Fluoroquinolone Resistance Associated with Target Mutations and Active Efflux in Oropharyngeal Colonizing Isolates of Viridans Group Streptococci

Abstract

Oropharyngeal samples from 60 hospitalized patients (30 fluoroquinolone [FQ]-treated and 30 non-FQ-treated patients) and 30 untreated nonhospitalized healthy control subjects yielded 20 isolates of viridans group streptococci with reduced susceptibility to FQ, mostly from the hospitalized patients. An efflux phenotype was commonly encountered, expressed either alone or with topoisomerase mutations. Interspecies transfer of the efflux phenotype was demonstrated via transformation of Streptococcus pneumoniae R6 with DNA from S. mitis and S. oralis.

Viridans group streptococci (VGS) are involved in two major types of infections, bacteremia in neutropenic patients and endocarditis. Fluoroquinolone (FQ) resistance in gram-positive bacteria, and especially in pneumococci and VGS, has been associated with mutations in both FQ targets, topoisomerase IV and DNA gyrase (8, 10, 11, 13, 16, 18). Low-level FQ resistance is essentially related to amino acid substitutions in the quinolone resistance-determining region (QRDR) of either the ParC subunit of topoisomerase IV or the GyrA subunit of the DNA gyrase, depending on the FQ used as selector (16, 18, 20). Such substitutions in topoisomerase IV and DNA gyrase contribute together to high-level resistance (11, 18, 20). An additional mechanism of FQ resistance is the increase in active FQ efflux which, in Streptococcus pneumoniae, has been shown so far to be encoded only by pmrA, a gene related to norA (7, 14, 21). Considering the existence of in vivo exchanges of chromosomal DNA between VGS and pneumococci and the demonstration of in vitro exchanges of mutated FQ targets via transformation (8, 10), it is likely that the emergence of FQ resistance in one or another streptococcal species would boost the in vivo spread of FQ resistance. For these reasons and since FQs are widely used for the treatment of various infections, we examined whether oropharyngeal colonizing VGS isolated from different patient populations, treated with FQ or not, harbored FQ resistance. Since these species are not supposed to be targeted by FQ treatment, FQ resistance in these species could serve as an indicator of the selective pressure exerted by these antibiotics.

Streptococcal strains used in this study were S. mitis 103335^T (SM), S. oralis 102922^T (SO), and S. sanguis 55128^T (SS) (all from Institut Pasteur, Paris, France). The clinical VGS strains were isolated between December 1998 and March 1999 from oropharyngeal samples (one sample per patient or control). Oropharyngeal samples were obtained from three groups of 30 individuals each: (i) hospitalized patients who received FQ treatment (ciprofloxacin, ofloxacin, or pefloxacin) for a mean duration of 12 ± 3 days, (ii) patients hospitalized during the same period in the same ward but in different rooms who had not received FQ for at least 5 months, and (iii) nonhospitalized healthy controls not exposed to FQs. VGS were identified by using the Rapid ID32 Strep system (bioMérieux, Marcy-l'Etoile, France) and, when necessary, by nucleotide sequencing of an internal fragment of sodA (19). Oropharyngeal samples were suspended in distilled sterile water and spread on Columbia agar (Difco, Paris, France) supplemented with 4% horse blood and colimycin (10 μg/ml) with or without ciprofloxacin (3 μg/ml). Plates were incubated at 37°C in the presence of 5% CO₂ for 48 h. To avoid false-positive results due to the possible selection of spontaneous mutants, samples which yielded less than 20 colonies on the FQ-containing plates were not considered.

Inocula of 5 × 10³ to 5 × 10⁴ CFU were spotted on Mueller-Hinton agar plates supplemented with 4% horse blood and containing one of the following antibiotics: sparfloxacin (Rhône-Poulenc Rohrer, Vitry-sur-Seine, France), ciprofloxacin (Bayer Pharma, Puteaux, France), and norfloxacin (Merck Sharp & Dohme-Chibret, Paris, France). MICs were read after 18 h. To detect the presence of an efflux phenotype, MICs were also determined in the presence of reserpine (10 μg/ml) (3, 7).

DNA extraction and PCR experiments were done as previously described (1, 11). Oligonucleotides PNC6 and PNC7 (11) and gyrB376 and gyrB512 (13) were used for the amplification of the QRDRs of gyrA and gyrB, respectively. Oligonucleotides PNC10 and PNC11 (11) were used to amplify the QRDR of parC, except for five strains of S. mitis, for which PARC56 (5′-GACAAGAGCTACCGTAAGTC-3′) and PARC117 (5′-GACAAACGIGCCTCIGTATAACG-3′) were used. Amplification of the corresponding parE region was done either with oligonucleotides PNC15 and PNC16 (11) or with parE398 and parE483 (8). Direct sequencing of DNA fragments, with the oligonucleotides used for amplification, was performed with the dRhodamine BigDye Terminator sequencing kit (Perkin-Elmer, Applied Biosystems Division). The nucleotide sequences were analyzed with Perkin-Elmer software (Sequence Analysis and Sequence Navigator). Multiple alignments of sequences were carried out using the CLUSTAL X program.

