Rinya Sugita

Low Concentrations of Mupirocin in the Pharynx following Intranasal Application May Contribute to Mupirocin Resistance in Methicillin-Resistant Staphylococcus aureus

ABSTRACT We describe a patient with methicillin-resistantStaphylococcus aureus (MRSA) colonizing the pharynx. The MIC of mupirocin was 0.25 μg/ml before treatment and increased after treatment to 8 μg/ml. Using pulsed-field gel electrophoresis, we confirmed that the genotypes of MRSA that colonized the pharynx before and after the use of mupirocin were identical. We measured the delivery of mupirocin to the pharynx in three normal volunteers and two patients. Low concentrations of mupirocin were present in the pharynx in all cases 10 min to 3 days after intranasal application. Our data suggested that low concentrations of the drug in the pharynx after intranasal application of mupirocin ointment might explain the selection of mupirocin resistance in MRSA.

High Rate of Transmission of Penicillin-Resistant Streptococcus pneumoniae between Parents and Children

ABSTRACT Transmission of Streptococcus pneumoniae between children and their parents was evaluated in 29 pairs from 25 families. The serotypes of 35 pneumococcal isolates from 18 (62.1%) of 29 child-parent pairs were identical. Of the 35 isolates, 23 showed intermediate resistance and 10 were fully resistant to penicillin G. PCR indicated that all 35 strains had at least one alteration in penicillin-binding protein genes pbp1a, pbp2x, and pbp2b and 33 strains had macrolide resistance genes mef(A) and/or erm(B). As a result, the PCR patterns of 16 of 18 pairs were identical. Molecular typing by pulsed-field gel electrophoresis showed that 12 pairs were indistinguishable, 3 pairs were closely related, 2 pairs were possibly related, and only one pair was different. Our data indicate the presence of a high rate of transmission of penicillin-resistant S. pneumoniae between children and their parents.

Serotype Distribution and Penicillin Resistance of Streptococcus pneumoniae Isolates from Middle Ear Fluids of Pediatric Patients with Acute Otitis Media in Japan

ABSTRACT Out of 175 pneumococcal isolates from middle ear fluids, 26.3% were penicillin-resistant S. pneumoniae (PRSP). Serotypes 19F and 23F occurred most frequently among PRSP strains. The 7-valent pneumococcal conjugate vaccine (PCV) showed better coverage of PRSP strains (87.0%). The 7-valent PCV may reduce the prevalence of PRSP in Japan.

Possible High Rate of Transmission of Nontypeable Haemophilus influenzae, Including β-Lactamase-Negative Ampicillin-Resistant Strains, between Children and Their Parents

ABSTRACT The possible transmission of nontypeable Haemophilus influenzae between children and their parents was evaluated in 18 pairs of subjects from 15 families. Of the 33 isolates, 31 were found to be β-lactamase negative, including 10 β-lactamase-negative, ampicillin (AMP)-resistant (BLNAR) strains (AMP MIC, ≥1.0 μg/ml) and 2 were β-lactamase producing. Molecular typing by pulsed-field gel electrophoresis (PFGE) showed that 10 BLNAR isolates had 6 patterns, 23 non-BLNAR isolates had 13 patterns, and these patterns were different from each other, except for 1 pattern. As a result, the PFGE patterns in 14 of 18 pairs were indistinguishable and those in 4 pairs were different. These data indicate a possible high rate of intrafamilial transmission of nontypeable H. influenzae, including BLNAR strains, between children and their parents.

Studies on anaerobic bacteria in chronic otitis media

In the 760 cases of chronic otitis media studied from 1970 to 1976, the detection ratio of anaerobic bacteria and the relation between the nature of anaerobic bacteria and the conditions of focus were investigated. In 8.2% of 760 cases 9 kinds and 81 strains of anaerobic bacteria was detected. Most of the anaerobic bacteria were Peptococcus sp. or Bacteroides sp. and in general non‐sporogenic anaerobic bacteria accounted for 97.5%. Anaerobes were frequently detected in cases where the middle ear cavity was filled with cholesteatoma or granulation and in cases of infection recurring after operation.

Evaluation of a rapid immunochromatographic ODK-0901 test for detection of pneumococcal antigen in middle ear fluids and nasopharyngeal secretions.

Since the incidence of penicillin-resistant Streptococcus pneumoniae has been increasing at an astonishing rate throughout the world, the need for accurate and rapid identification of pneumococci has become increasingly important to determine the appropriate antimicrobial treatment. We have evaluated an immunochromatographic test (ODK-0901) that detects pneumococcal antigens using 264 middle ear fluids (MEFs) and 268 nasopharyngeal secretions (NPSs). A sample was defined to contain S. pneumoniae when optochin and bile sensitive alpha hemolytic streptococcal colonies were isolated by culture. The sensitivity and specificity of the ODK-0901 test were 81.4% and 80.5%, respectively, for MEFs from patients with acute otitis media (AOM). In addition, the sensitivity and specificity were 75.2% and 88.8%, respectively, for NPSs from patients with acute rhinosinusitis. The ODK-0901 test may provide a rapid and highly sensitive evaluation of the presence of S. pneumoniae and thus may be a promising method of identifying pneumococci in MEFs and NPSs.

