1 Antimicrobial Research Laboratory, National Public Health Institute, PO Box 57, 20521 Turku; 2 Department of Ophthalmology, Turku City Hospital, Turku; 3 Department of Ophthalmology, Turku University Central Hospital, Turku, Finland
Received 19 December 2002; returned 23 January 2003; revised 5 July 2003; accepted 9 July 2003
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Abstract |
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Methods: In vitro susceptibilities of 16 antimicrobials were studied for 161 viridans streptococci (on average 5.8 isolates per person) from the normal flora of 28 elderly persons. Resistance mechanisms of erythromycin-resistant isolates were studied by the double disc test and PCR.
Results: In all, 16.8% of the isolates were non-susceptible (MIC 0.25 mg/L) to penicillin, but none showed high-level resistance (MIC
4 mg/L). Resistance to erythromycin, tetracycline, quinupristin/dalfopristin, levofloxacin and moxifloxacin was found in 22.4, 27.3, 13.0, 1.9 and 1.9% of the isolates, respectively. Combined resistance to erythromycin and tetracycline was found in 13.0% of the isolates. Erythromycin-resistant isolates were isolated from 57% of the study persons. Of the erythromycin-resistant isolates 80.6% were of the M phenotype and 19.4% were of the macrolidelincosamidestreptogramin B (MLSB) phenotype (one isolate with constitutive and six with inducible expression). Isolates with the M phenotype were the least susceptible to telithromycin, a new ketolide. The mef(A) gene was found in the isolates with the M phenotype and the erm(B) gene in the isolates with the MLSB phenotype.
Conclusions: The distribution of phenotypes among the viridans streptococci resembles that found in Streptococcus pyogenes, with predominance of the M phenotype. However, the coding gene for the MLSB phenotype, erm(B), is the same in viridans streptococci as in Streptococcus pneumoniae. Viridans group streptococci carrying different resistance traits provide a pool of resistant bacteria that may transfer resistance determinants to more pathogenic organisms.
Keywords: -haemolytic streptococci, erythromycin resistance, commensal flora, elderly persons
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Introduction |
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Resistance to penicillin and macrolides has been documented in viridans streptococcal isolates from clinical specimens in different parts of the world.24,615 Such resistance has also been found in viridans streptococci from normal oral flora of children and adults with and without infection.1722 In Finland, macrolide resistance has increased among S. pneumoniae and S. pyogenes23,24 and has also been found in group C and G ß-haemolytic streptococci.25 Three different resistance mechanisms have been shown to cause macrolide resistance in these streptococcal isolates: target site modification mediated by the erythromycin resistance methylases encoded by the erm(A) or erm(B) genes conferring resistance to macrolide, lincosamide and streptogramin B antibiotics (MLSB phenotype); active drug efflux mediated by the membrane-bound efflux protein encoded by the mef(A) gene conferring resistance to 14- and 15-membered macrolides only (M phenotype); and mutations in the streptococcal 23S rRNA or ribosomal protein genes leading to resistance to macrolide and streptogramin B antibiotics (MS phenotype).2527
In this study we investigated the in vitro activities of different antimicrobial agents against viridans streptococci from commensal flora of elderly persons, an age group among which viridans streptococci have been shown to have clinical importance.13 A problem encountered in normal flora studies is obtaining a representative group of isolates that would reflect the total normal flora of a species or a species group. We collected on average 5.8 viridans streptococcal isolates per study subject from persons without an ongoing infection, and who had not had any antibiotic treatment for at least 3 months preceding sampling, and obtained a unique heterogeneous sample of 161 normal flora isolates. We also studied the resistance mechanisms of macrolide-resistant isolates in order to determine whether the same mechanisms of resistance prevail among viridans and other streptococci in Finland.
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Materials and methods |
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Pharyngeal, nasopharyngeal and conjunctival swabs were taken between November 1999 and February 2001 from 28 persons (one swab from each site of each person) before cataract surgery in the Department of Ophthalmology of Turku University Central Hospital. The age of the study subjects ranged from 49 to 82 years (mean age 72.1 years). Written informed consent was obtained from every subject. The persons admitted to the study had no ongoing infection and had not received any antibiotic treatment for at least the previous 3 months. Permission for the study was obtained from the Ethical Committee of the Turku Central Hospital District and Turku University.
