Distribution of mef(A)-containing genetic elements in erythromycin-resistant isolates of Streptococcus pyogenes from Italy

In total, 124 Streptococcus pyogenes isolates were obtained from throat cultures of different symptomatic patients. All isolates showed M-phenotype macrolide resistance and contained the macrolide efflux gene mef(A). The isolates were screened for the presence and insertion site of mef(A)-containing genetic elements. In 25.8% of the isolates, mef(A) was found to be carried by elements belonging to the Tn1207.3/Phi10394.4 family inserted in the comEC gene, while 74.2% contained chimeric elements with a different genetic structure and chromosomal location, probably associated with the recently described 60-kb tet(O)-mef(A) element.     

Molecular epidemiology of erythromycin resistance in Streptococcus pneumoniae isolates from blood and noninvasive sites

Erythromycin-resistant isolates of Streptococcus pneumoniae from blood cultures and noninvasive sites were studied over a 3-year period. The prevalence of erythromycin resistance was 11.9% (19 of 160) in blood culture isolates but 4.2% (60 of 1,435) in noninvasive-site isolates. Sixty-two of the 79 resistant isolates were available for study. The M phenotype was responsible for 76% (47 of 62) of resistance, largely due to a serotype 14 clone, characterized by multilocus sequence typing as ST9, which accounted for 79% (37 of 47) of M phenotype resistance. The ST9 clone was 4.8 times more common in blood than in noninvasive sites. All M phenotype isolates were PCR positive for mef(A), but sequencing revealed that the ST9 clone possessed the mef(A) sequence commonly associated with Streptococcus pyogenes. All M phenotype isolates with this mef(A) sequence also had sequences consistent with the presence of the Tn1207.1 genetic element inserted in the celB gene. In contrast, isolates with the mef(E) sequence normally associated with S. pneumoniae contained sequences consistent with the presence of the mega insertion element. All MLS(B) isolates carried erm(B), and two isolates carried both erm(B) and mef(E). Fourteen of the 15 MLS(B) isolates were tetracycline resistant and contained tet(M). However, six M phenotype isolates of serotypes 19 (two isolates) and 23 (four isolates) were also tetracycline resistant and contained tet(M). MICs for isolates with the mef(A) sequence were significantly higher than MICs for isolates with the mef(E) sequence (P < 0.001). Thus, the ST9 clone of S. pneumoniae is a significant cause of invasive pneumococcal disease in northeast Scotland and is the single most important contributor to M phenotype erythromycin resistance.     

Prevalence of Streptococcus pneumoniae isolates bearing macrolide resistance genes in association with integrase genes of conjugative transposons in Japan

The relationship between susceptibility to macrolides and tetracycline, and the distribution of the resistance genes erm(B), mef(A) and tet(M) and the integrase gene of the conjugative transposon, Int-Tn, was examined in 43 isolates of Streptococcus pneumoniae from Gunma Prefecture, Japan. Thirty-five isolates were resistant to both macrolides and tetracycline and carried tet(M); erm(B) and mef(A) were detected in 19 and 16, respectively, of these isolates. Sixteen mef(A)-positive isolates were all identified as mef(E) variants. Int-Tn of Tn1545 was associated with 17 erm(B)- and 14 mef(A)-bearing isolates, and Int-Tn of Tn5252 was found in the remaining two mef(A)-carrying isolates. Pneumococcal strains with resistance to macrolides conferred by erm(B) or mef(A) in association with the integrase gene of conjugative transposon Tn1545 or Tn5252 appear to be prevalent in Japan.     

