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JAC Advance Access originally published online on April 25, 2007
Journal of Antimicrobial Chemotherapy 2007 59(6):1167-1170; doi:10.1093/jac/dkm106
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© The Author 2007. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oxfordjournals.org

Presence of plasmid pA15 correlates with prevalence of constitutive MLSB resistance in group A streptococcal isolates at a university hospital in southern Taiwan

Yi-Fang Liu1, Chih-Hung Wang2, Rajendra Prasad Janapatla2, Hsiu-Mei Fu2, Hsiu-Mei Wu2 and Jiunn-Jong Wu1,2,*

1 Institute of Basic Medical Sciences, College of Medicine, National Cheng Kung University, Tainan, Taiwan 2 Department of Medical Laboratory Science and Biotechnology, College of Medicine, National Cheng Kung University, Tainan, Taiwan

* Correspondence address. Department of Medical Laboratory Science and Biotechnology, College of Medicine, National Cheng Kung University, No. 1 University Road, Tainan 70101, Taiwan. Tel: +886-6-2353535 ext. 5775; Fax: +886-6-2363956; E-mail: jjwu{at}mail.ncku.edu.tw

Received 18 January 2007; returned 23 January 2007; revised 13 March 2007; accepted 19 March 2007


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Objectives: To investigate the role of a plasmid bearing the erm(B) gene on the prevalence of the macrolide, lincosamide and group B streptogramin (MLSB) phenotype of group A streptococci (GAS) and to characterize the plasmid and determine the clonal relation between the erythromycin-resistant isolates.

Methods: Two hundred and five erythromycin-resistant GAS isolates were collected from 1990 to 2006. Colony hybridization, PCR, plasmid curing and PFGE techniques were used to analyse the mechanisms behind the phenotypes.

Results: Among the 56 isolates with constitutive MLSB (cMLSB) resistance, 53 isolates harboured a plasmid, pA15, of 19 kb. erm(B) was on pA15 and it confered a cMLSB resistance phenotype. The prevalence rate of the pA15-containing isolates was 36.3% from 1993 to 1995, but the plasmid could not be detected from 2004 to 2006. To link the high-level resistance to pA15, clinical isolate A15 was selected and pA15 was cured by novobiocin. In the plasmid-cured strain SW503, the erythromycin MIC decreased from 256 to 0.032 mg/L. By electroporation, pA15 was re-introduced into the plasmid-cured erythromycin-susceptible strain, and the high-level erythromycin resistance was restored. Plasmid pA15 was also transferred to group B streptococci and group C streptococci by electroporation. In all the pA15-containing isolates, emm1 type was present and pulse type J was predominant (48 of 54 isolates).

Conclusions: The plasmid pA15 mediated cMLSB resistance in the mid-1990s, but pA15 was not detected in the clinical isolates from 2004 onwards, which correlates with the absence of cMLSB resistance in this region.

Keywords: erythromycin , clindamycin , plasmids , group A streptococci , PFGE


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In Taiwan, the prevalence rate of erythromycin resistance in group A streptococci (GAS) was 40% to 70% before 2000.1,2 Currently, it is < 20%, mainly due to decreased erythromycin use after antimicrobial reimbursement restriction for undocumented bacterial upper respiratory tract infections.1 Two major mechanisms are involved in the resistance to erythromycin in GAS. The first involves a macrolide efflux pump encoded by mef(A), involved in active efflux of the antibiotic, causing resistance to 14- and 15-membered ring macrolides only (M phenotype).3 The second involves target site modification by the methylase enzymes encoded by erm(B) and erm(A) that methylate 23S rRNA, thereby altering the binding site for macrolide, lincosamide and streptogramin B antibiotics to ribosomes (MLSB phenotype).3

Generally, antibiotic resistance genes in streptococci are plasmid-borne or on transposons. Previously, our laboratory reported that erythromycin resistance with the cMLSB phenotype was being replaced by the M phenotype in the southern Taiwan region.2 Understanding these phenotypes is an important factor in treating GAS infections. The purpose of the present study was (i) to investigate the role of a plasmid bearing the erm(B) gene on the prevalence of the cMLSB phenotype and (ii) to characterize the plasmid and determine the clonal relation between the erythromycin-resistant isolates.


