Primary laryngeal cryptococcosis resembling laryngeal carcinoma.

A case of an 82-year-old female with primary laryngeal cryptococcosis who had undergone long-term corticosteroid therapy for chronic obstructive pulmonary disease and rheumatoid arthritis is reported. She complained hoarseness with swallowing pain and irritability of the larynx for over a month. Endoscopic examination revealed a white, exudative irregular region on right arytenoid that mimicked a laryngeal carcinoma. Histological examination showed pseudoepitheliomatous hyperplasia and severe submucosal inflammation with ovoid budding yeasts by Grocott's stain. A serological study indicated a high titer of cryptococcal antigen. After treating with oral fluconazole for 3 months, her primary lesion of larynx turned to be clear. We implicate a long-term use of steroids as the significant risk factor in developing cryptococcosis of the larynx.

Genetic characteristics of Haemophilus influenzae and Streptococcus pneumoniae isolated from children with conjunctivitis-otitis media syndrome.

Acute conjunctivitis is the most common ocular disorders among children and frequently concomitant with acute otitis media (AOM) as conjunctivitis-otitis syndrome. In this study, we evaluated prevalence of causative pathogens and PCR-based genotypes of Haemophilus influenzae and Streptococcus pneumoniae among children with conjunctivitis-otitis media syndrome. Nontypeable H. influenzae (NTHi) is identified most often at 61.8% in conjunctiva exudates followed by S. pneumoniae at 28.2% and Moraxella catarrhalis at 19.1%. Genetic ß-lactamase nonproducing ampicillin resistant (gBLNAR) strains of NTHi and genetic penicillin resistant S. pneumoniae (gPRSP) were identified at 72.1% and at 74.2% among conjunctiva isolates by polymerase chain reaction (PCR), respectively. Pneumococcal strains having either ermB or mefE genes were identified at 93.5% among conjunctiva isolates. The restriction fragment of patterns of 89.7% pairs of H. influenzae isolates and 100% pairs of pneumococcal isolates from conjunctiva exudates, middle ear fluids (MEFs) and nasopharyngeal swabs were identical. In contrast to the previous reports, most prevalent strains from conjunctivitis-otitis media syndrome was BLNAR H. influenzae in this study. The causative pathogen responsible for acute conjunctivitis will be originated from the nasopharynx. In the absence of MEFs one can possibly rely on the nasopharyngeal culture to guide an appropriate treatment.

Activation of NF-κB via endosomal Toll-like receptor 7 (TLR7) or TLR9 suppresses murine herpesvirus 68 reactivation.

UNLABELLED: In order to understand and possibly treat B-cell malignancies associated with latent gammaherpesvirus infection, it is vital to understand the factors that control the balance between the two transcriptional states of gammaherpesviruses: latency and lytic replication. We used murine gammaherpesvirus 68 (MHV 68) as a model system to investigate how engagement of endosomal Toll-like receptors (TLRs) impacts reactivation from latency in vitro and establishment of latent infection in vivo. We found that treatment with TLR7 ligand R848 or TLR9 ligand CpG oligodeoxynucleotide (ODN) suppresses reactivation of MHV 68 in vitro. These suppressive effects correlated with the ability to activate cellular transcription factor NF-κB. Downregulation of TLR9 by RNA interference in vitro led to a reduction of nuclear levels of NF-κB p65 and consequently to an increase of spontaneous reactivation in cells latently infected with MHV 68, indicating that the TLR9 pathway suppresses spontaneous reactivation events. In vivo, sustained stimulation of TLR7 by repeated R848 treatment led to an increased frequency of infected splenocytes compared to mock-treated control results. Frequencies of infected splenic B cells in tlr7-/- or tlr9-/- mice after establishment of latency did not differ from those seen with their wild-type counterparts. Nevertheless, MHV 68-infected B cells from tlr9-/- mice showed a higher frequency of reactivation than B cells from wild-type or tlr7-/- mice in ex vivo reactivation assays. Thus, we show a suppressive effect of TLR7 or TLR9 triggering on MHV 68 reactivation that correlates with NF-κB activation and that the mere presence of a functional TLR9 signaling pathway contributes to dampen lytic gammaherpesvirus reactivation in infected cells. IMPORTANCE: A hallmark of gammaherpesviruses is their establishment of latency in B cells that is reversible through lytic reactivation. Latency can result in B-cell malignancies. Activation of the innate immune system is thought to contribute to controlling the switch between the transcriptional states of latency and reactivation. Nevertheless, the mechanisms involved are not clear. Here, we show that engagement of Toll-like receptor 7 (TLR7) and TLR9 suppresses reactivation of murine gammaherpesvirus MHV 68 in vitro and that stimulation of TLR7 in vivo increases the frequency of infected cells. TLR7 and TLR9 are innate immunity sensors of nucleic acids localized in endosomes. Additionally, we demonstrate that impairment of TLR9 signaling in latently infected B cells leads to increased reactivation. Thus, activated endosomal TLR7 and TLR9 pathways play an important role in promoting establishment of latent gammaherpesvirus infection. Counteracting signaling of these pathways allows reactivation and could represent treatment targets in gammaherpesvirus-associated malignancies.