Distribution of fibronectin-binding protein genes ( prtF1 and prtF2 ) and streptococcal pyrogenic exotoxin genes ( spe ) among Streptococcus pyogenes in Japan

Two hundred and seventy-two strains of Streptococcus pyogenes isolated from patients with invasive and noninvasive infections in Japan were evaluated for the prevalence of fibronectin-binding protein genes (prtF1 and prtF2). The possible associations of the genes with streptococcal pyrogenic exotoxin genes, macrolide resistance genes, and emm types were also evaluated. Overall, about 50% of S. pyogenes isolates carried fibronectin-binding protein genes. The prevalence of the prtF1 gene was significantly higher among isolates from noninvasive infections (71.4%) than among isolates from invasive infections (30.8%; P = 0.0037). Strains possessing both the prtF1 and prtF2 genes were more likely to be isolates from noninvasive infections than isolates from invasive infections (50.6% vs 15.4%; P = 0.019). S. pyogenes isolates with streptococcus pyrogenic exotoxin genes (speA and speZ) were more common among isolates without fibronectin-binding protein genes. The speC gene was more frequently identified among isolates with fibronectin-binding protein genes (P = 0.05). Strains belonging to emm75 or emm12 types more frequently harbored macrolide resistance genes than other emm types (P = 0.0094 and P = 0.043, respectively). Strains carrying more than one repeat at the RD2 region of the prtF1 gene and the FBRD region of the prtF2 gene were more prevalent among strains with macrolide resistance genes than among strains negative for macrolide resistance genes. These genes (i.e., the prtF1, prtF2, and spe genes) may enable host-bacteria interaction, and internalization in the host cell, but may not enable infection complications such as invasive diseases.

Prevalence of Streptococcus Invasive Locus (sil) and Its Relationship with Macrolide Resistance among Group A Streptococcus Strains