Molecular epidemiology of multiresistant Streptococcus pneumoniae with both erm(B)- and mef(A)-mediated macrolide resistance

Of a total of 1043 macrolide-resistant Streptococcus pneumoniae isolates collected from 24 countries as part of PROTEKT 1999-2000, 71 isolates tested positive for both the mef(A) and erm(B) genes. Of 69 isolates subjected to further molecular investigations, all were resistant to tetracycline, 63 (91.3%) were resistant to penicillin, and 57 (82.6%) were resistant to trimethoprim-sulfamethoxazole. One isolate was also fluoroquinolone resistant, and another was resistant to quinupristin-dalfopristin. The ketolide telithromycin retained activity against all of the isolates. Of the 69 of these 71 isolates viable for further testing, 46 were from South Korea, 13 were from the United States, 8 came from Japan, and 1 each came from Mexico and Hungary. One major clonal complex (59 [85.5%] of 69 isolates) was identified by serotyping (with 85.5% of the isolates being 19A or 19F), pulsed-field gel electrophoresis, and multilocus sequence typing. The remaining isolates were less clonal in nature. Representative isolates were shown to carry the mobile genetic elements Tn1545 and mega, were negative for Tn1207.1, had tetracycline resistance mediated by tet(M), and contained the mef(E) variant of mef(A). All isolates were positive for mel, a homologue of the msr(A) efflux gene. These clones are obviously very efficient at global dissemination, and hence it will be very important to monitor their progress through continued surveillance. Telithromycin demonstrated high levels of activity (MIC for 90% of the strains tested, 0.5 micro g/ml; MIC range, 0.06 to 1 micro g/ml) against all isolates.     

Characterization and prevalence of MefA, MefE, and the associated msr(D) gene in Streptococcus pneumoniae clinical isolates

Recent work has shown that the efflux genes in Streptococcus pneumoniae that are responsible for acquired macrolide resistance can be distinguished as either mef(E) or mef(A). The genetic elements on which mef(A) and mef(E) are found also carry an open reading frame (ORF) that is 56% homologous to msr(A) in Staphylococcus. The prevalence of mef(A/E) and of the msr-like ORF [msr(D)] was evaluated in 153 mef(+) S. pneumoniae clinical isolates collected in North America, Europe, Africa, and Asia from 1997 to 2002. Clinical isolates were screened with PCR primers specific for either mef(A) or mef(E) and for msr(D). mef(A), mef(E), and msr(D) were cloned from mef(+) strains and transformed into a susceptible, competent strain of S. pneumoniae. The transformants were tested for antimicrobial susceptibilities and efflux pump induction. The results of this work demonstrated that mef(A) is more often isolated in parts of Europe, with some incidence in Canada, and that the msr-like gene alone can confer the efflux phenotype.     

Monoclonal antibodies that recognize a common pneumococcal protein with similarities to streptococcal group A surface glyceraldehyde-3-phosphate dehydrogenase.

Monoclonal antibodies (MAbs) against clinical isolates of Streptococcus pneumoniae were produced in a search for common pneumococcal proteins. One of the fusions generated two MAbs, 174,B-8 (immunoglobulin G2a) and 177,D-8 (immunoglobulin G1), which by Western blotting (immunoblotting) stained with a main band of 40 kDa found in all isolates of S. pneumoniae examined. Cross-reactivity studies with streptococci other than pneumococci revealed very weak or moderate reactions with the MAbs. The 40-kDa protein was isolated by immunoaffinity chromatography and subsequent preparative Western blotting. N-terminal amino acid sequencing showed 90% amino acid sequence homology with a surface-located glyceraldehyde-3-phosphate dehydrogenase from Streptococcus pyogenes. This protein has also been reported to exhibit binding to mammalian proteins such as fibronectin, which may serve as host receptors. The epitopes for MAbs 174,B-8 and 177,D-8 reacting with the pneumococcal analog were not accessible to antibody binding in live bacteria but were exposed after heat killing. The MAbs showed negligible cross-reactions with S. pyogenes.     