Clinical and microbiological impact of human bocavirus on children with acute otitis media.

Human Bocavirus (HBoV) as a newly discovered parvovirus has been commonly detected in respiratory tract infections. However, its role in acute otitis media (AOM) has not been well studied. We examined HBoV in Japanese children with AOM and evaluated the virus prevalence together with clinical manifestations and bacterial findings. Overall, 222 nasopharyngeal swabs and 176 middle ear fluids (MEF) samples were collected from 222 children with AOM (median age, 19 months) between May 2006 and April 2007. HBoV detection was performed by PCR and bacterial isolation by standard culture methods. HBoV was found in the nasopharyngeal aspirates of 14 children (6.3%) and in the MEF of six children (2.7%). When HBoV detection results were evaluated with clinical characteristics of children, resolution time of AOM was significantly longer (p=0.04), and rate of fever symptom was also higher in HBoV-positive group (p=0.04). Furthermore, we found positive correlation between detection of HBoV and Streptococcus pneumoniae in the MEF (p=0.004). Nevertheless, nasopharyngeal proportion of S. pneumoniae was similar between virus positive and negative groups. Furthermore, S. pneumoniae was detected as a single pathogen in all MEF of HBoV-positive cases but one, while it presents mixed with other pathogenic bacteria in nasopharynx. In conclusion, HBoV may worsen the clinical symptoms and prolong the clinical outcome of AOM in pediatric population. Finally, HBoV may prime the secondary bacterial infection in the middle ear in favor of S. pneumoniae.

Protection against systemic fatal pneumococcal infection by maternal intranasal immunization with pneumococcal surface protein A (PspA).

Streptococcus pneumoniae is a leading causative pathogen responsible for various types of bacterial infectious diseases in children. The aim of this study was to evaluate the protection conferred against fatal pneumococcal infections during infancy by maternal intranasal immunization with pneumococcal surface protein A (PspA). Four-week-old female BALB/c mice were immunized with PspA mixed with, or without, cholera toxin B (CTB) intranasally twice a week for 3 weeks. After the final immunization, they were mated with male mice to obtain offspring. Offspring at 10 days old were intraperitoneally inoculated with a pneumococcus strain, TIGR4, serotype 4. After the infections their survival periods were monitored. Anti-PspA-specific IgG antibody was induced in sera and breast milk at birth and maintained for 14 days during nursing periods in the PspA-immunized mother mice. At birth, offspring delivered from PspA-immunized mother mice had levels of anti-PspA-specific IgG antibody in sera same to those in their mothers on the day of birth. The survival times to death of offspring delivered from PspA-immunized mother mice after systemic fatal pneumococcal infections were significantly extended compared to those of controls. These findings suggest that maternal intranasal immunization with PspA could be an attractive procedure to employ against pneumococcal infections in early childhood.

Determination of pneumococcal serotypes/genotypes in nasopharyngeal secretions of otitis media children by multiplex PCR.

The appropriate clinical applications of pneumococcal polysaccharide vaccines against recent increases in antimicrobial resistant Streptococcus pneumoniae (S. pneumoniae) urgently require accurate analytical methodologies for determining and characterizing the serotypes. The results of current immunological determinations of serotypes with anti-capsular polysaccharide-specific sera are difficult to interpret in terms of quellung changes of the pneumococci. In this study, we applied the multiplex PCR technique for the rapid identification of pneumococci and simultaneous rapid determinations of their serotypes and genotypes that directly correlated with antimicrobial susceptibilities from nasopharyngeal secretions (NPS). Serogroups 6, 19F and 23F were the predominant capsular types of S. pnuemoniae in the NPS samples. Strains of serotypes 19F and 23F frequently had mutations in pbp1a, pbp2x and pbp2b and expressed ermB and mefA; they also were mostly resistant to both penicillin G (PCG) and clarithromycin (CAM). Two NPS samples contained the strain of serotype 19F together with the strain of serotype 23F, although only the strain of serotype 19F was identified by a conventional bacterial culture. Pneumococci were identified in six NPS samples and their serotypes determined by the multiplex PCR, while a conventional bacterial culture failed to identify the pathogens. Our findings suggest that PCR-based serotyping and genotyping can provide an accurate and rapid distribution of pneumococcal serotypes and antimicrobial resistance. The relatively minor populations in the nasopharynx may be determined using molecular techniques.