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Bacterial strains, conditions and susceptibility testing

A total of 612 GAS isolates were collected from 1990 to 2006, including 99 isolates collected randomly from 1990 to 1995, in the Department of Pathology, National Cheng Kung University (NCKU) Hospital in southern Taiwan. The agar dilution method was used to determine the MICs of erythromycin and clindamycin (Sigma Chemical Co., St Louis, MO, USA). To confirm the MICs for resistant isolates, Etests (AB Biodisk, Solna, Sweden) were also performed. A double-disc diffusion agar inhibitory test (D test) was performed to identify the inducible MLSB (iMLSB) phenotype, in accordance with the CLSI (formerly NCCLS) guidelines.4 The presence of a D-shaped zone was reported as a positive test for the iMLSB phenotype and the absence of a D-shaped zone was considered as a negative test for the iMLSB phenotype. In-house reference strain GAS10 was used as a D test positive control and GAS04 was used as a negative control.

Erythromycin resistance gene screening

Screening for the erythromycin resistance genes erm(B), erm(A) (subclass ermTR) and mef(A) in the erythromycin-resistant GAS isolates was performed by PCR.5 For each resistant determinant, an in-house positive control was used in our study: GAS10 for erm(A), GAS15 for erm(B) and GAS04 for mef(A). GASA20 was used as a negative control for erm(A), erm(B) and mef(A). Molecular grade water was substituted for template DNA and used as a negative control for every single run. After observing the correct size of erm(A), erm(B) and mef(A) genes, PCR fragments were randomly selected, purified and confirmed by sequencing. Hybridization by an internal probe was performed to erm(B) only. The GAS chromosomal DNA and plasmid DNA were prepared as described previously.6

Plasmid curing, transformation and colony hybridization

Plasmid curing and transformation of GAS, group B streptococci (GBS) and group C streptococci (GCS) were performed according to the method described by Schalen et al.7 GAS isolates were also screened for the presence of plasmid pA15 by colony hybridization according to the method described by Sambrook and Russell.8 The zeta gene (GI:63021982, locus_tag = ‘pSM19035_009’) was used as a probe to screen the GAS isolates for the presence of plasmid pA15. The probe was labelled with Gene Images AlkPhos Direct Labelling and Detection System according to the manufacturer's instructions (Amersham Biosciences, Piscataway, NJ, USA). After hybridization, CDP-StarTM chemiluminescent detection reagent (Amersham Biosciences) was used for signal detection.

PFGE and emm typing and plasmid pA15 sequencing

Genetic relatedness of erythromycin-resistant isolates was determined by PFGE of SmaI-digested genomic DNA as described previously.2 Banding patterns were analysed by visual inspection and by computer-assisted analysis with GelCompar II (version 1.01) software (Applied Maths, Kortrijk, Belgium). Isolates were assigned different pulse types when the banding patterns revealed more than three band differences. Patterns with 0–3 band differences were considered to be one pulse type.

To identify the emm type in the macrolide-resistant isolates, the emm gene was amplified by PCR with the primers recommended by the Centers for Disease Control and Prevention (CDC), Atlanta, GA, USA. All the amplicons were sequenced at the core laboratory of NCKU. The emm sequences obtained were compared with those deposited in the GenBank database and also confirmed with the CDC Streptococcus pyogenes emm sequence database (http://www.cdc.gov/ncidod/biotech/strep/strepblast.htm). An isolate was considered to be of a given emm type if it had 95% identity over the first 160 bases obtained (http://www.cdc.gov/ncidod/biotech/strep/assigning.htm).9