Transformation experiments were carried out as previously described (10, 11) using DNA from viridans streptococci and the wild-type S. pneumoniae strain R6 as a recipient (15). Chromosomal DNA of viridans streptococci was added to the mixture to a concentration of 0.5 to 1 μg/ml, and transformants were selected on blood agar plates containing 4 μg of ciprofloxacin per ml.

Twenty VGS isolates from 20 samples (Table 1) which grew on ciprofloxacin-containing agar at a concentration of 3 μg/ml showed ciprofloxacin MICs of ≥4 μg/ml and were considered of reduced susceptibility to the FQs tested when compared to the respective reference strains. Most of the FQ-resistant VGS were isolated from the oropharynges of hospitalized patients, whether or not the patients received FQs for the treatment of infections other than of the respiratory tract (Table 1 and 2). In contrast, FQ-resistant VGS were isolated from only 3 of 30 healthy controls.

TABLE 1.

Repartition of FQ-resistant VGS among oropharyngeal samples

Parameter measured	Source
	Hospitalized patients		Healthy controls	Total
	FQ treateda	Non-FQ treated
No. of samplesb	30	30	30	90
No. of samples with FQ-resistant VGSc	9	8	3	20

^aPatients were treated for infections other than those of the upper respiratory tract. 

^bOne oropharyngeal sample per patient or healthy control. 

^cAnalysis by χ² test comparing the number of samples containing FQ-resistant VGS (for which ciprofloxacin MICs were ≥4 μg/ml) from hospitalized patients and healthy controls showed a statistical difference (P = 0.0486). 

TABLE 2.

MICs of fluoroquinolones for VGS and mutations in topoisomerases

Strain	Origina	MIC (μg/ml) ofb:			Amino acid (nucleotides) at indicated positionc
					GyrA		GyrB 494	ParC		ParE
		NOR	CIP	SPX	81	85		79	83	435	474
SM103335T		16 (4)	2 (2)	0.5	S (TCC)	E (GAA)	S (TCA)	S (AGT)	D (GAT)	D (GAT)	E (GAG)
SMB8	HQ	32 (2)	4 (2)	1	—	—	ND	—	—	— (GAC)	— (GAA)
SMB9	HQ	128 (8)	16 (8)	1	—	—	—	—	—	ND	ND
SMB11	H	32 (2)	4 (1)	1	— (TCA)	—	ND	—	—	ND	ND
SMB12	C	32 (2)	4 (1)	1	— (TCA)	—	ND	—	—	ND	ND
SMB13	C	64 (2)	16 (4)	2	—	—	ND	I (ATC)	—	—	—
SMB14	H	128 (4)	64 (4)	2	F (TTC)	—	—	—	—	— (GAC)	K (AAG)
SO102922T		32 (4)	2 (2)	0.5	S (TCC)	E (GAA)	T (ACT)	S (TCT)	D (GAT)	D (GAT)	E (GAA)
SOB3	H	64 (4)	4 (2)	0.5	—	—	S (TCA)	R (AGA)	—	—	— (GAG)
SOB4	C	64 (8)	4 (2)	0.5	— (TCG)	— (GAG)	—	— (AGT)	—	ND	ND
SOB5	HQ	128 (16)	16 (8)	1	—	—	—	—	—	— (GAC)	— (GAG)
SOB6	HQ	64 (4)	8 (2)	1	—	—	S (TCA)	—	N (AAT)	— (GAC)	—
SOB7	H	128 (4)	8 (2)	1	—	—	—	—	H (CAT)	—	—
SOB8	HQ	128 (4)	8 (2)	1	—	—	—	Y (TAT)	—	ND	ND
SOB9	HQ	128 (4)	16 (4)	1	—	—	ND	F (TTT)	—	ND	ND
SOB14	HQ	256 (4)	16 (4)	2	—	—	—	Y (TAT)	—	— (GAC)	—
SOB10	HQ	256 (8)	64 (4)	16	Y (TAC)	—	S (TCA)	— (AGT)	N (AAT)	ND	ND
SOB11	HQ	256 (4)	128 (2)	64	— (TCA)	G (GGA)	—	F (TTT)	—	—	— (GAG)
SOB12	H	256 (2)	128 (2)	32	Y (TAC)	—	—	F (TTT)	—	— (GAC)	—
SOB13	H	256 (2)	128 (2)	64	Y (TAC)	—	—	— (AGT)	N (AAT)	— (GAC)	—
SS55128T		32 (4)	2 (2)	0.5	S (TCT)	E (GAA)	ND	S (AGC)	D (GAT)	ND	ND
SSB1	H	64 (2)	4 (1)	0.5	— (TCC)	—	ND	— (AGT)	Y (TAT)	ND	ND
SSB2	HQ	128 (16)	16 (8)	0.5	— (TCC)	—	ND	—	—	ND	ND

^aH, hospitalized patient not treated with FQs; HQ, hospitalized patient treated with FQs; C, nonhospitalized, untreated control subject. 

