recombination rates of streptococcus pneumoniae isolates with both erm(b) and mef(a) genes

R E S E A R C H L E T T E R

Recombination ratesofStreptococcuspneumoniae isolateswithboth erm(B)andmef (A)genesJi-Young Lee1, Jae-Hoon Song2,3 & Kwan Soo Ko1,2

1Department of Molecular Cell Biology, Sungkyunkwan University School of Medicine, Suwon, Korea; 2Asian-Pacific Research Foundation for Infectious

Diseases (ARFID), Seoul, Korea; and 3Samsung Medical Center, Division of Infectious Diseases, Sungkyunkwan University School of Medicine, Seoul,

Korea

Correspondence: Kwan Soo Ko,

Department of Molecular Cell Biology,

Sungkyunkwan University School of

Medicine, Suwon 440-746, Korea. Tel.:

182 31 299 6223; fax: 182 31 299 6229;

e-mail: [email protected]

Received 7 May 2010; revised 31 May 2010;

accepted 1 June 2010.

Final version published online 2 July 2010.

DOI:10.1111/j.1574-6968.2010.02032.x

Editor: Anthony George

Keywords

multidrug resistance; pneumococci;

recombination; mutation; erythromycin.

Abstract

Erythromycin-resistant Streptococcus pneumoniae isolates containing both erm(B)

and mef(A) genes have a higher rate of multidrug resistance (MDR). We

investigated the relationships between the presence of erythromycin resistance

determinants and the recombination rate. We determined the mutation and

recombination frequencies of 46 S. pneumoniae isolates, which included 19 with

both erm(B) and mef(A), nine with only erm(B), six with only mef(A), and 11

erythromycin-susceptible isolates. Mutation frequency values were estimated as

the number of rifampin-resistant colonies as a proportion of total viable count.

Genotypes and serotypes of isolates with the hyper-recombination phenotype were

determined. Twelve S. pneumoniae isolates were hypermutable and four isolates

were determined to have hyper-recombination frequency. Streptococcus pneumo-

niae isolates with both erm(B) and mef(A) genes did not show a high mutation

frequency. In contrast, all isolates with a hyper-recombination phenotype con-

tained both erm(B) and mef(A) genes. In addition, the recombination rate of

isolates with both erm(B) and mef(A) genes was statistically higher than the rate of

other isolates. The dual presence of erm(B) and mef(A) genes in some pneumo-

coccal isolates may be associated with high recombination frequency. This may be

one of the reasons for the frequent emergence of MDR in certain pneumococcal

isolates.

Introduction

Streptococcus pneumoniae, one of the best examples of the

global emergence of resistance, is an important pathogen of

community-acquired pneumoniae, bacterial meningitis, oti-

tis media, and sinusitis (Adam, 2002). In particular, macro-

lide as well as penicillin resistance in S. pneumoniae are

serious concerns worldwide. Macrolide resistance in

S. pneumoniae occurs mainly due to modification of the drug-

binding site by erm(B) gene or active efflux of the drug by

mef(A) gene. Although erm(B) gene mediates high-level

resistance and mef(A) gene correlates with low-level resis-

tance, the rate of erythromycin-resistant S. pneumoniae

isolates containing both genes is growing worldwide (Song

et al., 2004a, b; Farrell et al., 2005). As the single presence of

erm(B) gene determines a high macrolide resistance level,

the dual presence of erm(B) and mef(A) genes may not be

advantageous in terms of bacterial survival. Thus, we

postulated that pneumococcal isolates with both erm(B)

and mef(A) genes originated from strains with only mef(A)

gene in which the erm(B) gene was introduced; this has been

supported by multilocus sequence typing (MLST) analysis

(Ko & Song, 2004). However, the characteristics of pneu-

mococcal isolates containing both erm(B) and mef(A) genes

have not been investigated.