The conjunctival and nasopharyngeal swabs were inoculated directly onto blood agar and heated blood agar (chocolate agar) prepared with Columbia agar base (Oxoid, Basingstoke, UK). The pharyngeal swabs were transported in charcoal medium, diluted in brainheart infusion broth within 2 h of collection, and inoculated from four serial dilutions on blood and chocolate agars with and without 5 mg/L of gentamicin and on blood agar with 8% thallium acetate. The plates were incubated up to 42 h at 37°C in an atmosphere of 5% CO2. The isolates were selected from the plates on the basis of -haemolysis, colony morphology, Gram staining and the optochin test. Optochin resistance (zone of inhibition around the optochin disc 0 mm) was considered to indicate a Streptococcus other than S. pneumoniae. Different isolates from the same patient were differentiated by colony morphology on blood agar on a visual basis; all different colonies meeting the above criteria were collected. Thus, three to eight different isolates per person, comprising a total of 172 isolates, were isolated.
Identification of the 172 isolates to species level was done by determination of 16S rRNA gene sequences with pyrosequencing28 using primers bio-strep V1 (5'-AGTTTGATCCTGGCTCAGGACG-3') and strep V1rev (5'-TTACCTACGCGTTACTCACCCG-3'). The control strains used are shown in Table 1. Of the 172 -haemolytic optochin-resistant isolates, 161 belonged to Streptococcus spp., nine to Enterococcus spp. and two were Granulitacella adiacens isolates. Of the streptococcal isolates, 43 were Streptococcus mitis, 25 Streptococcus oralis, 23 Streptococcus parasanguinis, 22 Streptococcus cristatus, 18 S. pneumoniae, nine Streptococcus gordonii or Streptococcus anginosus, 16 Streptococcus sanguinis, Streptococcus salivarius or Streptococcus vestibularis (see Table 2), and five could not be identified. Because the 18 S. pneumoniae isolates (that were isolated from 14 persons) were atypically optochin resistant, they were considered to belong to viridans group streptococci. One hundred and fifty-one of the viridans streptococci were from the pharynx, seven from the nasopharynx and three from the conjunctiva (see Table 2).
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Determination of the MICs of the antibiotics was carried out using the agar dilution method described by the NCCLS.29 An inoculum of 107 cfu/mL was placed on MuellerHinton II agar (Becton Dickinson Microbiology Systems, Cockeysville, MD, USA), pH 7.3, containing 5% sheep blood, with a Denley Multipoint Inoculator (Denley Instruments Ltd, Billingshurst, UK) delivering final inocula of 1 µL (104 cfu/spot). The plates, which contained doubling dilutions of different antibiotics, were incubated for 18 h at 35°C in air. The antibiotics tested were tobramycin, tetracycline, chloramphenicol, penicillin, vancomycin, cefalothin, clindamycin, lincomycin, norfloxacin, (SigmaAldrich Chemie, Gmbh, Steinhein, Germany), levofloxacin, erythromycin, azithromycin, telithromycin, spiramycin (Aventis Pharma, Romainville Cedex, France), quinupristin/dalfopristin (Rhone-Poulanc Rorer, Vitrysur-Seine, France) and moxifloxacin (Bayer AG, Leverkusen, Germany). Isolates that had tobramycin MICs > 64 mg/L were further tested with tobramycin Etest strips (AB Biodisk, Solna, Sweden).
The breakpoints for non-susceptibility by NCCLS30 were as follows (mg/L): penicillin 0.25 (
4 for high resistance); erythromycin and clindamycin
1; azithromycin
2; levofloxacin
4 (
8 for resistance); moxifloxacin (breakpoint given for S. pneumoniae)
2 (
4 for resistance); norfloxacin (breakpoint given for Enterococcus spp.)
8 (
16 for resistance); tetracycline
4 (
8 for resistance); chloramphenicol
8 (
16 for resistance); vancomycin
2; and quinupristin/dalfopristin
2 (
4 for resistance). S. pyogenes ATCC 10389 and S. pneumoniae ATCC 49619 were used as controls in MIC determinations. High-level resistance to tobramycin was defined as MIC > 256 mg/L in the Etest.