Increased prevalence of erythromycin resistance in streptococci: substantial upsurge in erythromycin-resistant M phenotype in Streptococcus pyogenes (1979-1998) but not in Streptococcus pneumoniae (1985-1999) in Taiwan

A total of 394 nonduplicate isolates of Streptococcus pyogenes collected from 1979 to 1998 and 267 nonduplicate isolates of Streptococcus pneumoniae collected from October, 1998, to May, 1999, in Taiwan were evaluated. Among the 220 erythromycin-resistant (MIC, > or =1 microg/ml) S. pyogenes isolates, 35% had an M phenotype and 65% had an ML phenotype (inducible resistance [iML], 0.5%, and constitutive resistance [cML], 64.5%). Among the 243 erythromycin-resistant S. pneumoniae isolates, the majority (65.4%) had an ML phenotype (iML, 0.4%, and cML, 65%) and 34.6% had an M phenotype. A substantial upsurge in the incidence of M-phenotype erythromycin-resistant isolates was found with time for S. pyogenes (0% in 1979-1984 and 100% in 1997-1998), and an increasing incidence of M-phenotype among erythromycin-resistant S. pneumoniae was also noted (<20% before 1994 and 45.4% in 1999). All S. pyogenes and all but four S. pneumoniae isolates exhibiting a cML or iML phenotype had harbored the ermAM gene. The presence of the mefA gene was demonstrated in all isolates of S. pyogenes and the mefE gene in all but four S. pneumoniae isolates exhibiting the M phenotype. Due to the increasing susceptibility of S. pyogenes and S. pneumoniae isolates to clindamycin, susceptibility tests of these two organisms to macrolides and clindamycin should be performed simultaneously in the clinical microbiology laboratory, particularly in areas with high rates of macrolide resistance.     

Tetracycline and macrolide co-resistance in Streptococcus pyogenes: co-selection as a reason for increase in macrolide-resistant S. pyogenes?

In Denmark, tetracycline resistance in Streptococcus pyogenes is frequent (>30%) whereas macrolide resistance is low (<5%). The aim of this study was to investigate the genetic background of tetracycline- and macrolide resistance in macrolide-resistant S. pyogenes (MRSP) and to investigate the correlation between the use of macrolide and tetracycline and macrolide resistance using international data. A total of 133 MRSP isolates were received at Statens Serum Institut from nine Danish clinical microbiology laboratories between. November 2000, and November 2002. The macrolide-resistance genes, erm(B), erm(A), and mef(A) were detected in 46%, 18%, and 32% of the tested MRSP isolates, respectively. In 4% of MRSP isolates, none of the MR genes were detected. Tetracycline resistance was found in 52% of MRSP. Tetracycline resistance was encoded by either tet(M) or tet(O). erm(B) and mef(A) were associated with tet(M). Sixteen different T types were detected among the 133 MRSP. Analysis of the importance of antibiotic use for development of macrolide resistance in S. pyogenes showed no correlation with macrolide use alone (p = 0.15) but a significant correlation (p = 0.03) for the combination of macrolide and tetracycline use. The frequency of macrolide resistance in Danish S. pyogenes was low and mainly due to erm genes. A high frequency of macrolide-tetracycline coresistance in S. pyogenes is found in many countries including Denmark, hence tetracycline use must be considered as a co-factor in selection of MRSP.     

Is There an Ecological Relationship between Rates of Antibiotic Resistance of Species of the Genus Streptococcus?

The relationship between resistance to antibiotics on the part of Streptococcus pneumoniae and Streptococcus pyogenes was studied by comparing different prevalences of resistance among hospitals obtained from a recent microbiological surveillance of community-acquired respiratory tract infections. A high correlation for erythromycin resistance was found between S. pneumoniae isolates from lower respiratory tract infections and S. pyogenes isolates collected from pharyngeal swabs.     

National surveillance programme on susceptibility patterns of respiratory pathogens in South Africa: moxifloxacin compared with eight other antimicrobial agents