Efficacy of a novel oral carbapenem, tebipenem pivoxil (TBM-PI), against experimental otitis media caused by penicillin resistant Streptococcus pneumoniae in chinchilla.

An animal model of otitis media using chinchillas was developed to evaluate the efficacy of tebipenem pivoxil (TBM-PI) against experimental otitis media. Chinchillas inoculated via the transbullar approach with Streptococcus pneumoniae serogroup 6 were included in the efficacy study with TBM-PI, amoxicillin (AMX) or untreated as controls. TBM-PI resulted in survival rate of 83%, compared with 25% survival for AMX and 0% survival for controls (p<0.01). Quantitative cultures in the middle ear effusions at day 5 of the TBM-PI group yielded 3.5+/-2.4log(10)CFUs/ml. TBM-PI is a promising antibiotic for the treatment of acute otitis media.

Rapid identification of nontypeable and serotype b Haemophilus influenzae from nasopharyngeal secretions by the multiplex PCR.

OBJECTIVE: Heamophilus influenzae (H. influenzae) is an important pathogen responsible for both invasive and non-invasive infectious diseases. While encapsulated type b strain recognized as a major cause of severe invasive diseases, nontypeable strains are the major causes of non-invasive infectious diseases. Detection of this pathogen from nasopharyngeal secretions (NPS) is important. METHODS: We developed a multiplex polymerase chain reaction (PCR) for rapid identification of nontypeable and serotype b H. influenzae from nasopharyngeal secretions. RESULTS: A total 25 nasopharyngeal secretions were evaluated in this study. The multiplex PCR provided rapid and unequivocal results for determining either nontypeable or encapsulated typeable especially type b strains including a determination of beta-lactamase productions. CONCLUSION: The multiplex PCR based serotyping provided more reliable results than slide agglutination test (SAT) and is a valuable and expeditious method for identification of H. influenzae with determining capsular serotypes.

Maternal intranasal immunization with outer membrane protein P6 maintains specific antibody level of derived offspring.

Acute otitis media is one of the most common infectious diseases in children younger than 2 years of age. Immunological studies in young children have revealed that immature antibody-responses to major pathogens, such as S. pneumoniae, H.influenzae, would cause the vulnerability to upper respiratory tract infections. Thus, it is very important to induce effective protective immunity among children younger than 2 years of age. In this study, we evaluated the capacity of maternal immunization with P6 of H. influenzae to evoke specific antibody to P6 and to transfer it to offspring. We intranasally immunized mother mice with P6 and investigated the induction of specific antibody in sera and breast milk. The specific antibody among offspring delivered by immunized mother was also investigated according to the nursing status to evaluate the importance of breast feedings by immunized mothers. Anti-P6 specific IgG in sera were high at delivery and maintained during nursing periods among P6-immunized mother mice. Anti-P6 specific IgG were predominantly induced in breast milk. IgG subclass induced in sera and breast milk from P6-immunized mother mice were IgG2b, followed by IgG1 and IgG2a subclass. Offspring delivered by P6-immunized mothers had anti-P6 specific IgG in sera at the birth. The levels of anti-P6 specific IgG in sera from offspring breast-fed by P6-immunized mothers were then increased until day 14 and then decreased on day 21. The anti-P6 specific IgG in sera from offspring breast-fed by sham-immunized mothers were rapidly decreased after birth. The current findings strongly suggest that maternal intranasal immunization with P6 would be an attractive strategy against NTHi infections during early childhood. It can supply protective antibodies via transplacental transfer during pregnancy and via breast milk after birth.

Kinetics and product analysis of the reaction catalysed by recombinant homoaconitase from Thermus thermophilus.

