Abstract

One hundred and twenty-four erythromycin-resistant pneumococcal isolates were examined for the presence of macrolide resistance genes. The erm(B) gene was detected in 118 (95.2%) isolates and the mef(A) gene in 83 (66.9%) isolates. Both the mef(A) and erm(B) genes were detected in 77 (62.1%) isolates. DNA macrorestriction analysis of these isolates identified them as belonging to a single multi-resistant clone.

Received 15 January 2002; returned 7 May 2002; revised 18 June 2002; accepted 8 July 2002

Introduction

The two most common mechanisms of macrolide resistance reported in Streptococcus pneumoniae are target modification and drug efflux, mediated by the erm and mef genes, respectively. The erm(B) gene product methylates a specific adenine residue within the 23S rRNA, which conveys cross-resistance to macrolides, lincosamides and streptogramin B compounds (MLSB phenotype). The mef(A) gene encodes an efflux pump, which is specific only for 14- and 15-membered macrolides (M phenotype).1

The prevalence of either mechanism tends to vary with geographical location. In the USA, the M phenotype is more prevalent,2 while in Europe the MLSB phenotype dominates.3 Previous studies have determined the prevalence of pneumococcal resistance to erythromycin to be 11% in New Zealand,4 but studies on the mechanisms of resistance have not yet been performed. The current study describes the prevalence of two macrolide resistance determinants [erm(A) and mef(B)] in New Zealand, as assessed by PCR analysis. Furthermore, DNA macrorestriction profiling was performed on selected isolates to determine whether clonal expansion was responsible for increased resistance.

Materials and methods

S. pneumoniae isolates were recovered between August 1997 and May 1999 from a community medical laboratory in Christchurch, New Zealand. The collecting laboratory serves primarily Christchurch and the Canterbury province (population 481 431) of the South Island of New Zealand. During the study period all pneumococcal isolates were screened for both penicillin and erythromycin resistance. One hundred and twenty-four erythromycin-resistant isolates were recovered, each representing a unique patient. Isolates were recovered predominantly from eye (27.5%), ear (27.5%), sputum (23%) and nasal (11%) swabs. The remaining 11% were isolated from assorted skin and respiratory sites. Disc diffusion testing was used initially to assess susceptibility to oxacillin, erythromycin, tetracycline, co-trimoxazole and chloramphenicol in accordance with NCCLS guidelines.5 MICs were subsequently determined by agar dilution testing in accordance with NCCLS guidelines. Erythromycin was purchased commercially (Sigma, St Louis, MO, USA), and antibiotic discs were from Oxoid (Basingstoke, UK).

erm(B) and mef(A) genes were amplified by PCR using primers and conditions described previously.6,7 PFGE was performed essentially as described elsewhere,8 and interpreted in accordance with published criteria.9 The ∼330 bp PCR products of each of the erm(B) and mef(A) genes were sequenced to confirm their identities, and subsequently used as probes for Southern hybridization analysis. Southern hybridizations were performed using the DIG Easyhyb kit (Roche) in accordance with the manufacturer’s instructions.

Results and discussion

Of the 124 erythromycin-resistant isolates, 117 (94.3%) had high-level erythromycin resistance (MIC ≥ 128 mg/L). The remaining seven (5.7%) isolates had MICs ranging from 4 to 8 mg/L. All isolates showed decreased susceptibility to penicillin; 22 (17.7%) isolates had intermediate resistance (MIC 0.1–1 mg/L); and 102 (82.3%) were highly resistant (MIC ≥ 2 mg/L). The range of penicillin MICs was 0.12– 8 mg/L. One hundred and seventeen (94.4%) isolates were resistant to co-trimoxazole, 112 (90.3%) were resistant to tetracycline and 24 (19.4%) were resistant to chloramphenicol.