AIMS: The susceptibility patterns of Streptococcus pneumoniae, Haemophilus influenzae, Moraxella catarrhalis, Klebsiella pneumoniae, and Streptococcus pyogenes isolated from specimens submitted to 12 private laboratories in South Africa were determined. METHODS: Minimum inhibitory concentration (MIC) determinations were performed on the isolates in the microbiology laboratory at Tygerberg Hospital according to the recommendations of the National Committee for Clinical Laboratory Standards (NCCLS). RESULTS: According to the NCCLS breakpoints, 24% of 729 S pneumoniae isolates were sensitive, 30% intermediate, and 46% resistant to penicillin. Rates of macrolide resistance were high, with 61% of the pneumococci being resistant to clarithromycin and azithromycin. Co-trimoxazole resistance was also high, with 28% of pneumococcal strains being sensitive, 21% intermediate, and 51% resistant. beta Lactamase was produced by 7% of 736 H influenzae isolates and 91% of 256 M catarrhalis isolates. The quinolones, moxifloxacin and levofloxacin, were universally active against all isolates tested, which included S pneumoniae, H influenzae, M catarrhalis, K pneumoniae, and S pyogenes. CONCLUSIONS: Haemophilus influenzae and S pneumoniae were the most commonly isolated organisms. Resistance to penicillin was one of the highest reported in the world (76%) in S pneumoniae, as was macrolide resistance in pneumonocci, although surprisingly, only 14% of S pyogenes were resistant. The quinolones, moxifloxacin and levofloxacin, were active against all organisms tested, including the penicillin and macrolide resistant strains and moxifloxacin was more active than levofloxacin against pneumococci.     

Macrolide efflux genes mef(A) and mef(E) are carried by different genetic elements in Streptococcus pneumoniae

Susceptibilities to macrolides were evaluated in 267 Streptococcus pneumoniae isolates, of which 182 were from patients with invasive diseases and 85 were from healthy carriers. Of the 98 resistant isolates, 20 strains showed an M phenotype and carried mef. Strains that carried both mef(A) and mef(E) were found: 17 strains carried mef(A) and 3 carried mef(E). The characteristics of the strains carrying the mef genes and the properties of the mef-containing elements were studied. Strains carrying mef(A) belonged to serotype 14, were susceptible to all the antibiotics tested except erythromycin, and appeared to be clonally related by pulsed-field gel electrophoresis (PFGE). The three mef(E) strains belonged to different serotypes, showed different susceptibility profiles, and did not appear to be related by PFGE. The sequences of a fragment of the mef-containing element, which encompassed mef and the msr(A) homolog, were identical among the three mef(E)-positive strains and among the three mef(A)-positive strains, although there were differences between the sequences for the two variants at 168 positions. In all mef(A)-positive strains, the mef element was inserted in celB, which led to impairment of the competence of the strains. In line with insertion of the mef(E) element at a different site, the competence of the mef(E)-positive strains was maintained. Transfer of erythromycin resistance by conjugation was obtained from two of three mef(A) strains but from none of three mef(E) strains. Due to the important different characteristics of the strains carrying mef(A) or mef(E), we suggest that the distinction between the two genes be maintained.     

The effect of carbon dioxide on susceptibility testing of azithromycin, clarithromycin and roxithromycin against clinical isolates of Streptococcus pneumoniae and Streptococcus pyogenes by broth microdilution and the Etest: Artemis Project—first-phase study

Objective: To evaluate the effect of carbon dioxide on the susceptibility testing, using broth microdilution and the Etest (AB Biodisk, Solna, Sweden), of azithromycin, clarithromycin and roxithromycin against Streptococcus pneumoniae and Streptococcus pyogenes .
Methods: Fresh clinical isolates collected from 36 hospital laboratories in 12 countries were evaluated using the Etest in the presence of carbon dioxide. The isolates were retested under ambient conditions (absence of carbon dioxide) using broth microdilution and/or the Etest.
Results: Carbon dioxide falsely elevated azithromycin, clarithromycin and roxithromycin MIC₉₀S for S. pneumoniae , determined by the Etest, approximately 12-fold. Also, the azithromycin MIC₉₀ for S. pyogenes was increased fourfold; the effect was less marked for clarithromycin and roxithromycin. When isolates were retested in the absence of carbon dioxide, using the Etest or microdilution, susceptibilities to azithroymycin were comparable to those to clarithromycin ( S. pneumoniae , 93.4% versus 91.3%; S. pyogenes , 96.4% versus 95.8%). Both organisms were less susceptible to roxithromycin ( S. pneumoniae , 71.3%; S. pyogenes , 85.7%). An internal standard control, consisting of 50 isolates each of S. pneumoniae, S. pyogenes and Haemophilus influenzae , confirmed that azithromycin susceptibility testing resulted in falsely elevated MICs.
Conclusions: Carbon dioxide falsely elevated azithromycin MICs for S. pneumoniae and S. pyogenes , with an apparent reduction in susceptibility. When the in vitro activity of azithromycin and other macrolides against S. pneumoniae and S. pyogenes is being evaluated, awareness of the pH effect is essential.     

