Microbiology 40, 774–8.[Abstract/Free Full Text]

6 . Farrell, D., Morrissey, I., Bakker, S. et al. (2004). Molecular epidemiology of multiresistant Streptococcus pneumoniae with both erm(B)- and mef(A)-mediated macrolide resistance. Journal of Clinical Microbiology 42, 764–8.[Abstract/Free Full Text]

7 . Pantosti, A., Boccia, D., D'Ambrosio, F. et al. (2003). Inferring the potential success of pneumococcal vaccination in Italy: serotypes and antibiotic resistance of isolates from invasive diseases. Microbial Drug Resistance 9, S61–8.[ISI][Medline]

8 . Dicuonzo, G., Gherardi, G., Gertz, R. E. et al. (2002). Genotypes of invasive pneumococcal isolates recently recovered from Italian patients. Journal of Clinical Microbiology 40, 3660–5.[Abstract/Free Full Text]

9 . National Committee for Clinical Laboratory Standards. (2002). Performance Standards for Antimicrobial Susceptibility Testing; Twelfth Informational Supplement M100-S12. NCCLS, Wayne, PA, USA.

10 . Pantosti, A., D'Ambrosio, F., Bordi, E. et al. (2001). Activity of quinupristin-dalfopristin in invasive isolates of Streptococcus pneumoniae from Italy. Clinical Microbiology and Infection 7, 503–6.[CrossRef][ISI][Medline]

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