pA15 was sequenced either by primer walking using primers erm(B)-forward (5'- TATTTGGTTGAGTACCTTTTC-3') and erm(B)-reverse (5'-GTAAACAATTTAAGTACCGTTACT-3'), or pA15 was digested with HincII and HindIII enzymes and cloned into pUC18 (New England Biolabs, MA, USA) and sequenced with primers pUC18-forward (5'-AGCGGATAACAATTTCACACAGGA-3') and pUC18-reverse (5'-GTTTTCCCAGTCACGAC-3'). After sequencing pA15 at the core laboratory of NCKU, sequence analysis was performed online using BLAST (www.ncbi.nlm.nih.gov/BLAST/).


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Of the 612 clinical isolates from 1990 to 2006, 205 (33.5%) were resistant to erythromycin (Figure 1). Among them, 56 isolates had a cMLSB phenotype and the erythromycin MIC was > 256 mg/L and 140 isolates had an M phenotype and the MIC was ≤ 32 mg/L. Only nine isolates had an iMLSB phenotype, and the MIC was ≤ 32 mg/L in eight isolates and > 256 mg/L in one isolate. Plasmid was isolated from 54 of the 56 isolates with the cMLSB phenotype. A high percentage of the clinical isolates had a plasmid in 1993 (40%), 1994 (36%) and 1995 (33%), but a plasmid could not be detected in isolates from 2004 to 2006. The mechanism of erythromycin resistance was determined by PCR. The erm(B) gene was present in all 56 cMLSB isolates, the mef(A) gene was present in all 140 M phenotype isolates and the erm(A) gene was present in all 9 iMLSB isolates.


Figure 1
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  		Figure 1.. Distribution of GAS clinical isolates by erythromycin resistance, phenotype and plasmid from 1990 to March 2006 (except 1996). White shading, erythromycin-susceptible isolates; light grey shading, plasmid-positive cMLSB isolates; dark grey shading, plasmid-negative M isolates; horizontal lines, plasmid-negative iMLSB isolates; vertical lines, plasmid-negative cMLSB isolates. With time, the number of plasmid-positive and erythromycin-resistant isolates has decreased.

 
The most extensively studied group of large replication plasmids in Gram-positive bacteria is the inc18 family consisting of pAMß1, pIP501 and pSM19035.10 Plasmid pSM19035 (28 975 bp) of GAS is a low-copy-number plasmid (two to five copies per cell) carrying erythromycin resistance that was studied in detail and the DNA sequence is contained in GenBank accession number AY357120 [GenBank] .10,11 In order to confirm whether plasmid pA15 present in the isolates is similar to pSM19035, the plasmid was partially sequenced. Plasmid pA15 sequence analysis revealed that of the 35 genes present in pSM19035, only 9 genes are similar in pA15, including erm(B) (function: N-methyltransferase), cops (plasmid copy number control), repS (initiation of plasmid replication), beta (site-specific recombinase), gamma (topoisomerase), delta (hypothetical protein, similar to ATPases involved in plasmid active partitioning), omega (transcriptional repressor), epsilon (antidote of epsilon-zeta post-segregational killing system) and zeta (toxin of epsilon-zeta post-segregational killing system). In pSM19035, epsilon and zeta genes encode an antitoxin and a toxin, respectively, a unique system in GAS.12 To screen the isolates for the presence of pA15, the zeta gene was used as a probe in colony hybridization. Among the 56 cMLSB isolates, 54 were zeta gene positive and 2 were negative. Plasmid pA15 could not be detected in the isolates with M and iMLSB phenotypes and in 22 erythromycin-susceptible isolates that were randomly selected. All the 54 zeta gene positive plasmids were verified by a restriction enzyme digestion pattern with HindIII, HincII, SpeI and PvuII; in 53 isolates, the size of the plasmid pA15 was ~19 kb and only in one isolate, plasmid pA768 (16 kb) showed a different pattern from pA15. The digestion pattern of pA15 with restriction enzymes did not match the pSM19035 digestion pattern predicted by software NEBcutter V2.0.