Characterization of erythromycin-resistant Streptococcus pneumoniae in Korea, and In Vitro activity of telithromycin against erythromycin-resistant Streptococcus pneumoniae

To characterize the phenotypes and genotypes of erythromycin-resistant clinical isolates of Streptococcus pneumoniae in Korea and to evaluate the in vitro activity of telithromycin against these erythromycin-resistant isolates, we tested a total of 676 isolates of S. pneumoniae collected from 1997 to 2002 in a tertiary hospital in Seoul, Republic of Korea. MICs for erythromycin and telithromycin were determined by the agar dilution method. The macrolide resistance phenotypes of erythromycin-resistant isolates were determined by the erythromycin- clindamycin-rokitamycin triple disk (ECRTD) and MIC induction tests, whereas their macrolide resistance genotypes were determined by PCR for the erm(B), erm(A), subclass erm(TR), and mef genes. To discriminate between mef(A) and mef(E), PCR-restriction fragment length polymorphism (RFLP) analyses were performed. Of the 676 S. pneumoniae isolates, 459 (67.9%) were resistant to erythromycin. Of the 459 erythromycin-resistant isolates, 343 (74.7%) were assigned to the cMLS phenotype, 48 (10.4%) to the iMcLS phenotype, 4 (0.9%) to the iMLS phenotype, and 64 (14.0%) to the M phenotype. The erm(B) gene was detected in 251 (54.6%) isolates, the mef gene was detected in 64 (14.0%), and both the erm(B) and mef genes were detected in 144 (31.4%) isolates. All of the mef genes detected were identified as mef(E). Of the 459 erythromycin- resistant isolates, all but one were susceptible to telithromycin. The MIC(50)/MIC(90) to telithromycin of isolates carrying erm(B), mef(E), and both genes was 0.06/0.5 microg/ml, 0.03/0.125 microg/ml, and 0.5/1.0 microg/ml, respectively. Although the MICs of telithromycin for the erythromycin-resistant isolates varied according to genotype, telithromycin was very active against these erythromycin-resistant S. pneumoniae.     

A common clone of erythromycin-resistant Streptococcus pneumoniae in Greece and the UK

Objective  To investigate the possible genetic relationship among erythromycin-resistant Streptococcus pneumoniae strains isolated in Greece and the UK.
Methods  During 1995–97, 140 S. pneumoniae strains were isolated from clinical specimens submitted to the microbiology departments of the two main children's hospital in Athens. All erythromycin-resistant strains were further studied with respect to the presence of genes encoding for the two major mechanisms of macrolide resistance, their serotypes, and pulsed-field gel electrophoresis (PFGE) types, in comparison to a previously characterized UK erythromycin-resistant clone.
Results  Eleven of the 140 isolates (7.9%) were resistant to erythromycin; nine of these were susceptible to penicillin. Serotyping allocated seven, three and one isolates to serotypes 14, 19F and serogroup 6, respectively. The mef A gene was detected in seven isolates (five serotype 14 and two serotype 19F), erm B in two (one serotype 19F and the serogroup 6 isolate), whilst in the remaining two isolates no resistance gene could be detected by polymerase chain reaction (PCR). Pulsed-field gel electrophoresis of genomic DNA showed that five Greek serotype 14 isolates belonged to the same chromosomal type as the serotype 14 erythromycin-resistant UK clone.
Conclusions  The present study showed that erythromycin resistance among the S. pneumoniae isolates was mostly owing to the efflux mechanism and suggested a possible clonal spread of serotype 14 erythromycin-resistant S. pneumoniae strains between Greece and the UK.     