HACN (homoaconitase) is a member of a family of [4Fe-4S] cluster-dependent enzymes that catalyse hydration/dehydration reactions. The best characterized example of this family is the ubiquitous ACN (aconitase), which catalyses the dehydration of citrate to cis-aconitate, and the subsequent hydration of cis-aconitate to isocitrate. HACN is an enzyme from the alpha-aminoadipate pathway of lysine biosynthesis, and has been identified in higher fungi and several archaea and one thermophilic species of bacteria, Thermus thermophilus. HACN catalyses the hydration of cis-homoaconitate to (2R,3S)-homoisocitrate, but the HACN-catalysed dehydration of (R)-homocitrate to cis-homoaconitate has not been observed in vitro. We have synthesized the substrates and putative substrates for this enzyme, and in the present study report the first steady-state kinetic data for recombinant HACN from T. thermophilus using a (2R,3S)-homoisocitrate dehydrogenase-coupled assay. We have also examined the products of the reaction using HPLC. We do not observe HACN-catalysed 'homocitrate dehydratase' activity; however, we have observed that ACN can catalyse the dehydration of (R)-homocitrate to cis-homoaconitate, but HACN is required for subsequent conversion of cis-homoaconitate into homoisocitrate. This suggests that the in vivo process for conversion of homocitrate into homoisocitrate requires two enzymes, in simile with the propionate utilization pathway from Escherichia coli. Surprisingly, HACN does not show any activity when cis-aconitate is substituted for the substrate, even though other enzymes from the alpha-aminoadipate pathway can accept analogous tricarboxylic acid-cycle substrates. The enzyme shows no apparent feedback inhibition by L-lysine.

High prevalence of Streptococcus pneumoniae with mutations in pbp1a, pbp2x, and pbp2b genes of penicillin-binding proteins in the nasopharynx in children in Japan.

OBJECTIVE: To evaluate the resistances of Streptococcus pneumoniae to beta-lactams developed by stepwise alterations in high-molecular-weight penicillin-binding proteins (PBPs) with a reduced binding affinity of beta-lactams. Among the numerous mutations in pbp genes that alter the affinity for beta-lactams, the decreased affinity of PBP1A, 2X and 2B is especially important in the development of resistances to beta-lactams. STUDY DESIGN: Retrospective review. METHODS: In this study, we investigated the mutations in pbp1a, pbp2x, and pbp2b genes evaluated by polymerase chain reaction (PCR) in 866 pneumococcal isolates collected from the nasopharynx of Japanese children with acute otitis media. RESULTS: 210 strains (24.3%) exhibited no mutations in the three pbp genes. 333 strains (38.5%) had mutations in the three pbp genes, 78 (9.0%) in two pbp genes, whereas 245 (28.3%) displayed mutations in only one pbp gene. Among the 656 strains with mutations in pbp genes, 620 (94.5%) strains had mutations in pbp2x. The annual prevalence of antimicrobial-resistant S. pneumoniae showed a gradual increase in strains with mutations in the three pbp genes and a parallel decrease in strains without mutations. CONCLUSIONS: PCR-based genotyping can characterize the antimicrobial resistances in pneumococci along with minimal inhibitory concentrations (MICs). Physicians should pay attention to the recent increase in antimicrobial-resistant S. pneumoniae when treating pediatric acute otitis media.

Evaluation of mutations in penicillin binding protein-3 gene of non-typeable Haemophilus influenzae isolated from the nasopharynx of children with acute otitis media.

CONCLUSION: Younger children tend to harbor more resistant strains because they are exposed to these pathogens more often through contacts with siblings or attendance at day-care centers and are frequently treated with antibiotics. The high prevalence of BLNAR strains should be taken into account in the treatment of AOM in young children. OBJECTIVE: Non-beta-lactamase-producing ampicillin-resistant (BLNAR) strains with mutations in penicillin-binding protein (PBP) genes of Haemophilus influenzae have been prevalent recently among younger children. MATERIAL AND METHODS: We investigated mutations in the ftsI gene encoding PBP-3 of H. influenzae isolated from the nasopharynx of children with acute otitis media (AOM) using polymerase chain reaction (PCR). RESULTS: Strains containing the bla gene (beta-lactamase-producing ampicillin-resistant) were identified in 4.7% of cases. Strains with mutations in the ftsI gene (BLNAR) were identified in 23.3% of cases. Strains without mutations in the ftsI gene and that did not contain the bla gene (non-beta-lactamase-producing ampicillin-susceptible) were identified in 70.7% of cases. Strains with both expression of the bla gene and mutations in the ftsI gene (beta-lactamase-producing amoxicillin clavulanate-resistant) were identified in 1.3% of cases. The MICs of ampicillin against the strains evaluated in this study were 0.5-2.0 microg/ml. Cefditoren-pivoxil had the lowest MIC90 against the strains (0.06 microg/ml). Strains with mutations in the ftsI gene (BLNAR) were broadly identified among young children.