To characterize the role of plasmid pA15 in erythromycin resistance, GAS clinical isolate A15 was selected and plasmid pA15 was cured using novobiocin. In the plasmid-cured isolate SW503, the erythromycin MIC decreased from > 256 to 0.032 mg/L. In the plasmid-complemented isolate SW513, high erythromycin resistance (MIC > 256 mg/L) was restored. pA15 was also used to transform clinical isolates of GBS and GCS. After transformation, GBS and GCS isolates SW514 and SW515, respectively, acquired erythromycin resistance. The MICs of erythromycin for SW514, SW515 and SW516 (GAS M1 strain with pA15) were over 256 mg/L and the plasmid digestion pattern of these strains was similar to pA15. In addition, plasmid DNA was isolated from the transformants and the erm(B) gene was detected by PCR and also by an erm(B) probe in Southern blotting.

To determine the clonal diversity of erythromycin-resistant isolates with and without plasmid from 1990 to 2006, PFGE and emm typing were performed. Twenty-three PFGE type patterns and 14 emm types were identified in the 187 isolates studied. In the M phenotype isolates, 19 pulse types and 14 emm types were present. Whereas, in the 54 plasmid-containing cMLSB isolates, pulse type J was predominant (48 of 54 isolates) and prevalent from 1990 to 2003. In the remaining six isolates, pulse types D (in two isolates), E (one), I (two) and L (one) were present. Only emm1 type was found in all the cMLSB isolates.


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Yan et al.2 reported that the mef(A) gene had replaced the erm(B) gene between 1995 and 1998 in GAS in southern Taiwan. Interestingly, we observed that the erm(B) determinant was present on plasmid pA15 and the prevalence rate of the plasmid-containing erythromycin-resistant isolates was 27% from 1990 to 1995, but the prevalence rate was only 7.6% from 1997 to 2001 and 0% from 2004 to March 2006. This remarkable decrease in plasmid-positive isolates correlated with the shift in predominance from the cMLSB phenotype to the M phenotype and a decrease in erythromycin resistance.

Although it was reported that erm(B) was present on a plasmid, few publications looked at the relationship between plasmid prevalence and the plasmid-mediated erythromycin resistance mechanism.7,13 We demonstrated that plasmid pA15 can transform erythromycin-susceptible GAS and other Streptococcus species in vitro, including GBS and GCS, conferring erythromycin resistance. Our study is the first such publication from Taiwan and we show that the plasmid pA15 may spread easily to other strains and contribute to an increase in erythromycin resistance prevalence under constant antibiotic selection. Besides pA15, we also found a plasmid, pA768, from clinical isolate A768 with different enzyme digestion patterns.

Our study also shows the clonal diversity between strains with cMLSB and M phenotypes. In all the pA15-containing isolates, emm1 type was present and pulse type J was predominant and prevalent from 1990 to 2003, but the pulse type J strain decreased dramatically and correlated with the reduced consumption of erythromycin from 1997 to 2006.1 Previously, it was reported that single or multiple clones mediate the spread of erythromycin resistance in GAS.13,14 We show that in Taiwan under antibiotic pressure, the cMLSB resistance observed in GAS was caused by the spread of a single clone and a particular serotype, signifying the importance of epidemiological surveillance and continuous monitoring of the resistance characteristics of GAS.

Hsueh et al.1 reported that the overall erythromycin resistance rate in Taiwan was 40% to 70% prior to 2000, and it was 15% in 2003. Our study found that in 2004, the resistance rate was 18.6%, in 2005 it was 3.6% and until March 2006 it was 0%. In Taiwan, it is widely accepted that the decrease in erythromycin resistance was significantly associated with the restrictive governmental policy implemented in 2001 to deny reimbursement for the costs of antibiotics used for acute upper respiratory tract infections without evidence of bacterial involvement.1

In conclusion, we observed that in southern Taiwan, pA15 with erm(B) mediated the high erythromycin resistance rate prior to 1997, but later, mef(A) was the predominant resistance mechanism. Plasmid pA15 can be transferred to GAS, GBS and GCS and confer erythromycin resistance.