Phenotyping
In addition to MIC determinations of erythromycin, azithromycin, spiramycin and clindamycin, the macrolide resistance phenotypes were determined using a modification of the double disc method used in phenotyping of S. pyogenes with erythromycin (diffusible content 78 µg) and clindamycin (diffusible content 25 µg) discs (Neo-sensitabs; A/S Rosco, Taastrup, Denmark).31 The discs were placed near each other (distance 4 mm instead of 1520 mm used for S. pyogenes) on MuellerHinton agar plates containing 5% sheep blood, after the plates had been inoculated with a swab from a bacterial suspension with a turbidity equal to that of a 0.5 McFarland standard. After 1824 h of incubation at 35°C in 5% CO2, the interpretation of the double disc test was performed as described previously.31
Detection of macrolide resistance genes
Preparation of DNA for PCR was done using the High Pure DNA isolation kit (Roche Diagnostics GmbH, Mannheim, Germany) according to the manufacturers instructions. For detection of erythromycin resistance genes the DNAs of erythromycin-resistant isolates were amplified with previously described primers24 that were specific for the erm(B), erm(TR) and mef(A) genes; E. coli with plasmid pJIR226, S. pyogenes A200 and S. pyogenes A569 were used as positive controls, respectively.
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Results |
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The number of resistant isolates in MIC testing of erythromycin and azithromycin was equal; 36 (22.4%) of the isolates were resistant (Table 3). The proportion of resistant isolates was highest among S. mitis, S. oralis and S. parasanguinis isolates (30.3%, 28.0% and 30.4% resistant isolates, respectively). Seven (19.4%) of the 36 macrolide-resistant isolates expressed the MLSB resistance phenotype in the MIC and double disc testing (one with constitutive and six with inducible expression) and harboured the erm(B) gene as determined by PCR. Twenty-nine (80.6%) of the macrolide-resistant isolates expressed the M phenotype. In 28 of these the presence of the mef(A) gene could be confirmed by PCR; one gave negative results. Resistance to erythromycin was found in 13 (48.1%) of the penicillin non-susceptible isolates and isolates resistant to both erythromycin and penicillin comprised 8.1% of all the isolates.
For both macrolide-resistant and -susceptible isolates, erythromycin MICs were somewhat lower than those of azithromycin (Table 3). Clindamycin, lincomycin and spiramycin had good potency against the macrolide-resistant isolates with the M phenotype and the erythromycin-susceptible isolates (Table 3), but all the isolates with the MLSB phenotype had elevated MICs (the inducibly resistant strains had MIC ranges of 0.5>64, 2>64 mg/L and 8>64 mg/L, and the constitutively resistant strain had MICs of >64, >64 and >64 mg/L, respectively). Telithromycin MICs were within the range 0.008 1 mg/L (Table 3). However, they were lower for isolates with the MLSB phenotype (range 0.0160.125 mg/L) and erythromycin-susceptible (range
0.0080.125 mg/L) isolates as compared with isolates with the M phenotype (range 0.0631 mg/L). The MICs of quinupristin/dalfopristin were within the range of 0.254 mg/L (Table 3); 62 (38.5%) and 21 (13.0%) of the isolates were intermediately resistant and resistant to this agent, respectively. Only four of the quinupristin/dalfopristin-resistant isolates were also resistant to erythromycin.
Resistance to tetracycline was found in 44 (27.3%) of all the isolates. The rate of tetracycline resistance was highest (47.8%) among S. parasanguinis isolates (Table 2). Tetracycline resistance was found in 21 (58.3%) of the isolates resistant to erythromycin and in 15 (55.6%) of the penicillin-non-susceptible isolates. Isolates with combined resistance to tetracycline and erythromycin, to tetracycline and penicillin, and to tetracycline, erythromycin and penicillin comprised 13.0, 9.3 and 5.6% of all the isolates, respectively (Table 2).
Levofloxacin and especially moxifloxacin showed good activity against the viridans streptococci, with 90% inhibition at MICs of 2 and 0.25 mg/L, respectively (Table 3). Resistance to these agents was detected only in three S. oralis isolates, which had MICs of 16 and 4 mg/L for levofloxacin and moxifloxacin, respectively. In addition, two S. parasanguinis isolates showed intermediate resistance to levofloxacin (MIC 4 mg/L). In contrast, an older fluoroquinolone, norfloxacin, showed poor activity, with an MIC50 of 16 mg/L (Table 3), and all but nine isolates were classified as intermediately resistant (34.2% of the isolates) or resistant (60.2%). The three S. oralis isolates that were resistant to levofloxacin and moxifloxacin also had the highest norfloxacin MICs (>64 mg/L), and one of them was also resistant to tetracycline.
