Telithromycin-Nonsusceptible Clinical Isolates of Streptococcus pneumoniae from Europe

Abstract

Telithromycin-nonsusceptible pneumococcal clinical isolates (n = 17) were analyzed for their antimicrobial susceptibility, macrolide resistance mechanisms, and genetic relatedness. All strains showed the erm(B) genotype and showed a wide range of combinations of macrolide resistance mechanisms. The predominant clone (n = 7) was serotype 14, sequence type 143.

Streptococcus pneumoniae continues to be a significant cause of morbidity and mortality in humans (14). The worldwide increase in antibiotic resistance in this species has become a serious problem within the last 20 years (2). Macrolide resistance in S. pneumoniae is usually caused by methylation of A2058 of the 23S rRNA mediated by the erm(B) gene or by an efflux mechanism mediated by the mef gene (13). In addition, other mechanisms of macrolide resistance have been described (4, 20, 21). Ketolides form a new class of semisynthetic agents derived from erythromycin A designed to overcome erythromycin A resistance in S. pneumoniae. Ketolide compounds inhibit bacterial protein synthesis by interacting with the peptidyltransferase site of the 50S ribosomal subunit and interact closely with domains II, at A752, and V, at A2058 and A2059, of the 23S rRNA (1). Ketolides show good activity against gram-positive bacteria responsible for respiratory tract infections, including penicillin G- and erythromycin A-resistant S. pneumoniae (11). The ketolide-resistant pneumococci described usually show the erm(B) genotype, and some, but not all, strains show additional alterations in the L4 ribosomal protein (1, 16). The scope of the present paper was to investigate the occurrence of these mechanisms of resistance in a collection of 17 telithromycin-nonsusceptible pneumococcal isolates from Europe.

(This work was presented in part at the 45th Interscience Conference on Antimicrobial Agents and Chemotherapy, 16 to 19 December 2005, Washington, D.C. [1a]).

MIC testing was performed using the broth microdilution method recommended by the Clinical Laboratory Standards Institute (formerly the National Committee for Clinical Laboratory Standards) (5). PCR of macrolide resistance genes was performed as described previously (17). Sequencing of the 23S rRNA and the genes encoding the ribosomal proteins L4 (rplD) and L22 (rplV) was performed as reported earlier by Tait-Kamradt et al. (4, 21) and Walsh et al. (22). The erm(B) gene and the erm(B) promoter region were amplified by PCR using the primers published by Walsh et al. (22). Pneumococcal strains were serotyped by Neufeld's Quellung reaction using type and factor sera provided by the Statens Serum Institut, Copenhagen, Denmark. Multilocus sequence typing (MLST) was carried out as described by Enright and Spratt (8). Phylogenetic analysis was performed as described before (18). Nucleotide sequences of the upstream regions of the erm gene were aligned using the program ClustalW. Sequence types (STs) of the telithromycin-nonsusceptible isolates were compared with those of all isolates available in the MLST database.

Demographic data and data on antibiotic resistance are presented in Table 1. Isolates were collected between 2001 and 2005 from patients with invasive and noninvasive pneumococcal disease, mostly in France (10 out of 17 strains). Telithromycin MICs ranged between 2 and 8 μg/ml. All strains were multiply resistant and besides being telithromycin nonsusceptible showed reduced susceptibility to β-lactams and resistance to macrolides, lincosamides, and tetracycline. One strain additionally showed high-level fluoroquinolone resistance.

TABLE 1.