PCR detection of the macrolide resistance determinants (Table 1) detected mef(A) in 83 (66.9%) isolates and erm(B) in 118 (95.2%) isolates. Both mef(A) and erm(B) were detected in 77 (62.1%) isolates. All 77 isolates containing both genes were multi-resistant; the most frequently associated combination of resistances was penicillin, erythromycin, co-trimoxazole and tetracycline, which was noted in 74 (96%) of these isolates. In all but one instance, when both genes were detected in the same isolate, the erythromycin MIC was ≥128 mg/L. This high-level resistance was presumably imparted by a functional erm(B) gene. The single isolate with low-level erythromycin resistance in which both resistance genes were detected (isolate 413) was also susceptible to clindamycin. This isolate may contain a deleted or otherwise defective erm(B) gene, and the low level of resistance resulted from the product of a functional mef(A) gene, imparting the M phenotype.

Typically, erythromycin-resistant pneumococci from any given geographical location possess only one of the two most commonly described resistance mechanisms. In the USA, the mef(A) gene is more dominant, being identified in 61% of 114 macrolide-resistant isolates examined.2 In contrast, in Europe erm(B) has been found in >80% of erythromycin-resistant isolates.3 In Christchurch, the predominant macrolide resistance genotype is both erm(B) and mef(A). This genotype was identified in 62.1% of the Christchurch isolates examined in this study.

Although an uncommon genotype, a recent report from South Africa found 36 of 118 (30.5%) erythromycin-resistant isolates tested contained both erm(B) and mef(A) genes, using PCR.10 DNA fingerprinting of these 36 serotype 19F isolates revealed that 30 isolates belonged to a single clone. To determine whether the Christchurch isolates were clonal, selected isolates were examined by macrorestriction analysis of chromosomal DNA. Of the 77 isolates from which both the erm(B) and mef(A) genes had been amplified, the first 40 consecutive isolates were analysed by PFGE; 38 belonged to a clonal group (Figure 1), and two (isolates 221 and 4) were unique. Of the 38 isolates assigned to a clonal group, 37 profiles were identical, whereas one was a subtle variant of the major clone (isolate 28). Serotype data were available for only 16 of the 40 isolates; 15 isolates belonging to the major clonal group were found to be serogroup 19 (four typed as 19F), and one isolate not belonging to the clonal group (isolate 4) was found to be 23F.

The presence of both erm(A) and mef(A) in the major clonal group was further analysed. To confirm the identity of the amplified products, the nucleotide sequences of the PCR amplicons from a single isolate were determined. In each case, the erm(B) and mef(A) genes had 98% and 100% sequence identity, respectively, to the synonymous genes in the database (data not shown). To verify the presence of these genes in the genome, Southern hybridization experiments were performed on SmaI-digested chromosomal DNA separated by PFGE. In each case, probes for both genes hybridized with the same 120 kb DNA fragment (data not shown). The presence of both macrolide resistance genes localized to the same ∼5% of the pneumococcal genome raises the possibility that the two genes may be linked. We are currently investigating the genetic elements on which these determinants are borne to establish whether such a link exists.

The results from this study show that the most predominant erythromycin resistance genotype in Christchurch is erm(B) mef(A). This combination of genes imparts a phenotype essentially identical to that imparted by erm(B) alone. The majority of the erm(B) mef(A) isolates belonged to a multi-resistant serotype 19F clone, which has much in common with a multi-resistant 19F clone recently described in South Africa.10 This report suggests the possible global spread of this clone.

Acknowledgements

We are grateful to Rosemary Ikram of Medlab South, Christchurch, for kindly providing pneumococcal isolates. This work was supported in part by a grant from SmithKline Beecham. D.C.B. was the recipient of an ESR scholarship, which is gratefully acknowledged.

*

Corresponding author. Tel: +064-03-364-2987 (ext. 7169); Fax: +064-364-2083; E-mail: d.bean@botn.canterbury.ac.nz

§

Present address. School of Molecular Biosciences, Washington State University, Pullman, WA, USA.

Figure 1.SmaI-digested genomic DNA separated by PFGE. Profiles of multi-resistant New Zealand isolates harbouring both erm(B) and mef(A) genes. Lanes 1 and 10, λ DNA ladder; lanes 2–6, isolates 2, 3, 13, 16 and 19, respectively; lane 7, isolate 28; lane 8, isolate 221; and lane 9, isolate 4.