^bNOR, norfloxacin; CIP, ciprofloxacin; SPX, sparfloxacin. Values in parentheses are the MIC fold decreases in the presence of 10 μg of reserpine per ml. 

^cAmino acid position corresponds to that of amino acid numbering of topoisomerase subunits from S. pneumoniae (2, 12, 17). —, no change with respect to type strain; ND, not determined. 

Compared to the reference strain S. mitis SM103335^T, strains SMB8 through -14 showed 2- to 32-fold increases in the MICs of norfloxacin, ciprofloxacin, and sparfloxacin. Compared to the reference strain S. oralis SO102922^T, strains SOB3 to SOB14 showed 2- to 128-fold increases in the MICs of norfloxacin, ciprofloxacin, and sparfloxacin. For the S. sanguis strains, the MICs of the FQs were increased up to eightfold compared to that showed for reference strain S. sanguis SS55128^T.

Among the FQ-resistant S. mitis strains, only SMB13 and SMB14 showed amino acid substitutions in the QRDRs of ParC, GyrA, or ParE (Table 2). SMB13 showed a Ser79Ile substitution in ParC. SMB14 showed no substitution in ParC, but a Ser81Phe substitution in GyrA and a Glu474Lys substitution in ParE were both associated with a 4- to 32-fold increase in the MICs of the FQs tested.

The presence of an active efflux was tested for with the hydrophilic FQs norfloxacin and ciprofloxacin, known to facilitate the detection of this mechanism of resistance (3, 7, 21). The efflux phenotype was considered when the MICs of norfloxacin or ciprofloxacin were decreased at least fourfold in the presence of reserpine. Such a phenotype was observed for strains SMB9, SMB13, and SMB14 and for the type strain SM103335^T. The most noticeable decrease was found for SMB9, which harbored no amino acid substitution in the QRDRs of ParC, GyrA, and GyrB.

A striking observation was an efflux phenotype in 10 of 12 strains of S. oralis, including the type strain SO102922^T (Table 2). This phenotype was characterized by a 4- to 16-fold decrease in the MICs of norfloxacin or ciprofloxacin in the presence of reserpine. The efflux phenotype was particularly obvious in SOB5, for which the MICs of norfloxacin and ciprofloxacin in the presence of reserpine dropped to those observed for the type strain SO102922^T and in which no amino acid substitution was found in the different QRDRs.

In addition, various amino acid substitutions were found at position 81 or 85 in GyrA and at position 79 or 83 in ParC. Compared to the type strain SO102922^T, substitutions in ParC led to a decrease in susceptibility to the FQs tested (strains SOB6 to SOB9 and SOB14), while a higher level of resistance, particularly to sparfloxacin, was observed when substitutions were simultaneously present in ParC and GyrA (strains SOB10 to SOB13).

The S. sanguis strain SSB1 had an Asp83Tyr substitution in ParC, while strain SSB2, with no substitution in the QRDRs of ParC and GyrA, had a pronounced efflux phenotype characterized by an 8- and 16-fold decrease in the MICs of ciprofloxacin and norfloxacin, respectively, in the presence of reserpine.

For the VGS for which nucleotide sequences were obtained (Table 2), no amino acid substitution was found in the QRDR of GyrB, except at position 494 as previously reported (8).

Using total DNA from strains S. mitis SMB9 and S. oralis SOB5, which in the absence of mutations in the ParC and GyrA QRDRs had the most noticeable efflux phenotype, we were able to transform pneumococcal strain R6 to efflux-mediated FQ resistance at frequencies of ca. 3 × 10⁻³ and 3 × 10⁻⁴, respectively. The MICs of norfloxacin and ciprofloxacin for the transformants were 32 and 8 μg/ml, respectively, and were decreased eightfold in the presence of reserpine (Table 3). In contrast, no transformants were obtained using the total DNA from S. sanguis SSB2.

TABLE 3.

Susceptibility to FQs of efflux-mediated resistant transformants of S. pneumoniae strain R6

Strain or transformantb	MIC (μg/ml) ofa:
	NOR	NOR + RES	CIP	CIP + RES	SPX
SMB9	128	16	16	2	1
SOB5	128	8	16	2	1
R6	4	4	1	1	0.25
R6TrSMB9	32	4	8	1	0.25
R6TrSOB5	32	4	8	1	0.25

^aNOR, norfloxacin; CIP, ciprofloxacin; RES, reserpine; SPX, sparfloxacin. 