None of the isolates was resistant to vancomycin and chloramphenicol, but three (1.9%) of the isolates (one S. mitis, one S. parasanguinis and one belonging to identification group S. sanguinis/salivarius/vestibularis) had a chloramphenicol MIC of 8 mg/L, indicating intermediate resistance. High-level resistance to tobramycin was rare, as only six isolates (belonging to four different species) had MICs of >64 mg/L (Table 3) in the agar dilution test, and only one of these (S. oralis) had an MIC > 256 mg/L in the Etest.
Erythromycin-resistant isolates were isolated from 16 (57.1%) of the 28 persons studied. Although one to four erythromycin-resistant isolates (belonging to up to three different species) were found per person, an additional one to seven erythromycin-susceptible isolates per person were isolated from the same subject (data not shown). There were 12 subjects whose erythromycin-resistant isolates were only of the M phenotype, one subject with erythromycin-resistant isolates only of the MLSB phenotype and three subjects with erythromycin-resistant isolates with both phenotypes. From one of the subjects, an erythromycin-resistant isolate (S. mitis) was found only from the eye conjunctiva.
All three S. oralis isolates resistant to levofloxacin and moxifloxacin were isolated from the same person, who also carried three other viridans streptococci, two of which were erythromycin resistant.
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Discussion |
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In our study, resistance to penicillin was noted in 16.8% of the viridans streptococci, the level of the resistance being only intermediate (MICs 0.251 mg/L). In previous studies, higher proportions of penicillin-resistant (MICs 0.25 mg/L) isolates have been reported among isolates from the normal flora (2262.5%) of healthy children17,18 and children with pharyngitis or haematological disorder20 and among isolates from blood cultures (2240%).25,8,10,11,13,15,16,32 The cause so far found for penicillin resistance in viridans streptococci is the presence of mosaic penicillin-binding protein (PBP) genes encoding altered PBPs with decreased affinity to ß-lactam antibiotics.33
Of the 161 viridans streptococcal isolates of this study, 22.4% were resistant to erythromycin. Higher proportions have been noted elsewhere; in a study from Spain the frequency of erythromycin-resistant viridans streptococci was highest among isolates from pharyngeal normal flora of patients with symptoms of pharyngitis (76.4%), followed by isolates from normal flora of healthy children (55.2%) and healthy adults (40.5%).19 In a study among healthy Greek children, 38.5% of the viridans streptococci isolated from the oropharynx were resistant to erythromycin.17 Recent studies of blood culture isolates have shown an erythromycin resistance rate of 3050% in most studies2,3,5,8,1012,34 and a lower rate of
15% in only a few reports.4,13
The cause of macrolide resistance was confirmed to be a methylase responsible for the MLSB phenotype in 19.4% of the 36 erythromycin-resistant isolates, as the erm(B) gene was found both in the single isolate with constitutive expression and in all the six isolates with inducible expression of the MLSB phenotype. In all, 80.6% of the isolates were of the M phenotype, suggesting an efflux pump as the cause of resistance, and in all but one of the M phenotype isolates the presence of the mef(A) gene was confirmed by PCR. As regards the ratio of viridans streptococcal isolates with the MLSB phenotype and isolates with the M phenotype in the normal flora, the results of our study are consistent with those reported by Ioannidou et al.17 and Aracil et al.,19 but different from the results of the studies by Trallero et al.21 and King et al.,22 who reported a higher proportion (50% in Spain and 38% in England, respectively) of viridans streptococci with the MLSB phenotype. On the other hand, as regards the ratio of inducible and constitutive isolates within the MLSB phenotype, a different distribution from ours has been noted in previous studies among viridans streptococcal isolates from the oropharynx, the constitutive type being more common than the inducible type,17,19,21 but the coding gene for the methylase, erm(B), has been the same.21,22 Among viridans group streptococcal isolates from blood cultures and other non-respiratory sites a predominance of isolates with the constitutive MLSB phenotype harbouring the erm(B) gene has also been found.3436 In testing of the phenotypes of erythromycin-resistant viridans streptococci with the double disc test, we found that it was important that the distance between the erythromycin and clindamycin discs was shorter (4 mm) than the 1520 mm used for testing of S. pyogenes, otherwise the D-shape of the clindamycin zone of inhibition seen in isolates with inducible expression of the MLSB phenotype31 would have been missed.