Increase of macrolide-resistant Streptococcus pneumoniae-expressing mefE or ermB gene in the nasopharynx among children with otitis media.

OBJECTIVE: To evaluate prevalence of macrolide resistant strains and the genotypes of the resistance among Streptococcus pneumoniae isolated from the nasopharynx of children with otitis media. STUDY DESIGN: Retrospective review. METHODS: A total of 858 S. pneumoniae isolates were collected from the nasopharynx of pediatric patients with acute otitis media at the clinics of Otolaryngology-Head and Neck Surgery, Wakayama Medical University Hospital and six affiliated hospitals in Wakayama prefecture between January 1998 and December 2002. The antibiotic susceptibility patterns were analyzed for penicillin, erythromycin, and clindamycin according to the National Committee for Clinical Laboratory Standards. Macrolide resistance genes of mefE and ermB were determined by polymerase chain reaction of all S. pneumoniae. RESULTS: Of 858 clinical isolates, 259 (30.1%) were strains without ermB or mefE gene, 279 (32.5%) carrying mefE, 292 (34.0%) carrying ermB, and 28 (3.4%) carrying both genes. There was a strong correlation between phenotypes and the presence of macrolide resistance genes. The macrolide resistance genes were especially frequently identified among penicillin-resistant S. pneumoniae. Strains carrying ermB gene gradually increased from 25% in 1998 to 45% in 2002, with a concurrent decrease in strains carrying mefE from 36% in 1998 to 1999 to 19% in 2002. Strains having mefE were frequently identified among children younger than 2 years old. The current finding suggested that high-level ermB-mediated macrolide resistance in S. pneumoniae is increasing at an alarming rate in pediatric patients with otitis media, especially among young children. Physicians should pay close attention to such macrolide-resistant bacterial pathogens in the antimicrobial treatment of pediatric patients with otitis media.

Current status of antimicrobial susceptibility of clinical isolates of Streptococcus pyogenes in Japan: report of a countrywide surveillance study.

The growing number of macrolide-resistant strains of Streptococcus pyogenes is an increasing problem worldwide. In this study, we evaluated 62 clinical isolates of S. pyogenes obtained from the upper respiratory tract. Susceptibilities to penicillins, cephalosporins, fluoroquinolones, macrolides, and carbepenems were determined by minimal inhibitory concentrations (MICs). Expressions of macrolide-resistance genes (mefA, ermB, and ermTR) were also examined, by polymerase chain reaction (PCR). All strains were susceptible to beta-lactams. On the other hand, of the 62 S. pyogenes isolates, 6.5%, 6.5%, 6.5%, and 3.2% of the strains were resistant to azithromycin (AZM), roxithromycin (RXM), clarithromycin (CAM), and telithromycin (TEL), respectively. Four (6.5%) strains had a type of macrolide-resistance gene; there were two strains with ermB and two strains with ermTR, and these four strains were resistant to AZM, CAM (one strain was intermediately resistant), and RXM. Strains having ermB were resistant to TEL (MIC, > or = 8 microg/ml), while strains having ermTR were susceptible to TEL. Physicians and researchers need to take into consideration the macrolide resistance of some strains of S. pyogenes.

A recombinant P4 protein of Haemophilus influenzae induces specific immune responses biologically active against nasopharyngeal colonization in mice after intranasal immunization.