Several investigators have reported that S. pneumoniae

isolates with both erm(B) and mef(A) gene show resistance

against more antimicrobial agents (Farrell et al., 2004;

Jenkins et al., 2008). As multidrug resistance (MDR) is

linked to an increased risk of treatment failure, increased

prevalence of S. pneumoniae isolates containing both erm(B)

and mef(A) genes may represent a serious public health

threat. Although MDR of S. pneumoniae isolates with both

erm(B) and mef(A) genes is documented, it is not known

FEMS Microbiol Lett 309 (2010) 163–169 c� 2010 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

MIC

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mailto:[email protected]

why they confer high MDR. Instead, it has been suggested

that mutators are associated with the emergence of anti-

microbial resistance in several pathogenic bacterial species

such as Escherichia coli, Pseudomonas aeruginosa, Neisseria

meningitidis, Helicobacter pylori, and Staphylococcus aureus

(Chopra et al., 2003). Mutators (hypermutable strains) are

defined as bacterial strains with greater than normal muta-

tion frequencies. Mutators are generally defective in the

methyl-directed mismatch repair system, with mutations in

mutS or mutL genes (Oliver et al., 2000).

The relationship between antimicrobial resistance and

frequency of mutation in S. pneumoniae has been investi-

gated (Morosini et al., 2003; del Campo et al., 2005; Gould

et al., 2007). However, whereas most studies have focused on

fluoroquinolone resistance and point mutations in hyper-

mutable S. pneumoniae, the present study investigated the

relationships between the presence of macrolide resistance

determinants and the recombination rate.

Materials and methods

Bacterial isolates

A total of 89 S. pneumoniae isolates were collected in a

tertiary-care hospital in Korea, and antimicrobial suscept-

ibility testing was performed. In addition, we determined

erythromycin resistance determinants, erm(B) and mef(A)

genes, by the duplex PCR method (Ko & Song, 2004). Of

these, 46 S. pneumoniae isolates were selected and used for

further research. Thirty-five isolates were erythromycin-

resistant and the others were erythromycin-susceptible. Of

the 27 erythromycin-resistant S. pneumoniae isolates, 20

isolates contained both erm(B) and mef(A) genes (Group I),

nine isolates contained only the erm(B) gene (Group II), and

six isolates contained only the mef(A) gene (Group III).

Erythromycin-susceptible S. pneumoniae isolates were cate-

gorized as Group IV.

In vitro susceptibility testing

Minimum inhibitory concentration (MIC) was determined

by the Clinical and Laboratory Standards Institute (CLSI)

(2008) broth microdilution method. In vitro susceptibility

was tested for 19 antimicrobial agents including erythromy-

cin, penicillin, amoxicillin–clavulanate, ceftriaxone, cefur-

oxime, cefixime, cefprozil, cefdinir, imipenem, ertapenem,

ciprofloxacin, levofloxacin, moxifloxacin, gatifloxacin, clin-

damycin, tetracycline, trimethoprim–sulfamethoxazole, ri-

fampin, and vancomycin.

Determination of mutation frequency

Three to five colonies from an overnight culture on 5%

sheep blood agar plates (Becton-Dickinson, Sparks, MD)

were resuspended in 10 mL of brain–heart infusion (BHI)

broth (Difco Laboratories, Detroit, MI) and incubated for

6–8 h at 35 1C without shaking. For total viable count

determination, a 100-mL aliquot was diluted in

1� phosphate-buffered saline (PBS) and plated onto 5%

sheep blood agar plates. The remaining culture was centri-

fuged for 5 min at 2500 g. The pellet was resuspended

in 200mL of BHI broth and plated onto 5% sheep blood

agar plates containing 2 mg mL�1 rifampin (Sigma-Aldrich,

St. Louis, MO). Mutation frequency values are reported as

the proportion of rifampin-resistant colonies (detected after

48–72 h of incubation in a 5% CO2 atmosphere) vs. total

viable cell counts (O’Neill & Chopra, 2002). Results corre-

spond to the mean value obtained in triplicate experiments.

An isolate was considered a mutator strain when its

frequency was Z7.5� 10�8 (Morosini et al., 2003).