As compared with other streptococci, the distribution of the erythromycin resistance phenotypes found in this study correlates with that previously found in Finland among clinical isolates of S. pyogenes.23 However, the gene coding for the inducible MLSB phenotype differed in that the erm(A) gene, subclass erm(TR), was found in the vast majority of S. pyogenes isolates with the inducible MLSB phenotype.23 In Finland, the erm(B) gene has frequently been found in penicillin non-susceptible S. pneumoniae with MLSB resistance.24 It is of note that erm(A) was not at all found among the viridans streptococci. So far, erm(A), subclass erm(TR), has been found in viridans streptococci only in The Netherlands, in two S. anginosus isolates with the inducible MLSB phenotype isolated from clinical samples of non-respiratory sites.36 In streptococci other than S. pyogenes and the viridans group, this gene has been found in Finland also in group G ß-haemolytic streptococci and in other countries among Peptostreptococcus spp.,37 in a few isolates of group B, C and G ß-haemolytic streptococci38 and in one S. pneumoniae clone of serotype 11A.39
In accordance with previous results among clinical isolates of viridans streptococci,10,16,40 telithromycin had good potency against isolates from the normal flora (MIC90 0.25 mg/L). Higher MICs were found among erythromycin-resistant isolates with the M phenotype (MIC90 0.5 mg/L) than those with the MLSB phenotype and erythromycin-susceptible isolates, which correlates to the results of our previous study among S. pneumoniae and S. pyogenes in which the cause of MLSB resistance was erm(B) and erm(A), subclass erm(TR), respectively.41 It seems that telithromycin is a poor substrate for the efflux pump in viridans streptococci, but it is also known that ketolides with a C11/12 carbamate lactone ring extension remain active against most strains with an altered target site because of their stronger interaction with the hairpin 35 in domain II of the 23S rRNA, which is enough to prevent protein synthesis in MLSB-resistant ribosomes.41 However, in our pervious study, S. pyogenes with the constitutive MLSB phenotype caused by the erm(B) gene were resistant to telithromycin.41 This variation in the effect of erm(B) derived methylation on telithromycin susceptibility is possibly due to the structural similarity of ribosomes of viridans streptococci and S. pneumoniae, and the dissimilarity of the S. pyogenes ribosomes from these.
Erythromycin-resistant isolates were found in 57% of the subjects. This is a lower carrier rate than noted in recent studies among adult patients from England and Spain, in which 75.5% of patients with symptoms of a respiratory tract infection and 94% of healthy persons and patients with pharyngitis carried erythromycin-resistant viridans streptococci in the normal oral flora, respectively.21,22
In our study, resistance to tetracycline was found in 27.3% of the isolates, and combined resistance to tetracycline and erythromycin was noted in half of the erythromycin-resistant isolates. These results are in agreement with previous reports of isolates from normal flora17 and clinical infections.2,5,6,8
Decreased activity of quinupristin/dalfopristin was noted, as 51.5% of the isolates of this study were non-susceptible (MIC 2 mg/L) to this agent. These results agree with previous results among clinical isolates from Taiwan42 and the USA,3 where 51% and 70.2% of quinupristin/dalfopristin non-susceptible viridans streptococci were noted, respectively. Despite a mode of action that is similar to that of macrolides, non-susceptible isolates were found both among erythromycin-resistant and -susceptible isolates in the present study, and in a study from The Netherlands.43 In contrast to these results, studies among blood culture and other clinical isolates collected from the USA,15 Spain,44 Germany,13 North and South America11 and different continents14,40 have indicated much lower proportions (117%) of quinupristin/dalfopristin-non-susceptible isolates. In a study from Spain, higher MICs of quinupristin/dalfopristin correlated with higher MICs of erythromycin.44 This variation in quinupristin/dalfopristin MICs among viridans streptococci in various studies suggests a between-laboratory variation in the performance of MIC testing. For our control strain, S. pneumoniae ATCC 49619, the MICs obtained for this agent in different series were 0.51 mg/L, and the acceptable quality control limits given by the NCCLS are 0.251 mg/L.30
As in previous studies of viridans streptococci from blood cultures,11,15,16 the newer quinolones, levofloxacin and moxifloxacin, showed better activity against these Gram-positive organisms compared with older compounds (norfloxacin in this study). Moxifloxacin was more active than levofloxacin (MIC90 0.25 versus 2 mg/L). Resistance to these newer agents was found in only three isolates. Mechanisms involved in quinolone resistance in viridans streptococci include mutations primarily in parC, but also in parE and gyrA genes, and enhanced drug efflux.45,46
All the isolates in this study were susceptible to vancomycin, and 97.1% were susceptible to chloramphenicol. So far, vancomycin resistance has not been found in viridans streptococci. In other studies of the activity of chloramphenicol against viridans streptococci, the proportion of resistant isolates among blood culture isolates was 27.4% in Taiwan,5 but considerably lower (03%) elsewhere,13,10,11 and among isolates from oral flora a proportion of 1.5% has been reported.17
Various antimicrobial resistance determinants are carried by viridans streptococci of the normal flora of elderly persons. These antibiotic-resistant organisms of the normal flora are capable of causing significant infections, especially in immunocompromised hosts, and also provide a pool of resistant bacteria that may mediate resistance determinants to more pathogenic organisms through gene transfer.1,21 Identification of the viridans streptococcal species is easier and more accurate when complex and often unspecific biochemical testing can be replaced by exact modern DNA sequencing methods, which will also make reliable comparison of results between species from different laboratories possible.