Outer membrane protein P4, together with P6, is highly conserved among all typeable and nontypeable strains of Haemophilus influenzae (H. influenzae). Thus, the protein is an attractive antigen for the inclusion in a vaccine against nontypeable H. influenzae (NTHi). However, the ability of P4 to induce antibodies protective against NTHi infections is still controversial. In this study, we investigated the specific mucosal immune responses against NTHi induced by intranasal immunization with the lipidated form of recombinant P4 protein (rP4) and non-fatty acylated recombinant P6 protein (rP6) with or without cholera toxin (CT) in BALB/c mice model. Intranasal immunization with either rP4+CT, a mixture of rP4 and rP6+CT, or rP4 and rP6 without CT elicited anti-rP4 specific IgG antibody in serum of mice. Intranasal immunization with either rP4+CT or a mixture of rP4, rP6+CT elicited anti-rP4 specific IgA antibody in nasopharyngeal washing (NPW), while intranasal immunization with rP4 and rP6 without CT did not induced anti-rP4 specific IgA antibody responses in NPWs. Sera from mice intranasally immunized with rP4+CT and a mixture of rP4, rP6+CT also showed bactericidal activity. Significant clearance of NTHi in nasopharynx was seen 3 days after the inoculation of live NTHi in mice intranasally immunized with rP4+CT. The current findings suggested that P4 would be a useful antigen as the component of the vaccine to induce protective immune responses against NTHi. The use of an intranasal vaccine composed of the different surface protein antigens is an attractive strategy for the development of a vaccine against NTHi.

Genotyping of Streptococcus pneumoniae and Haemophilus influenzae isolated from paired middle ear fluid and nasopharynx by pulsed-field gel electrophoresis.

Twenty-eight isolates of Streptococcus pneumoniae and 30 isolates of Haemophilus influenzae from paired nasopharynx and middle ear fluids of 21 children with acute otitis media (AOM) were evaluated to determine genotypes by polymerase chain reaction and pulsed-field gel electrophoresis (PFGE). Among the 28 isolates of S. pneumonaie, 21 isolates (75.0%) possessed mutations in the pbp1a,pbp2x, and pbp2b genes, and 7 isolates (25%) had mutations in the pbp2x gene. Nineteen isolates (67.9%) expressed the mefE gene, and 5 isolates (17.9%) possessed the ermB gene. Among the 30 isolates of H. influenzae, 5 isolates (16.7%) had mutations in pbp3 genes, 3 isolates (10.0%) produced beta-lactamase, and 2 (6.7%) isolates possessed mutations both in the pbp3 gene and the beta-lactamase gene. Ten out of the 14 pairs (71.4%) of the restriction fragment patterns of S. pneumoniae from paired nasopharynx and middle ear fluids were indistinguishable following PFGE analysis. The same patterns were identified among 5 children of unrelated families. The restriction fragment patterns of H. influenzae isolated by PFGE were also indistinguishable in 13 out of the 15 pairs (86.7%) of nasopharynx and middle ear fluids. The genetic similarity between nasopharyngeal and middle ear isolates suggests that the causative bacteria migrate from the nasopharynx into the middle ear cavity via the Eustachian tube. Some resistant strains might be prevalent. In children with AOM, the nasopharynx could have been colonized by a virulent strain of bacteria that replaced the benign, commensal bacteria and then progressed to the middle ear, where they caused AOM.

Macrolide-resistant genes of Streptococcus pyogenes isolated from the upper respiratory tract by polymerase chain reaction.

The growing number of macrolide-resistant strains of Streptococcus pyogenes is an increasing problem worldwide. This study evaluated 300 clinical isolates obtained from the upper respiratory tract. Minimal inhibitory concentrations (MICs) of erythromycin (EM), azithromycin (AZM), and clindamycin (CLDM), serotypes, and macrolide resistance genes of mefA, ermB, and ermTR were determined. The genetic relationship of EM-resistant and susceptible strains were also analyzed by pulsed-field gel electrophoresis (PFGE). Twenty-nine (9.7%) EM-resistant S. pyogenes were identified. Of the 29 strains showing resistance to EM, 22 isolates (7.3%, MIC 3.13-12.5 microg/ml) expressed the mefA gene. The predominant serotypes among the mefA-positive isolates were T12, emm9 or T25, emm75-1. The two isolates (0.1%) that possessed the ermB gene were highly resistant to EM (MIC > 100 microg/ml). The remaining five strains (1.6%) possessed the ermTR gene (MIC 3.13-100 microg/ml). Restriction fragment polymorphism analyzed by pulsed-field gel electrophoresis (PFGE) by SmaI and ApaI digestions showed several clones among the mefA-positive S. pyogenes. Our findings suggest that the mefA gene is the predominant mechanism for macrolide resistance and that this gene is horizontally transmitted among M phenotype strains of S. pyogenes. Consequently, macrolides would not be the first drug of choice for treatment of tonsillitis and other S. pyogenes-related diseases. Physicians and researchers need to take into consideration the macrolide resistance of some strains of S. pyogenes.