Characteristics, MICs, and resistance phenotypes of 17 telithromycin-nonsusceptible S. pneumoniae strains from Europe

Strain	Country	Source	Yr of isolation	Patient age (yr)	MIC (μg/ml) ofa:									Phenotypeb
					TEL	PEN	AMX	CTX	CLR	CLI	TET	LVX	SXT
PW 778	Belgium	Sputum	2000	67	8	2	1	2	≥32	≥32	≥32	1	≥8/152	PMLTC
PW 521	France	BALc	2001	44	8	4	2	2	≥32	≥32	≥32	1	4/76	PMLTC
PW 868	France	BAL	2001	59	2	4	≥4	2	≥32	≥32	≥32	1	0.25/4.75	PMLT
PW 899	France	BAL	2001	64	4	4	4	2	≥32	≥32	≥32	1	2/38	PMLTC
PW 1760	France	Sinus	2001	31	4	2	2	2	≥32	≥32	≥32	0.5	1/19	PMLTc
PW 1765	France	BAL	2001	57	8	2	2	2	≥32	≥32	≥32	1	0.25/4.75	PMLT
PW 1791	France	Sputum	2002	53	8	1	0.5	1	≥32	≥32	≥32	1	1/19	pMLTc
PW 1840	France	Blood	2002	59	4	2	2	2	≥32	≥32	≥32	1	≥8/152	PMLTC
PW 1981	France	Sputum	2002	69	4	2	2	2	≥32	≥32	≥32	2	4/76	PMLTC
PW 1976	France	Sputum	2002	ND	2	2	2	≥2	≥32	≥32	≥32	2	8/152	PMLTC
PW 1996	France	Blood	2002	81	2	4	2	2	≥32	≥32	≥32	1	1/19	PMLTc
PN 302	Germany	Blood	2003	34	8	0.5	0.25	0.125	≥32	≥32	16	1	1/19	pMLTc
PS 5646	Germany	BAL	2005	58	8	2	2	1	≥32	≥32	≥32	1	4/76	PMLTC
PW 712	Spain	Sputum	2001	56	4	2	4	≥2	≥32	≥32	≥32	2	8/152	PMLTC
PW 715	Spain	Sputum	2001	70	2	2	2	1	≥32	≥32	≥32	1	0.25/4.75	PMLT
PW 735	Spain	Sputum	2001	75	4	2	2	≥2	≥32	≥32	≥32	≥32	≥8/152	PMLTCF
PW 1420	Switzerland	BAL	2002	30	8	1	0.5	1	≥32	≥32	≥32	2	1/19	pMLTc

^aBreakpoints (intermediate and resistant, respectively) according to the Clinical Laboratory Standards Institute: telithromycin (TEL), 2 and ≥4 μg/ml; penicillin G (PEN), 0.1 to 1 and ≥2 μg/ml; amoxicillin (AMX), 4 and ≥8 μg/ml; cefotaxime (CTX [nonmeningitis]), 2 and ≥4 μg/ml; clarithromycin (CLR), 0.5 μg/ml and ≥1 μg/ml; clindamycin (CLI), 0.5 and ≥1 μg/ml; tetracycline (TET), 4 and ≥8 μg/ml; levofloxacin (LVX), 4 and ≥8 μg/ml; trimethoprim-sulfamethoxazole (SXT), 1/19 to 2/38 μg/ml and ≥4/76 μg/ml. All isolates were susceptible to vancomycin (MIC of ≤1 μg/ml). ND, no data.

^bResistance phenotypes: p, penicillin G intermediate; P, penicillin G resistant; M, macrolide resistant; L, lincosamide resistant; T, tetracycline resistant; c, cotrimoxazole intermediate; C, cotrimoxazole resistant; F, fluoroquinolone resistant.

^cBAL, bronchoalveolar lavage.

Genotypic and phenotypic characteristics and results from MLST are presented in Table 2. The predominant serotype was type 14 (10 of 17 isolates). The predominant clone was ST 143 (7 of 17 isolates), which was found in France (n = 6) and Germany (n = 1). Telithromycin resistance has also spread to the Spain^23F-1 clone (ST 81; n = 1) and its 19A variant. All strains showed the erm(B) genotype and the cMLS_B phenotype and were negative for the mef gene. The isolates had multiple alterations in 23S rRNA. Analysis of the genes encoding ribosomal protein L22 showed a wild type for all isolates. Of note, in nine isolates an S20N alteration in ribosomal protein L4 was observed. Isolates belonging to one clone showed identical or quite similar resistance mechanisms. Of note, three clones were described for the first time in the present investigation (strain PW 778, ST 1569; PW 735, ST 1558; and PW 868, ST 2010) (Table 3).