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Acknowledgements |
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Footnotes |
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References |
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2 . Potgieter, E., Carmichael, M., Koornhof, H. J. et al. (1992). In vitro antimicrobial susceptibility of viridans streptococci isolated from blood cultures. European Journal of Clinical Microbiology and Infectious Diseases 11, 5436.[ISI][Medline]
3 . Doern, G. V., Ferraro, M. J., Brueggemann, A. B. et al. (1996). Emergence of high rates of antimicrobial resistance among viridans group streptococci in the United States. Antimicrobial Agents and Chemotherapy 40, 8914.[Abstract]
4 . Renneberg, J., Niemann, L. L. & Gutschik, E. (1997). Antimicrobial susceptibility of 278 streptococcal blood isolates to seven antimicrobial agents. Journal of Antimicrobial Chemotherapy 39, 13540.[Abstract]
5 . Wu, J. J., Lin, K. Y., Hsueh, P. R. et al. (1997). High incidence of erythromycin-resistant streptococci in Taiwan. Antimicrobial Agents and Chemotherapy 41, 8446.[Abstract]
6 . Teng, L. J., Hsueh, P. R., Chen, Y. C. et al. (1998). Antimicrobial susceptibility of viridans group streptococci in Taiwan with an emphasis on the high rates of resistance to penicillin and macrolides in Streptococcus oralis. Journal of Antimicrobial Chemotherapy 41, 6217.[Abstract]
7
.
Arpin, C., Canron, M. H., Maugein, J. et al. (1999). Incidence of mefA and mefE genes in viridans group streptococci. Antimicrobial Agents and Chemotherapy 43, 23356.
8
.
de Azavedo, J. C., Trpeski, L., Pong-Porter, S. et al. (1999). In vitro activities of fluoroquinolones against antibiotic-resistant blood culture isolates of viridans group streptococci from across Canada. Antimicrobial Agents and Chemotherapy 43, 2299301.
9
.
Ono, T., Shiota, S., Hirota, K. et al. (2000). Susceptibilities of oral and nasal isolates of Streptococcus mitis and Streptococcus oralis to macrolides and PCR detection of resistance genes. Antimicrobial Agents and Chemotherapy 44, 107880.
10
.
Alcaide, F., Benitez, M. A., Carratala, J. et al. (2001). In vitro activities of the new ketolide HMR 3647 (telithromycin) in comparison with those of eight other antibiotics against viridans group streptococci isolated from blood of neutropenic patients with cancer. Antimicrobial Agents and Chemotherapy 45, 6246.
11 . Diekema, D. J., Beach, M. L., Pfaller, M. A. et al. (2001). Antimicrobial resistance in viridans group streptococci among patients with and without the diagnosis of cancer in the USA, Canada and Latin America. Clinical Microbiology and Infection 7, 1527.[CrossRef][ISI][Medline]
12
.
Marron, A., Carratala, J., Alcaide, F. et al. (2001). High rates of resistance to cephalosporins among viridans-group streptococci causing bacteraemia in neutropenic cancer patients. Journal of Antimicrobial Chemotherapy 47, 8791.
13
.
Reinert, R. R. von Eiff, C., Kresken, M. et al. (2001). Nationwide German multicenter study on the prevalence of antibiotic resistance in streptococcal blood isolates from neutropenic patients and comparative in vitro activities of quinupristindalfopristin and eight other antimicrobials. Journal of Clinical Microbiology 39, 192831.