Activity of quinupristin–dalfopristin in invasive isolates of Streptococcus pneumoniae from Italy

Eighty-five recent isolates of Streptococcus pneumoniae from patients with invasive disease were examined for their susceptibility to erythromycin, clindamycin, penicillin and quinupristin-dalfopristin by E test. A novel duplex PCR assay was used to detect the presence of the erm (B) or mef (A) genes in all of the erythromycin-resistant isolates. All of the strains tested were susceptible to the combination quinupristin–dalfopristin, regardless of their susceptibility to penicillin or to erythromycin. By duplex PCR, two-thirds of the erythromycin-resistant strains harbored erm , and one-third harbored mef . The activity of quinupristin–dalfopristin was not influenced by the genetic determinant of erythromycin resistance. The in vitro susceptibility of S. pneumoniae to quinupristin–dalfopristin is promising for future use; however, it is important to monitor the possible emergence of resistance.     

Prophage Carriage as a Molecular Epidemiological Marker in Streptococcus pneumoniae

The great majority of clinical isolates of Streptococcus pneumoniae carry prophages that may be identified through their hybridization with a DNA probe specific for the pneumococcal lytA gene (M. Ramirez, E. Severina, and A. Tomasz, J. Bacteriol. 181:3618-3625, 1999). We now show that the lytA hybridization pattern of chromosomal SmaI digests is stable for a given strain during extensive serial culturing in the laboratory; the pattern is specific for the strain's clonal type, as defined by pulsed-field gel electrophoretis (PFGE) pattern, and variations in PFGE subtypes may be explained by changes in the number and chromosomal localization of this prophage(s). These observations indicate that the lytA hybridization pattern may be used as a molecular epidemiological marker that offers additional resolution of the genetic background of S. pneumoniae strains.     

Therapeutic failures of antibiotics used to treat macrolide-susceptible Streptococcus pyogenes infections may be due to biofilm formation

Streptococcus pyogenes infections often fail to respond to antibiotic therapy, leading to persistent throat carriage and recurrent infections. Such failures cannot always be explained by the occurrence of antibiotic resistance determinants, and it has been suggested that S. pyogenes may enter epithelial cells to escape antibiotic treatment. We investigated 289 S. pyogenes strains isolated from different clinical sources to evaluate their ability to form biofilm as an alternative method to escape antibiotic treatment and host defenses. Up to 90% of S. pyogenes isolates, from both invasive and noninvasive infections, were able to form biofilm. Specific emm types, such as emm6, appeared to be more likely to produce biofilm, although variations within strains belonging to the same type might suggest biofilm formation to be a trait of individual strains rather than a general attribute of a serotype. Interestingly, erythromycin-susceptible isolates formed a significantly thicker biofilm than resistant isolates (P < 0.05). Among resistant strains, those carrying the erm class determinants formed a less organized biofilm than the mef(A)-positive strains. Also, prtF1 appeared to be negatively associated with the ability to form biofilm (P < 0.01). Preliminary data on a selection of strains indicated that biofilm-forming isolates entered epithelial cells with significantly lower efficiency than biofilm-negative strains. We suggest that prtF1-negative macrolide-susceptible or mef(A)-carrying isolates, which are poorly equipped to enter cells, may use biofilm to escape antimicrobial treatments and survive within the host. In this view, biofilm formation by S. pyogenes could be responsible for unexplained treatment failures and recurrences due to susceptible microorganisms.     

Discrimination of Streptococcus pneumoniae from viridans group streptococci by genomic subtractive hybridization