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None to declare.


   Acknowledgements
 
This work was partly supported by grants NSC89-2320-B006-036 from the National Science Council, DOH89-TD-1006 from the Department of Health and NHRI-EX92-9027 SP from National Health Research Institutes, Taiwan.


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1 Hsueh PR, Shyr JM, Wu JJ. Decreased erythromycin use after antimicrobial reimbursement restriction for undocumented bacterial upper respiratory tract infections significantly reduced erythromycin resistance in Streptococcus pyogenes in Taiwan. Clin Infect Dis (2005) 40:903–5.[CrossRef][Web of Science][Medline]

2 Yan JJ, Wu HM, Huang AH, et al. Prevalence of polyclonal mefA-containing isolates among erythromycin-resistant group A streptococci in southern Taiwan. J Clin Microbiol (2000) 38:2475–9.[Abstract/Free Full Text]

3 Leclercq R. Mechanisms of resistance to macrolides and lincosamides: nature of the resistance elements and their clinical implications. Clin Infect Dis (2002) 34:482–92.[CrossRef][Web of Science][Medline]

4 Clinical and Laboratory Standards Institute. Performance Standards for Antimicrobial Susceptibility Testing: Sixteenth Informational Supplement M100-S16 (2006) Wayne, PA, USA: CLSI.

5 Amezaga MR, Carter PE, Cash P, et al. Molecular epidemiology of erythromycin resistance in Streptococcus pneumoniae isolates from blood and noninvasive sites. J Clin Microbiol (2002) 40:3313–8.[Abstract/Free Full Text]

6 Chiang-Ni C, Wang CH, Tsai PJ, et al. Streptococcal pyrogenic exotoxin B causes mitochondria damage to polymorphonuclear cells preventing phagocytosis of group A streptococcus. Med Microbiol Immunol (2006) 195:55–63.[CrossRef][Medline]

7 Schalen C, Gebreselassie D, Stahl S. Characterization of an erythromycin resistance (erm) plasmid in Streptococcus pyogenes. APMIS (1995) 103:59–68.[Web of Science][Medline]

8 Sambrook J, Russell D. Molecular Cloning: A Laboratory Handbook (2001) Third Edition. Cold Spring Harbor, NY, USA: Cold Spring Harbor Laboratory Press.

9 Beall B, Facklam R, Thompson T. Sequencing emm-specific PCR products for routine accurate typing of group A streptococci. J Clin Microbiol (1996) 34:953–8.[Abstract]

10 Ceglowski P, Lurz R, Alonso JC. Functional analysis of pSM19035-derived replicons in Bacillus subtilis. FEMS Microbiol Lett (1993) 109:145–50.[CrossRef][Web of Science][Medline]

11 Ceglowski P, Alonso JC. Gene organization of the Streptococcus pyogenes plasmid pDB101: sequence analysis of the orf eta-copS region. Gene (1994) 145:33–9.[CrossRef][Web of Science][Medline]

12 Zielenkiewicz U, Ceglowski P. The toxin–antitoxin system of the streptococcal plasmid pSM19035. J Bacteriol (2005) 187:6094–105.[Abstract/Free Full Text]

13 Cresti S, Lattanzi M, Zanchi A, et al. Resistance determinants and clonal diversity in group A streptococci collected during a period of increasing macrolide resistance. Antimicrob Agents Chemother (2002) 46:1816–22.[Abstract/Free Full Text]

14 Perez-Trallero E, Marimon JM, Montes M, et al. Clonal differences among erythromycin-resistant Streptococcus pyogenes in Spain. Emerg Infect Dis (1999) 5:235–40.[Web of Science][Medline]


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