TABLE 2.

Genotypic and phenotypic characterization and macrolide resistance mechanisms of 17 telithromycin-nonsusceptible S. pneumoniae strains from Europe

Strain	Serotype	STa	Genotype	Phenotype	Alteration(s) in:
					23S rRNAb	Ribosomal protein		Erm(B)
						L22	L4
Pn 302	15A	63	erm(B)	cMLSB	T107G, A1513T, T1514A, A1530T, T1531A, A1535G, A1745T	WTc	S20N	I75T, H118R
PW 712	19A	81	erm(B)	cMLSB	C150T, A1745T, C2216T	WT	S20N	N100S
PW 715	19A	81	erm(B)	cMLSB	C150T, A1745T, C2216T	WT	S20N	N100S
PW 1981	23F	81	erm(B)	cMLSB	C150T, A1745T	WT	S20N	I75T, H118R
PW 1976	14	143	erm(B)	cMLSB	A138G, A260G, A1745T	WT	WT	N100S
PW 1996	14	143	erm(B)	cMLSB	A138G, A260G, A1745T	WT	WT	E58A, I75T, N100S, H118R
PW 521	14	143	erm(B)	cMLSB	A138G, A260G, A1745T	WT	WT	N100S
PW 899	14	143	erm(B)	cMLSB	A138G, A260G, A1745T	WT	WT	N100S
PW 1760	14	143	erm(B)	cMLSB	C150T	WT	S20N	I75T, H118R
PW 1765	14	143	erm(B)	cMLSB	A138G, A260G, A1745T	WT	WT	N100S
PS 5646	14	143	erm(B)	cMLSB	T107G; A138G; A260G, C389T, A1745T	WT	WT	I75T, H118R
PW 1840	14	156	erm(B)	cMLSB	A138G; A260G, A1745T	WT	WT	I75T, N100S, H118R
PW 1420	14	276	erm(B)	cMLSB	C150T	WT	S20N	I75T, N100S, H118R, V171A
PW 1791	19A	276	erm(B)	cMLSB	C150T	WT	S20N	I75T, N100S, H118R, V171A
PW 735	19A	1558	erm(B)	cMLSB	C150T, A1745T, C2216T	WT	S20N	N100S
PW 778	19A	1569	erm(B)	cMLSB	A138G, A260G, A1745T	WT	S20N	I75T, N100S, H118R
PW 868	14	2010	erm(B)	cMLSB	A138G, A260G, A1745T	WT	WT	N100S

^aThe STs had the following alleles: ST 63, 2-5-36-12-17-21-14; ST81, 4-4-2-4-4-1-1; ST143, 7-5-10-18-6-8-1; ST 1569, 8-11-10-1-6-8-1; ST276, 2-19-2-17-6-22-14; ST 1558, 7-13-8-12-9-1-1; ST 2010, 7-5-140-18-6-8-215.

^bMutations were found in all four copies of the 23S rRNA.

^cWT, wild type.

TABLE 3.

Genetic relatedness of telithromycin-nonsusceptible S. pneumoniae strains from Europe

Strain(s) in present study	Sequence type	Country (present study)	Described serotype(s)a	Country or countries in other reportsb	Time
PN 302	63	Germany	15A	Portugal, Spain, Italy, Sweden	1992-1998
PW 712, PW 715, PW 1981	81	France, Spain	23F, 19A, 19F, 14	Worldwide	Since 1980s
PW 1976, PW 1996, PW 521, PW 899, PW 1760, PW 1765, PS 5646	143	France, Germany	14	Poland, Hungary, United States	1994-2002
PW 778	1569c	Belgium	14, 9V, 3, 9A	United Kingdom, Germany, France, Portugal	1997-2003
PW 1420, PW 1791	276	Switzerland, France	19A	The Netherlands	1997
PW 735	1558c	Spain	19A		2001
PW 1840	156	France	14	Worldwide	1988-2003
PW 868	2010c	France	14		2001

^aSerotypes observed in the present study are given in boldface.

