Ribosomal Mutations Conferring Resistance to Macrolides in Streptococcus pneumoniae Clinical Strains Isolated in Germany

Abstract

Among a collection of 4,281 pneumococcal isolates, 7 strains isolated in Germany had an unusual macrolide resistance phenotype. The isolates were found to have multiple mutations in the 23S rRNA and alterations in the L4 ribosomal protein. One strain had an amino acid alteration in the L22 ribosomal protein.

Resistance to macrolides is increasingly being reported among clinical isolates of Streptococcus pneumoniae worldwide (7). In Germany a dramatic increase in the rate of macrolide resistance among pneumococcal strains isolated from patients with invasive disease was observed only recently (17, 18).

The most prevalent mechanisms of macrolide resistance in S. pneumoniae are mediated by mef(A), a gene encoding an efflux pump, and erm(B), a 23S rRNA methylase that dimethylates the adenine at position 2058, resulting in macrolide-lincosamide-streptogramin B (MLS_B) resistance (19). MLS_B resistance can be expressed either constitutively (cMLS_B phenotype) or inducibly (iMLS_B phenotype) (12). In addition, mutations in 23S rRNA and ribosomal proteins L4 and L22 have been shown to account for resistance in pneumococci (8, 9, 11, 13, 14, 22). In rare cases the presence of an erm(A) gene in pneumococci has been reported (20).

Three surveillance studies on antibiotic resistance screened for strains with unusual macrolide resistance genotypes or phenotypes. Strains were included in the present investigation if they were macrolide resistant and showed the erm(B)-negative and mef(A)-negative genotype in repeated PCR assays (five strains) or a discrepancy between the macrolide resistance genotype and phenotype (two strains). The characteristics of the studies are presented in Table 1 (5, 17, 25). Each isolate was confirmed as S. pneumoniae by optochin sensitivity and bile solubility testing. MICs were determined by the broth microdilution method, as described by the NCCLS (15). Determination of macrolide-resistant phenotypes was performed as described previously (12). The primers described by Trieu-Cuot et al. (23) and Tait-Kamradt et al. (21) were chosen for the detection of erm(B) and mef(A). Sequencing of the 23S rRNA genes and ribosomal protein L4 and L22 genes was performed as reported earlier by Canu et al. (1) and Tait-Kamradt et al. (22). The number of mutated copies of the 23S rRNA was determined as described by Canu et al. (1). Strains were serotyped by Neufeld's Quellung reaction, and the genetic relatedness of strains was analyzed by multilocus sequence typing (MLST) (4).

TABLE 1.

Macrolide resistance and resistance genotypes of 4,281 pneumococcal strains documented by nationwide studies in Germany, 1992 to 2002^a

Study	Age group	Disease	Study period	No. of centers	No. of strains	No. (%) of strains				Reference
						Eryr	mef(A) positiveb	erm (B) positiveb	mef (A) and erm (B) negativeb
A	Children	IPD	1997-2000	123	690	122 (17.7)	85 (69.7)	35 (28.7)	2 (1.6)	von Kries et al. (25)c
B	Adults and children	IPD	1992-2000	86	3,186	226 (7.1)	117 (51.8)	107 (47.3)	2 (0.9)	Reinert et al. (17)
C	Adults	IPD, RTI	2001-2002	35	405	51 (12.6)	24 (47.1)	26 (51.0)	1 (2.0)	Felmingham et al. (5)
Total					4,281	399 (9.3)	226 (56.6)	168 (46.6)	5 (1.3)

^aAbbreviations: RTI, respiratory tract infections; IPD, invasive pneumococcal disease; Ery^r, erythromycin resistant.

^bThe denominator was the number of macrolide-resistant strains.

^cUpdated data.

The genotypes and phenotypes of these strains are presented in Table 2; and their serotypes, mutations in 23S rRNA, and changes in ribosomal proteins L4 and L22 are presented in Table 3.

TABLE 2.