Allelic replacement mutagenesisand transformation

Allelic replacement mutagenesis for determination of the

recombination rate of S. pneumoniae isolates was performed

and competent cells were prepared as described previously

(Song et al., 2005). A spr0476 gene that has already been

reported as a nonessential gene was used as a target gene for

homologous recombination (Thanassi et al., 2002). Ampli-

fication of the left and right flanking regions of spr0476 was

performed using two pairs of primers, 0476L-F/L-R (50-CAT

CAG TGG AAG GAA TGG TTG ACC-30/50-GAC GAA CTC

CAA TTC ACT GTT ATC TAC CCA CAA GAG CTT GA-30)

and 0476R-F/R-R (50-AGA TTT AGA TGT CTA AAA AGC

CAT GAA AAG CGT CGT TTG AC-30/50-GTT GCG ATT

GCG TCC ACC TCC TCA-30), generating PCR products of

500 and 430 bp, respectively. Primers 0476L-R and 0476R-F

contained 21 nucleotides that are identical to the 50- and

30-ends of the kanamycin resistance gene cassette, followed

by 23 bp of spr0476 gene-specific sequence. The resulting

fused PCR product of 1.8 kb was directly transformed into

each S. pneumoniae isolate, and homologous recombination

between the construct and spr0476 in the chromosome was

forced. Pneumococcal transformation was executed under

the conditions described previously (Gutierrez et al., 2004;

Song et al., 2005).

Determination of recombination rate

To estimate the rate at which the fused PCR products

recombine with the chromosomal spr0476 in S. pneumoniae,

a 100-mL aliquot was diluted in 1�PBS and plated onto 5%

sheep blood agar plates. The remaining 100 mL was plated on

Todd–Hewitt agar supplemented with 0.5% yeast extract

plus 400 mg L�1 kanamycin (Sigma-Aldrich) and incubated

at 35 1C for 48–72 h. Recombination rate values were

calculated as the proportion of kanamycin-resistant colonies

FEMS Microbiol Lett 309 (2010) 163–169c� 2010 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

164 J.-Y. Lee et al.

to total viable cell counts. Results correspond to the mean

value obtained in triplicate experiments. An isolate was

considered to be arbitrary to a strain with a high recombina-

tion rate, that is, hyper-recombination, when its frequency

was Z1.0� 10�4 (Hsieh et al., 2006).

Genotyping and serotyping

Genotypes and serotypes of S. pneumoniae isolates showing

high recombination frequency were determined using

MLST performed as described previously (Enright & Spratt,

1998). Serotypes were determined by the capsular Quellung

reaction with commercial antisera (Statens Serum Institute,

Copenhagen, Denmark) as recommended by the manufac-

turer.

Statistical analysis

Student’s t-test was used to compare continuous variables

and Pearson’s w2-test was used to compare categorical

variables. The SPSS for Windows software package (version

11.5; SPSS, Chicago, IL) was used for statistical analysis.

Results

Antimicrobial resistance

Among 89 S. pneumoniae isolates, 56 isolates (62.9%) were

resistant to erythromycin (Table 1), which was a somewhat

smaller proportion than in previous studies (Song et al.,

2004a, b). Among the 56 erythromycin-resistant isolates, 27

(48.2%) contained both the erm(B) and mef(A) genes.

Twenty-five (44.6%) and eight (14.3%) contained only the

erm(B) gene and mef(A) gene, respectively. The penicillin

resistance rate (MIC4 2 mg L�1) was 52.8%, but high

penicillin resistance (MIC4 8 mg L�1) was not found. Cef-

triaxone resistance was found only in pneumococcal isolates

with both erm(B) and mef(A) genes (Group I). Antimicro-

bial resistance rates of Group I were significantly higher than

those of erythromycin-susceptible isolates (Group IV) for

most antimicrobial agents except ciprofloxacin and ceftriax-

one. This was also case between Group I and Group III,

except for tetracycline. In addition, penicillin, amoxicillin–-

clavulanate, cefuroxime, cefixime, and cefdinir resistance

rates of Group I isolates were significantly higher than those

of Group II isolates. When the antimicrobial resistances

were compared between Group I and Groups II–IV, they

were shown to be significantly higher in Group I. In contrast

to the other antimicrobial agents, the ciprofloxacin resis-

tance rate was higher in Group IV isolates, but was not

significant (Table 1). Isolates displaying resistance to imipe-

nem, ertapenem, levofloxacin, moxifloxacin, gatifloxacin,

rifampin, and vancomycin were not found.