^bAs available in the MLST database (www.mlst.net) by April 2006.

^cFirst described in the present study.

The DNA sequences of the erm(B) upstream region of the telithromycin-nonsusceptible clinical isolates were compared to the upstream sequence region of erm(B) found in Tn1545 (accession no. X52632) (15), erm(B) (accession no. AJ972605) (7), pAM77 (accession no. K00551) (12), and Tn917-like LP-1 (accession no. AB111455) (15) (Fig. 1). In the region upstream of the erm(B) start codon, mutations were found at positions −5, −6, −7, −28, −33, and −90 [upstream of the ATG start codon of erm(B)]. These mutations were homologous to mutations found at positions 320, 319, 318, 297, and 292 in Tn1545, respectively. In the open reading frame of erm(B), the mutation E58A was observed in 1 isolate, I75T was observed in 9 isolates, N100S was observed in 13 isolates, H118R was observed in 9 isolates, and V171A was observed in 2 isolates. However, some of the mutations described in the present study are likely not involved in resistance and are outside of regions that are known to interact with the ribosome. Therefore, these mutations may simply represent heterogeneity in the erm(B) gene and its promoter region.

FIG. 1.

Schematic representation of alterations of the regulatory sequences of erm(B) genes from isolates of S. sanguis (pAM77); S. pneumoniae containing Tn1545; and the clinical isolates PW 1840, PW 1981, PW 712, PW 715, and PW 735. The other 12 isolates show the Tn1545-like organization (27 amino acids). SD1 and SD2 (Shine-Dalgarno sites) represent the ribosomal binding sites for the control peptide and the erm(B) gene. −10 and −35 represent promoter regions. The numbers in the boxes represent the length of the open reading frame of the leader peptide. The nucleotides in boldface given at the end of the leader peptides represent insertions as compared to pAM77. The resulting sequence up to the next stop codon is given.

To our knowledge, the present report describes the largest collection of telithromycin-nonsusceptible clinical isolates characterized for their resistance mechanisms and their epidemiological background to date. The global surveillance project PROTEKT (Prospective Resistant Organism Tracking and Epidemiology for the Ketolide Telithromycin) reported that only 0.2% of S. pneumoniae isolates collected between 1999 and 2003 were resistant to telithromycin and that all showed the cMLS_B phenotype (10). Concordantly, the present study and other studies on telithromycin resistance in S. pneumoniae report on resistance mainly in erm(B)-positive strains. In addition, a recent study showed that telithromycin can be removed from Streptococcus pyogenes by efflux, related to the presence of the mef gene (3). Faccone and coworkers have reported on a high-level telithromycin-resistant isolate from Argentina. The isolate was mef(E) positive, but erm(B) and erm(A) negative (9). In addition, substantial levels of telithromycin resistance were recently detected in a collection of soil isolates, possibly due to inactivation of the compound (6). Analysis of the clonal relatedness of the present collection of strains showed that telithromycin resistance was predominantly found among two clones, ST 81 and ST 143. ST 81 (Spain^23F-1 clone and its serotype 19A variant) is known to be responsible for the worldwide spread of resistance among pneumococci (19).

In summary, the present study demonstrates that mutations in the erm(B) gene, its promoter region, ribosomal protein L4, and 23S rRNA may be associated with resistance to telithromycin in clinical isolates of S. pneumoniae. Although the incidence of telithromycin resistance in pneumococci remains rare worldwide, the spread of telithromycin resistance to multidrug resistance clones with worldwide distribution is a worrisome finding of the present study.

Nucleotide sequence accession numbers.

The GenBank accession numbers of the ermB (upstream region and open reading frame) sequences of this study are DQ855638 (PW1791 and PW1420), DQ855639 (PW899, PW1765, PW1976, and PW521), DQ855640 (PW1840), DQ855641 (PW712 and PW715), DQ855642 (PW1760), DQ855643 (PW735), DQ855644 (PW788), DQ855645 (PW868), DQ855646 (Pn302), DQ855647 (PW1981), DQ855648 (PS5646), and DQ855649 (PW1996).