Patient demographic data and MICs for seven macrolide-resistant strains isolated in Germany, 1992 to 2000^a

Strains	Yr of isolation	German town	Age	Source	Macrolide resistance:		MIC (μg/ml)
					GT	PT	ERY	ROX	AZM	CLA	CLI	JOS	SPI	TEL	Q-D	PEN	CTX	LEV	TET	SXT
NRZ 288	1998	Chemnitz	5 yr	Blood	erm(B)−, mef(A)−	cMLSB	≥32	≥32	≥32	≥32	8	16	≥32	0.25	1	0.016	0.016	1	0.5	0.06/1.2
NRZ 796	2000	Kassel	3 mo	Blood	erm(B)−, mef(A)−	iMLSB	8	8	8	4	0.25	8	8	0.03	1	0.016	0.016	0.5	0.5	0.06/1.2
PS 2938	1999	Dillingen (Saar)	36 yr	Pleural puncture	erm(B)−, mef(A)−	cMLSB	≥32	≥32	≥32	≥32	≥32	≥32	≥32	0.06	1	0.25	0.06	2	32	0.25/4.75
PS 2909	1999	Munich	33 yr	BAL	erm(B)−, mef(A)−	M	16	16	32	16	0.06	4	8	0.06	1	8	4	1	32	0.25/4.75
PW 555	2000	Weingarten	28 yr	CSF	erm(B)−, mef(A)−	iMLSB	2	4	4	2	0.125	1	2	0.03	1	0.016	0.016	1	0.5	0.25/4.75
NRZ 462	1999	Heidelberg	8 yr	CSF	erm(B)+, mef(A)−	iMLSB	≥32	≥32	≥32	≥32	0.5	≥32	≥32	0.125	1	0.016	0.016	0.5	32	0.5/9.5
NRZ 810	2000	Frankfurt	5 yr	CSF	erm(B)−, mef(A)+	cMLSB	≥32	≥32	≥32	≥32	≥32	≥32	≥32	0.25	1	1	1	0.5	32	1/19

^aAbbreviations: GT, genotype; PT, phenotype; erm(B)−, erm(B) negative; erm(B)+, erm(B) positive; mef(A)−, mef(A) negative; mef(A)+, mef(A) positive; M phenotype, macrolide resistance; ERY, erythromycin A; ROX, roxithromycin; AZM, azithromycin; CLA, clarithromycin; CLI, clindamycin; JOS, josamycin; SPI, spiramycin; TEL, telithromycin; Q-D, quinupristin-dalfopristin; PEN, penicillin G; CTX, cefotaxime; LEV, levofloxacin; TET, tetracycline; SXT, sulfamethoxazole-trimethoprim; BAL, bronchoalveolar lavage; CSF, cerebrospinal fluid.

TABLE 3.

Mutations in 23S rRNA and changes in ribosomal proteins L4 and L22 in macrolide-resistant pneumococcal strains

Strain	Mutation in 23S rRNA genea	Change in protein L22b	Acquired macrolide resistance gene	Changes in protein L4b	Multilocus sequence typec	Serotype
NRZ 288	A138G, T389C, A2060Gd (A2058G)	WTe	None	S20N, A197V	392	17F
NRZ 796	A138G, A682G, A2061Gd (A2059G)	WT	None	S20N	New type	7F
PS 2938	G211A, T389C, A2937Gf	WT	None	R95H	New type	15A
PS 2909	T389C	C117T	None	Multiple	663	19F
PW 555	A2937G,f G2939T,f C3096Tg	WT	None	WT	62	11A
NRZ 462	A138G, A376T, T389C, G2135T, G2939Af	WT	erm(B)	WT	43	19F
NRZ 810	A138G, T139C, T389C, A2939Gf	WT	mef(A)	WT	New type	23F

^aThe nucleotide sequences of 23S rRNA are those of S. pneumoniae, and the corresponding Escherichia coli numbering is given in parentheses.

^bThe positions of L4 and L22 are according to the sequence of S. pneumoniae R6 (GenBank accession number AF126059).

^cThe following strains had the indicated multilocus sequence allele numbers: NRZ 288, 7-5-1-1-6-31-14; NRZ 796, 8-9-1-1-6-28-17; PS 2938, 10-13-14 variant-16-6-1-8; PS 2909, 7-13-8-6-6-6-99; PW 555, 2-5-29-12-16-3-14; NRZ 462, 1-10-4-1-9-3-8; NRZ 810, 15-29-4-21-30-1-8.

^dThe mutation was seen in four of four alleles.

^eWT, wild type.

^fMutations outside the 23S rRNA. The G2939A mutation is identical to the sequence of serotype 4 strain (tigr. org) but different from the sequence of S. pneumoniae R6. The mutations are found in the internal transcribed spacer between the 3′ end of the 23S rRNA and the 5′ start of the 5S rRNA, 35 and 37 bases, respectively, relative to the 3′ end of the 23S rRNA.