Mutation frequency

Among 46 S. pneumoniae isolates tested, 12 (26.1%) showed

the mutator phenotype (mutation frequency 4 7.5� 10�8)

(Table 2). Of these, six isolates contained both erm(B) and

mef(A) genes (Group I). Each single isolate of Groups II and

III was a mutator phenotype, and four mutators were

erythromycin susceptible (Group IV), i.e. 30.0% of Group

I, 11.1% of Group II, 16.7% of Group III, and 36.4% of

Group IV were mutators. The mean estimate of mutation

frequency was the highest in Group IV (1.37� 2.25� 10�7;

Table 3, Fig. 1a). Although mutation frequencies of Group I

pneumococcal isolates were significantly higher than those

Table 1. Antimicrobial resistances in each group of Streptococcus pneumoniae isolates with respect to erythromycin resistance determinants�

Antimicrobials Total (n = 89)

Groupsw P-valuesz

I (n = 27) II (n = 25) III (n = 8) IV (n = 29) II, III, IV (n = 62) I–II I–III I–VI I–others

Erythromycin 56 (62.9)

Penicillin 47 (52.8) 25 (92.6) 18 (72.0) 2 (25.0) 2 (6.9) 22 (35.5) 0.050 o 0.001 o 0.001 o 0.001

Amoxicillin–clavulanate 15 (16.9) 14 (51.9) 1 (4.0) – – 1 (1.6) o 0.001 0.009 o 0.001 o 0.001

Ceftriaxone 1 (1.1) 1 (3.7) – – – – 0.331 0.581 0.296 0.128

Cefuroxime 54 (60.8) 26 (96.3) 19 (76.0) 5 (62.5) 4 (13.8) 28 (45.2) 0.032 0.008 o 0.001 o 0.001

Cefixime 58 (65.2) 26 (96.3) 21 (84.0) 5 (62.5) 6 (20.7) 32 (51.6) 0.133 0.008 o 0.001 o 0.001

Cefprozil 52 (58.4) 25 (92.6) 18 (72.0) 5 (62.5) 4 (13.8) 27 (43.5) 0.050 0.033 o 0.001 o 0.001

Cefdinir 54 (60.8) 26 (96.3) 19 (76.0) 5 (62.5) 4 (13.8) 28 (45.2) 0.032 0.008 o 0.001 o 0.001

Ciprofloxacin 14 (15.7) 3 (11.1) 2 (8.0) 1 (12.5) 8 (27.6) 11 (17.7) 0.704 0.914 0.121 0.430

Clindamycin 51 (57.3) 26 (96.3) 24 (96.0) 1 (12.5) – 25 (40.3) 0.956 o 0.001 o 0.001 o 0.001

Tetracycline 70 (78.7) 26 (96.3) 25 (100) 8 (100) 11 (37.9) 44 (71.0) 0.331 0.581 o 0.001 0.007

Trimethoprim–

sulfamethoxazole

43 (46.1) 24 (88.9) 17 (68.0) – 2 (6.9) 19 (30.6) 0.065 o 0.001 o 0.001 o 0.001

�For imipenem, ertapenem, levofloxacin, moxifloxacin, gatifloxacin, rifampin, and vancomycin, no resistant isolates were found.wGroup I, S. pneumoniae with both erm(B) and mef(A) genes; Group II, with only erm(B) gene; Group III, with only mef(A) gene; Group IV, erythromycin-

susceptible S. pneumoniae.zThe significant differences are in bold-type.


165Streptococcus pneumoniae with high recombination frequency

of Group II isolates (P � 0.015), they were lower than those

of Group IV (Table 4, Fig. 1a). Thus, S. pneumoniae isolates

with both erm(B) and mef(A) genes may not show a high

mutation frequency.

Recombination rate

Recombination rates of 46 S. pneumoniae isolates ranged

from 3.0� 10�7 to 4.5� 10�4 (Table 2). When the cutoff of

high recombination rate was chosen as 1.0� 10�4, four

isolates displayed the hyper-recombination phenotype (Ta-

ble 2). These four isolates belonged to Group I, pneumo-

coccal isolates with both erm(B) and mef(A) genes. The

recombination rate in S. pneumoniae isolates of Group I

ranged from 1.9� 10�6 to 4.5� 10�4 (mean� SD,

1.01� 1.43� 10�4), which was the highest rate (Table 3;

Fig. 1b). The recombination rate of Group II was higher

than those of Groups III and IV. Statistical analysis indicated

that the recombination rate of Group I was significantly

higher than those of Groups III and IV (P � 0.043 and

0.006, respectively), although it was not significantly higher

than that of Group II (P � 0.394) (Table 4).