A recent study by Bidet et al. (1) reported the molecular epidemiology of the streptococcal invasive locus (sil) in the group A streptococcus (GAS), an organism which caused invasive infections in French children. The authors demonstrated the prevalence of emm type toxin genotypes among 74 invasive GAS isolates from French children. The authors PCR amplified and characterized the locus DNA of sil from invasive isolates, but there were no data concerning noninvasive isolates. It seems that the invasive locus was present not only in invasive isolates but possibly also in noninvasive isolates. Therefore, we conducted a study in which our aims were (i) to examine the prevalence of Streptococcus pyogenes exotoxins in relationship to the sil gene in invasive and noninvasive isolates of GAS, (ii) to define whether sil was predominantly present only in invasive isolates or also in noninvasive isolates of GAS, and (iii) to characterize the relationship between GAS and macrolide resistance. To set up our hypothesis, we examined 242 noninvasive isolates (tonsillitis, 170 isolates; rhinosinusitis, 51 isolates; and acute otitis media, 21 isolates) and 13 invasive isolates (septicemia, 5 isolates; purulent arthritis, 4 isolates; meningitis, 2 isolates; necrotizing fasciitis, 1 isolate; and peritoneal abscess, 1 isolate) of GAS, which were isolated from individual patients. emm typing of GAS strains was performed by DNA sequencing according to the recommendations of the Division of Bacterial and Mycotic Diseases, the Centers for Disease Control and Prevention, and the emm sequence database (http://www.cdc.gov/ncidod/biotech/strep/strepindex.htm). Multiplex PCR was used for toxin gene (speA, speB, speC, speF, speG, speH, speJ, ssa, and smeZ) profiling, as described by Schmitz et al. (5). PCR detection of the sil locus was performed according to the method described by Bidet et al. (1). Macrolide resistance genes of GAS were determined by the PCR methods described by Weber et al. (6). To study the degree of macrolide resistance, MICs of azithromycin to all strains were determined by broth microdilution, using the standard method (2). All the experiments were conducted in duplicate. Among the 242 noninvasive isolates, 11.98% (29/242) harbored the sil gene in their genomic DNA. The emm types and the toxin gene profiles of sil-positive isolates are shown in Table ​Table1.1. In noninvasive strains, the sil locus was detected in 9 out of 33 emm types found in the collection (27.27%), and 41.4% (12/29) of the sil-positive isolates belonged to emm type 4. emm type 4 (12 isolates), emm type 48 (3 isolates), and emm type 94 (6 isolates) represented 72.41% (21/29) of the sil-positive isolates. All of the sil-positive noninvasive isolates carried speB alleles, but 68.96% of strains carried speC. There were no significant differences between the toxin gene profile of the sil-positive isolates and that of the sil-negative isolates, except for smeZ, which was 10.3% of the sil-negative isolates but 31% of the sil-positive noninvasive isolates. Seventy-five percent of emm type 4, 75% of emm type 48, 100% of emm type 94, 100% of emm type 53, 100% of emm type 54, and 100% of emm type 102 isolates harbored the sil gene in their DNA. TABLE 1. Characteristics of streptococcal toxin gene profile of invasive and noninvasive sil-positive isolatesa Although we used limited numbers of invasive isolates, 15.4% of the invasive GAS isolates harbored the sil gene, which is consistent with data from a previous study of invasive strains, which showed that 16% carried the sil gene (1). One hundred percent of emm type 87 and 100% of emm sequence type 1732 were positive for the invasive locus. Thirty percent of the sil-negative invasive isolates carried speA alleles, but all sil-positive isolates were negative for the speA gene. All strains were positive for the speB gene. Fifty percent of the sil-positive isolates were positive for speC, but 30% of the sil-negative isolates were positive for speC. There is no statistical significance in the prevalence of the sil gene among invasive and noninvasive isolates (Fishers exact test, P = 0.499). Among 255 invasive and noninvasive isolates, 16.86% (3 were invasive, and 40 were noninvasive; total, 43/255) of the isolates were azithromycin resistant and were positive for macrolide-resistant genes (Table ​(Table2).2). Among these strains, 65.12% (28/43), 13.95% (6/43), and 20.93% (9/43) of the strains possessed the mef(A), erm(B), and erm(TR) genes, respectively. All sil-positive isolates were sensitive to azithromycin and were negative for macrolide resistance genes (Fishers exact test, P < 0.006). TABLE 2. Relationship between sil-positive and macrolide-resistant genes and invasive and noninvasive GASa From these result, we concluded that sil is present not only among invasive isolates but also among noninvasive isolates, with similar prevalences (15.4% versus 11.98%, respectively). To our knowledge, this is the first report to show the prevalence rates of sil in both invasive and noninvasive isolates of GAS in Japan. The predominant emm types that harbored sil were emm type 4, emm type 94, and emm type 48. Hidalgo-Grass et al. identified sil in the invasive serotype M14 clone, the organism that caused necrotizing fasciitis in Israel (3). In our study, sil was absent from emm type 3 isolates, a finding comparable to that in a previous study and associated with GAS invasive diseases worldwide (3). The sil locus was confirmed by direct sequencing of several representative PCR-amplified products and comparing those with the previous sequence. The overall prevalence of the sil locus in invasive isolates was the same as that from a previous study (16% versus 15.4%, respectively) (1). Up to now, there was no study which showed the status of noninvasive strains with the sil gene. When we examined noninvasive strains, the sil gene was found in 12% of isolates, which is not a remarkably different rate from that found in invasive isolates. All sil-positive isolates were negative for macrolide resistance genes, which were irreversibly important for clinical practice. Future studies should focus on a better understanding of the role of sil in the pathogenesis of GAS infection and its relationship with macrolide resistance. A recent candidate vaccine based on the M protein failed to elicit antibodies to serotype M4, and sil-encoded proteins might represent alternative vaccine targets for this serotype (4). The results of this study should contribute to a better understanding of the pathogenesis of GAS, as well as the epidemiology of GAS-associated disease, and to the establishment of methods for the prevention of diseases caused by GAS in Japan.

Bacteriological features and chemotherapy of adult acute purulent otitis media

Eighty-eight patients from 16 to 79 years old, with acute purulent otitis media, were bacteriologically examined at the Otorhinolaryngology Department of a primary care hospital in Tokyo from July 1979 to May 1983. Fifty-six patients underwent paracentesis, and 32 patients exhibited otorrhea due to previous spontaneous perforation of the tympanic membrane. Bacteriologic cultures revealed the presence of Streptococcus pneumoniae (62.5 per cent), including S. pneumoniae Type III (28.1 per cent), Haemophilus influenzae (10.5 per cent), Staphylococcus aureus (11.5 per cent), and Streptococcus pyogenes (7.3 per cent). S. pneumoniae Type III had a notably high detection rate in patients from 50 to 79 years old (50-75 per cent). Because Haemophilus influenzae was detected at a relatively high rate in patients of all ages, if can be considered as a major causative pathogen of AOM. In 44 patients, selected mainly from those who underwent paracentesis, a comparative study of bacteria found in middle ear fluid and naso-pharyngeal mucus revealed the same bacteria in 43 out of 44 cases (97.7 per cent), indicating the presence of bacterial infection through the auditory canal. Antibiotics were selected according to an Expected Efficacy Index (EEI), the antibiotic of first choice being Ampicillin or Cefaclor.

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 β-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.