^gMutation outside the 23S rRNA. The mutation is found in the internal transcribed spacer between the 3′ end of the 5S rRNA and the 5′ start of the t-RNA-valine 3; 94 bases relative to the 3′ end of the 23S rRNA.

Strain NRZ 288 exhibited three point mutations in the 23S rRNA gene and two amino acid alterations in ribosomal protein L4. The strain showed a cMLS_B phenotype, high levels of resistance to all macrolides, and a relatively low level of resistance to clindamycin compared to the resistance of the erm(B) strains with constitutive resistance (MIC for NRZ 288, 8 μg/ml). Strain NRZ 796 also showed three mutations in the 23S rRNA in combination with a change in protein L4 (S20N). Of note, this strain showed an inducible phenotype, and another efflux mechanism like the new efflux mechanism recently described in Streptococcus pyogenes may be present (6). Strain PS 2938 showed two mutations (G211A and T389C) in the 23S rRNA and one alteration (R95H) in ribosomal protein L4. This strain exhibited the cMLS_B phenotype and high-level resistance to all macrolides and clindamycin. Strain PS 2909 showed a single mutation (T389C) in the 23S rRNA gene. Among the strains in the collection, this was the only isolate showing a change (C117T) in protein L22. Multiple changes were found in ribosomal protein L4. Strain PW 555 showed mutations only downward to the 3′ end of the 23S rRNA. Strain NRZ 462 expressed the erm(B) macrolide resistance gene in repeated PCR assays and showed high-level resistance to all macrolides. Interestingly, this strain showed the iMLS_B phenotype. Sequencing of the 23S rRNA gene of strain NRZ 462 showed four mutations (A138G, A376T, T389C, and G2135T). Strain NRZ 810 showed three mutations (A138G, T139C, and T389C) in the 23S rRNA gene. This strain was mef(A) positive but showed high-level resistance to both macrolides and clindamycin (MICs, ≥32 μg/ml).

All strains were isolated from different geographic areas in Germany and from individuals in various age groups. The strains were genetically unrelated and, with one exception, represented different serotypes (Table 3). MLST demonstrated that all strains belonged to different sequence types. Interestingly, three of the seven strains belonged to new multilocus sequence types not reported in the MLST database to date (Table 3).

Most information available today is based on in vitro selection studies showing that certain structures involving domains II and V of 23S rRNA participate in the binding of macrolides (1-3). In clinical isolates most of the point mutations were identical to those found in in vitro selection studies, but new mutations were also observed (11). The A2058G and A2058U substitutions confer the highest level of MLS_B resistance, with the MICs of erythromycin and related macrolides being between 32 and >200 μg/ml (1, 14, 22, 24).

A clinical isolate that had an A2062C mutation and that exhibited high-level resistance to spiramycin and streptogramin B has recently been described by Depardieu and Courvalin (3). Other mutations reported to date include C2610 and C2611 (which confer low-level macrolide resistance) (1, 22).

Mutations in the L4 protein occur in a region of 32 amino acids and interfere with the binding of the protein to rRNA (8, 14, 16). A 3-amino-acid alteration of GTG to TPS at position 69 was also seen in strain PS 2909 in the present investigation.

Two isolates in the present study showed the S20N alteration in the L4 protein. The latter has been described in only one other clinical serotype 18F strain, which was isolated in 1988 in France and which exhibited the S20N alteration in the L4 protein in combination with an A2062C mutation in the 23S rRNA. However, the investigators demonstrated that the S20N mutation was not involved in the expression of macrolide resistance in this strain (3).

In the present study only one clinical isolate (PS 2909) showed a mutation in the L22 protein. Mutations in the L22 protein are usually located in a β-hairpin extension at the C terminus of the protein (1, 11). A prvevious study (13) reported the emergence of a mutant with an L22 protein mutation during treatment with azithromycin in a patient with fatal pneumococcal pneumonia.

In summary, the seven macrolide-resistant strains isolated in Germany showed some of the well-known mutations in the 23S rRNA and L4 ribosomal protein, but they also exhibited new mutations and new combinations of L4 and 23S rRNA mutations. A new alteration in the L22 protein which has not been reported before is described. Two strains showed a genotype that did not match the phenotype, which has been described in pneumococci before (10). In addition, the relevance of the newly described mutations for the development of macrolide resistance in pneumococci needs to be confirmed by future experiments.