Genotypes and serotypes

The four isolates displaying the hyper-recombination phe-

notype showed different sequence types (STs) in MLST

analysis: ST1439 (04-005; allelic profile, 5-5-6-1-9-14-14),

ST237 (04-018; 15-16-19-15-6-20-1), ST-new1 (04-058;

4-16-new-15-6-20-1), and ST-new2 (04-133; 4-16-19-15-

6-20-14). Whereas three isolates showed serotype 19F, the

serotype of one isolate (04-005) was nontypeable.

Discussion

Generally, bacterial resistance towards antimicrobial agents

emerges by three main genetic mechanisms: acquisition of

plasmids or other transposable elements including resis-

tance genes; recombination of DNA by transformation; and

point mutation events (Pope et al., 2008). In this study, we

focused on the relationships of recombination efficiency

with antimicrobial resistances in S. pneumoniae. Streptococ-

cus pneumoniae possesses a natural competence for genetic

transformation (Havarstein et al., 1995). Horizontal gene

transfer of S. pneumoniae due to this competence enables the

organism to adapt to environmental changes such as anti-

biotic pressure. Indeed, the high competence of S. pneumo-

niae may be one of causes of the emergence of MDR.

Penicillin-resistant S. pneumoniae strains, rather than peni-

cillin-susceptible strains, tend to acquire cross-resistance to

other antimicrobial agents (Song et al., 2006). However, the

competence of S. pneumoniae isolates is not significantly

related to penicillin resistance (Hsieh et al., 2006). Recently,

several studies reported an increased prevalence of erythro-

mycin-resistant S. pneumoniae isolates with both erm(B)

and mef(A) genes (Farrell et al., 2004, 2005; Song et al.,

2004a, b; Jenkins et al., 2008). Interestingly, such isolates

frequently display the MDR phenotype (Farrell et al., 2005),

which was also evident in this study (Table 1). We postulated

Table 2. Mutation and recombination frequencies of each Strepto-

coccus pneumoniae isolate

Groups

Isolate

no.

Mutation

frequency

Recombination

frequency

Group I erm(B)

and mef(A)