^bSuperscript indicates the genomic DNA donor strain. Tr, transformant. 

Resistance to FQs in gram-positive bacteria, especially S. pneumoniae, is becoming an increasing problem (4, 9). It seems to result from the frequent use of FQs for the treatment of infections not necessarily restricted to the upper respiratory tract. It was striking that about one-third of the hospitalized patients of this study, whether treated with FQs or not, carried FQ-resistant VGS in their oropharynges, while only 3 of 13 healthy controls was colonized with such strains (Table 1). The prevalence of FQ resistance among VGS may have been underestimated, since a sample was considered positive only if a minimum of 20 colonies with a similar morphology were detected on the FQ-containing plates. In addition, we did not test all colonies for the different FQ resistance phenotypes. Although we have no explanation for the presence of FQ-resistant VGS in patients not treated with FQs, we noted that none of the parC and gyrA sequences from FQ-resistant VGS strains, whether isolated from hospitalized patients or from healthy carriers, were perfectly identical (data not shown). This observation does not favor a clonal spread of FQ resistance among the VGS analyzed in this study. Interestingly, the DNA sequence of the topoisomerases genes, especially those of gyrA and parC, were rather heterogeneous in the S. mitis or the S. oralis group. However, since we sequenced only the QRDRs and an insufficient number of strains (including wild-type strains), it is difficult at this point to conclude if this heterogeneity reflects only the difficulty in species determination or mosaic genes.

Fourteen of the 20 FQ-resistant VGS studied apparently had an active efflux, which in 4 cases was not associated with a change in the determined QRDRs. Efflux was deduced to occur in at least 10 of the 12 FQ-resistant S. oralis isolates and in 3 of the 6 FQ-resistant S. mitis isolates. Compared to the FQ-susceptible type strains, an increase in active efflux was observed in one strain of S. mitis, three strains of S. oralis, and one strain of S. sanguis (Table 2). In a recent study (5), an active efflux was also found in a sample of FQ-resistant VGS isolates, mainly in S. mitis.

Interestingly, using DNA from S. mitis SMB9 and S. oralis SOB5 which expressed the highest efflux-mediated resistance, we could transform S. pneumoniae R6 and obtain the efflux phenotype. This is reminiscent of the transfer via transformation of the efflux phenotype from clinical isolates of S. pneumoniae to S. pneumoniae strain R6 (7, 20, 21). Since the level of efflux-mediated FQ resistance in R6 transformants could be reduced to that of wild-type strain R6 in the presence of reserpine, it therefore resembles the resistance mechanism associated with the pmrA efflux pump described for S. pneumoniae (7).

Mutations in topoisomerase IV, alone or associated with DNA gyrase mutations, were predominantly found in S. oralis. This was the case for 10 of 12 S. oralis strains studied, all of which were isolated from hospitalized patients. For nine of these strains (SOB6 to SOB14), the MICs of norfloxacin and ciprofloxacin did not decrease to the level of that for the control strain, SOB102922, in the presence of reserpine, as was expected considering the topoisomerase mutations. Among the other substitutions described in this study, Glu85Gly in GyrA, as well as Asp83His and Asp83Tyr in ParC, was not previously described in VGS (5, 8, 10). In S. mitis SMB14, the unusual Glu474Lys substitution in ParE combined with the Ser81Phe substitution in GyrA was associated with high-level resistance to all FQs. This particular substitution is adjacent to Asn473 and equivalent to Asn470 in GrlB of S. aureus, a position at which an Asn470Asp substitution was previously described (6). This single substitution in S. aureus was associated with an eightfold increase in the MIC of norfloxacin and a twofold increase in the MICs of ciprofloxacin and sparfloxacin. This was less than the apparent increase in MICs observed for S. mitis SMB14, suggesting that the two substitutions (Ser81Phe and Glu474Lys) combined had an enhanced effect.

In conclusion, the observation of colonizing FQ-resistant VGS in one third of an FQ-treated group of patients, and especially of strains with mutated topoisomerases, reemphasizes the role of these species as a reservoir of resistance genes and therefore the risk of diffusion, through horizontal exchange, of FQ resistance determinants between VGS and S. pneumoniae (8, 10), including now those of active efflux.

Nucleotide sequence accession numbers.

The nucleotide sequences reported in this paper have been assigned the following GenBank accession numbers: AF246721 to AF246740 (parC regions), AF246741 to AF246760 (gyrA regions), AF246761 to AF246774 (gyrB regions), and AF246775 to AF246785 (parE regions).