14 . Gordon, K. A., Beach, M. L., Biedenbach, D. J. et al. (2002). Antimicrobial susceptibility patterns of ß-hemolytic and viridans group streptococci: report from the SENTRY Antimicrobial Surveillance Program (19972000). Diagnostic Microbiology and Infectious Diseases 43, 15762.[CrossRef][ISI][Medline]
15
.
Anderegg, T. R., Biedenbach, D. J. & Jones, R. N. (2002). In vitro evaluation of AZD2563, a new oxazolidinone, tested against ß-haemolytic and viridans group streptococci. Journal of Antimicrobial Chemotherapy 49, 101921.
16
.
Gershon, A. S., de Azavedo, J. C., McGeer, A. et al. (2002). Activities of new fluoroquinolones, ketolides, and other antimicrobials against blood culture isolates of viridans group streptococci from across Canada, 2000. Antimicrobial Agents and Chemotherapy 46, 15536.
17 . Ioannidou, S., Tassios, P. T., Kotsovili-Tseleni, A. et al. (2001). Antibiotic resistance rates and macrolide resistance phenotypes of viridans group streptococci from the oropharynx of healthy Greek children. International Journal of Antimicrobial Agents 17, 195201.[CrossRef][ISI][Medline]
18 . Guiot, H. F., Corel, L. J. & Vossen, J. M. (1994). Prevalence of penicillin-resistant viridans streptococci in healthy children and in patients with malignant haematological disorders. European Journal of Clinical Microbiology and Infectious Diseases 13, 64550.[ISI][Medline]
19
.
Aracil, B., Minambres, M., Oteo, J. et al. (2001). High prevalence of erythromycin-resistant and clindamycin-susceptible (M phenotype) viridans group streptococci from pharyngeal samples: a reservoir of mef genes in commensal bacteria. Journal of Antimicrobial Chemotherapy 48, 5924.
20 . Mogi, A., Nishi, J. I., Yoshinaga, M. et al. (1997). Increased prevalence of penicillin-resistant viridans group streptococci in Japanese children with upper respiratory infection treated by ß-lactam agents and in those with oncohematologic diseases. Pediatric Infectious Disease Journal 16, 11404.[CrossRef][ISI][Medline]
21
.
Trallero, E., Vicente, D., Montes, M. et al. (2001). High proportion of pharyngeal carriers of commensal streptococci resistant to erythromycin in Spanish adults. Journal of Antimicrobial Chemotherapy 48, 2259.
22 . King, A., Bathgate, T. & Phillips, I. (2002). Erythromycin susceptibility of viridans streptococci from the normal throat flora of patients treated with azithromycin or clarithromycin. Clinical Microbiology and Infection 8, 8592.[CrossRef][ISI][Medline]
23 . Kataja, J., Huovinen, P., Muotiala, A. et al. (1998). Clonal spread of group A streptococcus with the new type of erythromycin resistance. Finnish Study Group for Antimicrobial Resistance. Journal of Infectious Diseases 177, 7869.[ISI][Medline]
24
.
Pihlajamaki, M., Kaijalainen, T., Huovinen, P. et al. (2002). Rapid increase in macrolide resistance among penicillin non-susceptible pneumococci in Finland, 19962000. Journal of Antimicrobial Chemotherapy 49, 78592.
25
.
Kataja, J., Seppälä, H., Skurnik, M. et al. (1998). Different erythromycin resistance mechanisms in group C and group G streptococci. Antimicrobial Agents and Chemotherapy 42, 14934.
26
.
Kataja, J., Huovinen, P., Skurnik, M. et al. (1999). Erythromycin resistance genes in group A streptococci in Finland. The Finnish Study Group for Antimicrobial Resistance. Antimicrobial Agents and Chemotherapy 43, 4852.
27
.
Pihlajamaki, M., Kataja, J., Seppälä, H. et al. (2002). Ribosomal mutations in Streptococcus pneumoniae clinical isolates. Antimicrobial Agents and Chemotherapy 46, 6548.
28
.
Pourmand, N., Elahi, E., Davis, R. W. et al. (2002). Multiplex pyrosequencing. Nucleic Acids Research 30, e31.
29 . National Committee for Clinical Laboratory Standards. (2000). Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria that Grow AerobicallyFifth Edition: Approved Standard M7-A5. NCCLS, Wayne, PA, USA.
30 . National Committee for Clinical Laboratory Standards. (2001). Performance Standards for Antimicrobial Susceptibility TestingEleventh Informational Supplement. NCCLS, Wayne, PA, USA.