04-005 1.78� 10�8 2.84� 10�4�

04-011 6.76� 10�9 6.5� 10�6

04-018 1.41� 10�8 4.5� 10�4�

04-036 3.09� 10�8 3.65� 10�5

04-041 9.17� 10�9 5.7� 10�5

04-046 1.45� 10�7w

4.49� 10�5

04-054 7.56� 10�9 5.06� 10�5

04-058 4.7� 10�8 1.08� 10�4�

04-079 4.64� 10�8 5.21� 10�5

04-093 7.15� 10�9 5.9� 10�6

04-105 3.16� 10�8 1.66� 10�5

04-121 1.12� 10�7w

1.9� 10�6

04-124 6.32� 10�8 3.77� 10�5

04-131 1.39� 10�7w

3.6� 10�6

04-133 3.03� 10�8 3.65� 10�4�

04-039 5.01� 10�7w

6.38� 10�5

04-051 8.9� 10�8w

2.23� 10�5

04-061 3.7� 10�8 3.75� 10�5

04-092 3.35� 10�8 1.23� 10�5

04-128 1.1� 10�7w

6.44� 10�5

Group II erm(B) 04-028 7.01� 10�9 3.01� 10�5

04-053 4.37� 10�9 8.6� 10�6

04-063 1.49� 10�8 6.0� 10�6

04-072 1.46� 10�8 8.29� 10�5

04-095 1.79� 10�8 7.05� 10�5

04-112 1.41� 10�8 2.95� 10�5

04-125 8.36� 10�9 4.36� 10�5

04-075 2.2� 10�9 1.45� 10�5

04-102 1.24� 10�7w

7.5� 10�6

Group III mef(A) 04-059 1.27� 10�8 1.52� 10�5

04-080 5.3� 10�8 1.16� 10�5

04-126 7.57� 10�9 2.3� 10�6

04-004 4.16� 10�8 1.3� 10�6

04-103 3.22� 10�7w

7.39� 10�5

04-020 1.16� 10�8 8.3� 10�6

Group IV erythromycin

susceptible

04-001 1.89� 10�8 4.8� 10�6

04-013 4.09� 10�9 1.88� 10�5

04-055 1.31� 10�7w

2.26� 10�5

04-056 5.3� 10�8 5.44� 10�5

04-077 3.3� 10�7w

3.92� 10�5

04-090 1.48� 10�8 3.0� 10�7

04-098 4.68� 10�8 1.8� 10�6

04-117 9.9� 10�8w

9.0� 10�7

04-120 7.57� 10�7w

1.8� 10�6

04-030 2.45� 10�8 2.55� 10�5

04-082 2.82� 10�8 6.2� 10�6

�Hyper-recombination rate (Z1.0�10�4).wHypermutation frequency (Z7.5� 10�8).



that the MDR phenotype of S. pneumoniae isolates with

both erm(B) and mef(A) genes may be associated with their

high recombination ability. We examined this hypothesis by

estimating the recombination frequency of S. pneumoniae

isolates. In addition, we estimated the mutation frequency

to investigate its relationship with antimicrobial resistance

and the dual presence of erm(B) and mef(A) genes.

Here, we demonstrate that mutation frequency was not

related with the uptake of both erm(B) and mef(A) genes. In

addition, mutators did not showed higher resistance rates

than nonmutators in most antimicrobial agents, except in

the case of ciprofloxacin (data not shown). In addition, we

did not observe an association between hexA and hexB

polymorphisms and the mutator phenotype, which agrees

with previous observations (Gutierrez et al., 2004). So far, it

has not been established whether mutators are related to the

emergence of antimicrobial resistance in bacteria (Chopra

et al., 2003). Studies involving E. coli have suggested that

mutators may be related to the acquisition of antimicrobial

resistance or to evolution of extended-spectrum b-lactamase

(Tanabe et al., 1999; Orentica et al., 2001; Miller et al., 2002).

In S. aureus, macrolide resistance is thought to result from

selective antibiotic pressure in cystic fibrosis (Prunier et al.,

2003). However, a previous study did not show any sig-

nificant correlation between antimicrobial resistance and

hypermutable phenotype, although it did identify a high

frequency of S. pneumoniae mutator phenotype from pa-

tients with cystic fibrosis (del Campo et al., 2005). In

addition, an association between hypermutation and anti-

microbial resistance was not observed in P. aeruginosa

(Gutierrez et al., 2004).

On the contrary, pneumococcal isolates with both erm(B)

and mef(A) genes displayed a high recombination frequency

in this study which was statistically significantly higher than

that of isolates possessing only the mef(A) gene and ery-

thromycin-susceptible isolates. Although not significant,

their recombination frequency was also higher than that of

Table 3. Mutation and recombination frequencies of each group

Groups�

Mutation frequency Recombination frequency

Range Mean� SD Range Mean� SD

I (n = 20) 6.8� 10�9�5.01�10�7 (7.39� 10.9)�10�8 1.9� 10�6�4.5� 10�4 (1.01� 1.43)� 10�4

II (n = 9) 2.2� 10�9�1.24�10�7 (2.30� 3.82)�10�8 6.0� 10�6�8.29� 10�5 (3.87� 2.92)� 10�5

III (n = 6) 7.57� 10�9�3.22� 10�7 (7.47� 12.2)�10�8 1.3� 10�6�7.39� 10�5 (2.09� 3.03)� 10�5

IV (n = 11) 4.09� 10�9�7.57� 10�7 (1.37� 2.25)�10�7 3.0� 10�7�5.44� 10�5 (1.61� 1.96)� 10�5

�Group I, Streptococcus pneumoniae with both erm(B) and mef(A) genes; Group II, with only erm(B) gene; Group III, with only mef(A) gene; Group IV,

erythromycin-susceptible S. pneumoniae.