Figure 1.SmaI-digested genomic DNA separated by PFGE. Profiles of multi-resistant New Zealand isolates harbouring both erm(B) and mef(A) genes. Lanes 1 and 10, λ DNA ladder; lanes 2–6, isolates 2, 3, 13, 16 and 19, respectively; lane 7, isolate 28; lane 8, isolate 221; and lane 9, isolate 4.

Table 1.

 Erythromycin resistance determinants detected in 124 isolates of S. pneumoniae from a clinical laboratory in Christchurch, New Zealand, and their corresponding erythromycin MICs

 Number (%) of isolates falling into MIC range (mg/L) 
Resistance determinant detected by PCR 4–8  ≥128  
erm(B) determinant only 41 (33.1) 
mef(A) determinant only 6 (4.8)  0 
Both erm(B) and mef(A) determinants 1 (0.8) 76 (61.3) 
 Number (%) of isolates falling into MIC range (mg/L) 
Resistance determinant detected by PCR 4–8  ≥128  
erm(B) determinant only 41 (33.1) 
mef(A) determinant only 6 (4.8)  0 
Both erm(B) and mef(A) determinants 1 (0.8) 76 (61.3) 

References

1.
Tait-Kamradt, A., Clancy, J., Cronan, M., Dib-Hajj, F., Wondrack, L., Yuan, W. et al. (
1997
). mefE is necessary for the erythromycin-resistant M phenotype in Streptococcus pneumoniae.
Antimicrobial Agents and Chemotherapy
 
41
,
2251
–5.
2.
Shortridge, V. D., Doern, G. B., Brueggemann, A. B., Beyer, J. M. & Flamm, R. K. (
1999
). Prevalence of macrolide resistance mechanisms in Streptococcus pneumoniae isolates from a multicenter antibiotic resistance surveillance study conducted in the United States in 1994–1995.
Clinical Infectious Diseases
 
29
,
1186
–8.
3.
Schmitz, F. J., Perdikouli, M., Beeck, A., Verhoef, J. & Fluit, A. C. (
2001
). Molecular surveillance of macrolide, tetracycline and quinolone resistance mechanisms in 1191 clinical European Streptococcus pneumoniae isolates.
International Journal of Antimicrobial Agents
 
18
,
433
–6.
4.
Brett, W., Masters, P. J., Lang, S. D., Ikram, R. B., Hatch, S. H. & Gordon, M. S. (
1999
). Antibiotic susceptibility of Streptococcus pneumoniae in New Zealand.
New Zealand Medical Journal
 
112
,
74
–8.
5.
National Committee for Clinical Laboratory Standards. (
1999
). Performance Standards for Antimicrobial Susceptibility Testing: Ninth Informational Supplement M100-S9. NCCLS, Wayne, PA, USA.
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.
7.
Shortridge, V. D., Flamm, R. K., Ramer, N., Beyer, J. & Tanaka, S. K. (
1996
). Novel mechanism of macrolide resistance in Streptococcus pneumoniae.
Diagnostic Microbiology and Infectious Disease
 
26
,
73
–8.
8.
Hall, L. M. C., Whiley, R. A., Duke, B., George, R. C. & Efstratiou, A. (
1996
). Genetic relatedness within and between serotypes of Streptococcus pneumoniae from the United Kingdom: analysis of multilocus enzyme electrophoresis, pulsed-field gel electrophoresis, and antimicrobial resistance patterns.
Journal of Clinical Microbiology
 
34
,
853
–9.
9.
Tenover, F. C., Arbeit, R. D., Goering, R. V., Mickelsen, P. A., Murray, B. E., Persing, D. H. et al. (
1995
). Interpreting chromosomal DNA restriction patterns produced by pulsed-field gel electrophoresis—criteria for bacterial strain typing.
Journal of Clinical Microbiology
 
33
,
2233
–9.
10.
McGee, L., Klugman, K. P., Wasas, A., Capper, T. & Brink, A. (
2001
). Serotype 19F multiresistant pneumococcal clone harboring two erythromycin resistance determinants [erm(B) and mef(A)] in South Africa.
Antimicrobial Agents and Chemotherapy
 
45
,
1595
–8.