31 . Seppälä, H., Nissinen, A., Yu, Q. et al. (1993). Three different phenotypes of erythromycin-resistant Streptococcus pyogenes in Finland. Journal of Antimicrobial Chemotherapy 32, 88591.[ISI][Medline]
32 . Alcaide, F., Liñares, J., Pallares, R. et al. (1995). In vitro activities of 22 ß-lactam antibiotics against penicillin-resistant and penicillin-susceptible viridans group streptococci isolated from blood. Antimicrobial Agents and Chemotherapy 39, 22437.[Abstract]
33
.
Amoroso, A., Demares, D., Mollerach, M. et al. (2001). All detectable high-molecular-mass penicillin-binding proteins are modified in a high-level ß-lactam-resistant clinical isolate of Streptococcus mitis. Antimicrobial Agents and Chemotherapy 45, 207581.
34
.
Rodriguez-Avial, I., Rodriguez-Avial, C., Culebras, E. et al. (2001). Distribution of mef(A) and erm(B) genes in macrolide-resistant blood isolates of viridans group streptococci. Journal of Antimicrobial Chemotherapy 47, 7278.
35
.
Poutanen, S. M., de Azavedo, J., Willey, B. M. et al. (1999). Molecular characterization of multidrug resistance in Streptococcus mitis. Antimicrobial Agents and Chemotherapy 43, 15057.
36
.
Jacobs, J. A., van Baar, G. J., London, N. H. et al. (2001). Prevalence of macrolide resistance genes in clinical isolates of the Streptococcus anginosus ("S. milleri") group. Antimicrobial Agents and Chemotherapy 45, 23757.
37
.
Reig, M., Galan, J., Baquero, F. et al. (2001). Macrolide resistance in Peptostreptococcus spp. mediated by ermTR: possible source of macrolidelincosamidestreptogramin B resistance in Streptococcus pyogenes. Antimicrobial Agents and Chemotherapy 45, 6302.
38
.
Portillo, A., Lantero, M., Olarte, I. et al. (2001). MLS resistance phenotypes and mechanisms in ß-haemolytic group B, C and G Streptococcus isolates in La Rioja, Spain. Journal of Antimicrobial Chemotherapy 47, 1156.
39
.
Syrogiannopoulos, G. A., Grivea, I. N.,Tait-Kamradt, A. et al. (2001). Identification of an erm(A) erythromycin resistance methylase gene in Streptococcus pneumoniae isolated in Greece. Antimicrobial Agents and Chemotherapy 45, 3424.
40 . Rospide, M. F., Biedenbach, D. J. & Jones, R. N. (2001). Comparative antimicrobial activity of ABT-773, a novel ketolide, tested against drug-resistant Gram-positive cocci and Haemophilus influenzae. International Journal of Antimicrobial Agents 17, 4515.[CrossRef][ISI][Medline]
41
.
Jalava, J., Kataja, J., Seppälä, H. et al. (2001). In vitro activities of the novel ketolide telithromycin (HMR 3647) against erythromycin-resistant Streptococcus species. Antimicrobial Agents and Chemotherapy 45, 78993.
42
.
Luh, K. T., Hsueh, P. R., Teng, L. J. et al. (2000). Quinupristindalfopristin resistance among gram-positive bacteria in Taiwan. Antimicrobial Agents and Chemotherapy 44, 337480.
43
.
Mouton, J. W., Endtz, H. P., den Hollander, J. G. et al. (1997). In-vitro activity of quinupristin/dalfopristin compared with other widely used antibiotics against strains isolated from patients with endocarditis. Journal of Antimicrobial Chemotherapy 39, Suppl. A, 7580.
44 . Alcaide, F., Carratala, J., Liñares, J. et al. (1996). In vitro activities of eight macrolide antibiotics and RP-59500 (quinupristindalfopristin) against viridans group streptococci isolated from blood of neutropenic cancer patients. Antimicrobial Agents and Chemotherapy 40, 211720.[Abstract]
45
.
Gonzalez, I., Georgiou, M., Alcaide, F. et al. (1998). Fluoroquinolone resistance mutations in the parC, parE, and gyrA genes of clinical isolates of viridans group streptococci. Antimicrobial Agents and Chemotherapy 42, 27928.
46
.
Ferrandiz, M. J., Oteo, J., Aracil, B. et al. (1999). Drug efflux and parC mutations are involved in fluoroquinolone resistance in viridans group streptococci. Antimicrobial Agents and Chemotherapy 43, 25203.