–5(a) (b)

–3

–7

–6

–5

–4

–8

CF

U)

quen

cy (

Log1

0M

utat

ion

fre

–6

–9Group I Group II Group III Group IV

–7Group I Group II Group III Group IV

10 C

FU

)re

quen

cy (

log

com

bina

tion

fR

ec


Fig. 1. Mutation (a) and recombination (b) frequencies of Streptococcus pneumoniae isolates. The mean values, SDs, and ranges of each group

are presented.

Table 4. Statistical analyses of mutation and frequency frequencies

between groups (P-values)�


Group I Group II Group III Group I Group II Group III

Group II 0.015 – 0.394 –

Group III 0.685 0.161 – 0.043 0.014 –

Group IV 0.551 0.023 0.489 0.006 0.066 0.741

�The significant differences are in bold-type.



isolates possessing only the erm(B) gene. In addition, all

four isolates showing the hyper-recombination phenotype

(recombination frequency 4 10�4) contained both the

erm(B) and mef(A) genes. Although these four isolates with

the hyper-recombination phenotype did not show a signifi-

cantly higher antimicrobial resistance rate, probably due to

the limited number of isolates examined (data not shown),

pneumococcal isolates with both erm(B) and mef(A) genes

exhibited significantly higher resistance rates than isolates of

other groups in most antimicrobial agents (Table 1). Sig-

nificantly higher resistance rates were particularly evident

with penicillin, amoxicillin–clavulanate, cefuroxime, cefpro-

zil, and cefdinir, even when compared with pneumococcal

isolates possessing only the erm(B) gene. Only one isolate

was resistant to ceftriaxone, and resistance to imipenem,

ertapenem, levofloxacin, moxifloxacin, gatifloxacin, rifam-

pin, and vancomycin was not found. Therefore, only cipro-

floxacin resistance was not related to the dual presence of the

erm(B) and mef(A) genes. Fluoroquinolone resistance in-

cluding ciprofloxacin is mainly due to point mutations of

the genes encoding DNA gyrase or topoisomerase IV (Jones

et al., 2000). Thus, ciprofloxacin resistance may not be

related to the uptake of foreign materials, as is the case with

erythromycin resistance from the acquisition of erm(B) or

mef(A) genes.

As only the erm(B) gene bestows a high-level resistance

against erythromycin, the dual presence of erm(B) and

mef(A) may not be advantageous, and may pose a burden

for growth. However, we have shown that pneumococcal

isolates with both erm(B) and mef(A) genes may have

originated from isolates possessing only the mef(A) gene

and acquiring the erm(B) gene in a certain clonal complex,

CC271 (Ko & Song, 2004). Compared with isolates that

possess only the mef(A) gene, and which exhibit low-level

erythromycin resistance, pneumococcal isolates with both

erm(B) and mef(A) genes may have some advantages in

certain environments, such as antibiotic pressure. Pneumo-

coccal strains show differences in recombination frequency

(Samrakandi & Pasta, 2000; Hsieh et al., 2006). Hsieh et al.

(2006) reported that certain serotypes such as 6B, 14, 19F,

9V, 23F, 3, and 18C showed a high competence for plasmid

uptake. It is noteworthy that these serotypes are included in

the seven-valent pneumococcal conjugate vaccine (PCV7)

because of public health concerns due to high antimicrobial

resistance and prevalence.

The dual presence of erm(B) and mef(A) genes and

uptake of foreign resistance determinants in certain pneu-

mococcal isolates may be due to the same traits, such as their

high competency and recombination rates. Thus, certain

isolates with high potency to transform and recombine

foreign genes have a strong possibility of acquiring anti-

microbial resistance determinants and to become MDR. The

present results demonstrate that the dual presence of erm(B)

and mef(A) genes in some pneumococcal isolates may be

associated with a high recombination frequency, high anti-

microbial resistance, and even a high prevalence of those

isolates.

Acknowledgement

This study was partly supported by a grant from

the Samsung Biomedical Research Institute (SBRI, Seoul,

Korea).

Authors’contribution

J.-Y.L. and J.-H.S. contributed equally as joint first authors.

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recombination rates of streptococcus pneumoniae isolates with both erm(b) and mef(a) genes

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