Two oligonucleotide primer sets for the discrimination of Streptococcus pneumoniae from "pneumococcus-like" oral streptococcal isolates by PCR were developed. Genomic subtractive hybridization was performed to search for differences between Streptococcus pneumoniae strain WU2 and the most closely related oral streptococcus, Streptococcus mitis strain 903. We identified 19 clones that contained S. pneumoniae-specific nucleotide fragments that were absent from the chromosomal DNA of typical laboratory strains of S. mitis and other oral bacteria. Subsequently, oligonucleotide PCR primers for the detection of S. pneumoniae were designed from the sequences of the subtracted DNA fragments, and the specificities of the 19 primer sets were evaluated by PCR using chromosomal DNAs extracted from four S. pneumoniae clinical isolates and from 20 atypical organisms classified as S. mitis or S. oralis, which harbored genes encoding the pneumococcal virulence factors autolysin (lytA) or pneumolysin (ply), as templates. Of the 19 primer sets, two (Spn9802 and Spn9828) did not amplify PCR products from any of the pneumococcus-like streptococcal strains that we examined. The genes containing the Spn9802 and Spn9828 sequences encoded proteins of unknown function that did not correspond to any previously described proteins in other bacteria. These new oligonucleotide primers may be very useful for early and correct diagnosis of S. pneumoniae infections.     

DNA probe for identification of Streptococcus pneumoniae

A total of 287 clinical isolates of Streptococcus pneumoniae (pneumococcus) were tested for their ability to undergo autolysis when treated with sodium deoxycholate. The test was positive for all but one isolate, strain DOC-1. This autolysis required the activity of an enzyme which is unique and characteristic of S. pneumoniae: a choline-dependent N-acetylmuramoyl-L-alanine amidase, the gene product of the lytA gene. We used lytA as a DNA probe to test the distribution of the autolysin gene among clinical isolates of S. pneumoniae. In dot blot hybridization experiments our probe reacted with the DNA of 60 of 60 strains tested, including the autolysis-deficient clinical isolate DOC-1. No hybridization occurred when strains of Streptococcus sanguis, Streptococcus mutans, Streptococcus pyogenes, Streptococcus (Enterococcus) faecalis, Streptococcus (Enterococcus) faecium, Streptococcus agalactiae, and Streptococcus bovis were tested. The lytA gene appears to be an ideal candidate for use as a DNA probe for the identification of S. pneumoniae.     

Differentiation of resistance phenotypes among erythromycin-resistant streptococci

Although macrolide antibiotics have proved to be a valuable alternative to beta-lactam antibiotics in the treatment of respiratory tract infections, resistance to these agents is now becoming established in streptococci, especially among Streptococcus pneumoniae isolates. Of particular concern is the emergence of cross-resistance to 14-, 15- and 16-membered macrolides, licosamides and group B streptogramins (MLSb phenotype). MLS resistance can be expressed either constitutively (cMLS phenotype) or inducibly (iMLS phenotype). MLS resistance is mediated by two classes of methylase genes--the conventional erm(B) and recently described erm(A) determinants. A new macrolide efflux mechanism has been described for streptococci, in which it is associated with a new resistance pattern (M phenotype) characterized by resistance to 14- and 15-membered macrolides, and susceptibility to 16-membered macrolides, lincosamides and streptogramin B. The recognition of the prevalence of M phenotype in streptococci has implications for sensitivity testing and may have an impact on the choice of antibiotic therapy in clinical practice. While M resistance is similar in S. pyogenes and S. pneumoniae being mediated by mef(A) and mef(E), respectively, MLS resistance in both species appears to be genotypically and phenotypically more varied. Differentiation of M and MLS phenotypes of erythromycin-resistant strains can be performed using the erythromycin-clindamycin double-disc method (ECDD). Distinguishing not only M resistance but also constitutively or inducibly MLS phenotype by ECDD in S. pyogenes is easily and reliably achieved. Inducible MLS phenotype S. pyogenes strain is genotypically and phenotypically heterogeneous and is further subdivided into three recently described subtypes, iMLS-A, iMLS-B and iMLS-C, by a triple-disk test with erythromycin plus clindamycin and josamycin. While distinguishing M from MLS resistance in S. pneumoniae by ECDD test is easily and reliably achieved, the differentiation between constitutive and inducible MLS resistance is by far more uncertain. The meaning of inducible MLS resistance appears to be different in S. pneumoniae from that in S. pyogenes. In order to easily differentiate, within erythromycin-resistant pneumococci, not only the strains of the M phenotype from those with MLS resistance but also among the latter, cMLS from iMcLS strains, a triple-disk test has been set up by adding a rokitamycin disk to the conventional