Detection of Bartonella quintana (Hyphomicrobiales: Bartonellaceae) Among Day Laborers in Osaka, Japan, 2009-2010.

Bartonella quintana is a gram-negative bacterium causing trench fever, an illness historically acquired by soldiers during World War I. More recently, outbreaks of trench fever have been reported in those experiencing homelessness in the United States, France, Russia, and Tokyo, as well as in children in Nepal and persons in Ethiopia. Reports of B. quintana infection outside of Tokyo are rare in Japan. The aim of this study was to examine body lice and blood obtained from people staying in shelters in Osaka (2009-2010) for B. quintana via polymerase chain reaction and enzyme-linked immunosorbent assays. Day laborers were defined as homeless individuals and shelter residents in this study. We detected genes of B. quintana in body lice by PCR and antibodies against B. quintana. The positive rate of B. quintana genes was 6/10 (60%) in body lice and the seroprevalence (IgG) of B. quintana was 4/10 (40%). This demonstrates that trench fever was endemic in people staying in shelters in Osaka in 2009-2010.

Immunogenic Peptides from Pap31 and SCS-α of Bartonella bacilliformis: One Step Closer to a Rapid Diagnostic Tool for Carrion's Disease.

Bartonella bacilliformis is the causal agent of Carrion's disease, an overlooked illness endemic in the Andean Mountains with Peru being the most affected country. The diagnostic of this illness is a challenge due to the limited resources and the common symptomatology with other infectious diseases. The goal of this study was to identify immunogenic peptides from Pap31 and succinyl-CoA synthetase α (SCS-α) of B. bacilliformis that might be suitable for developing a serologic tool. The immunodominant character of Pap31 and SCS-α was determined by Western blotting and in-silico analysis. Subsequently, 35 peptides were selected for epitope mapping and their immunoreactivity was tested by enzyme-linked immunosorbent assay (ELISA). A total of 30 sera were tested including pre-exposed people with high IgM levels for Pap31/SCS-α (23 sera), patients (2 sera) as well as 5 sera with no reactivity to Pap31/SCS-α. The results indicate that Pap31-8 (187QAIGSAILKGTKDTGT202) and SCS-α-12 (59IFASVAEGKEKTGANA74) are the most immunogenic peptides, with Pap31-8 showing potential to discriminate between B. bacilliformis and the remaining Bartonella spp., and SCS-α-12 differentiating Bartonella spp. from other microorganisms.

Detection of Bartonella quintana Infection among the Homeless Population in Tokyo, Japan, from 2013-2015.

Several outbreaks of trench fever caused by Bartonella quintana occurred in soldiers during World Wars I and II. Although trench fever cases have been decreasing worldwide, the disease was reported among the homeless population in developing and developed countries. The current prevalence of B. quintana infection in Japan is unclear. Blood and body louse (Pediculus humanus humanus) samples were obtained from homeless inpatients with body lice during emergency hospitalization in Tokyo from January 2013 to March 2015. Patients were tested for B. quintana infections using the culture method, polymerase chain reaction, and indirect immunofluorescence assay (IFA). Among the 29 patients tested, the presence of Bartonella spp. was confirmed by genomic sequencing of DNA extracted from two samples from blood culture performed for 15 out of 29 patients and from body louse samples of 20 patients (69%). Immunoglobulin G against B. quintana was detected in 10 patients (34.5%) at a cut-off titer of 1:256 in IFA. B. quintana infection was detected in samples obtained between 2013 and 2015 in Tokyo and needs to be on the list of differential diagnoses performed for febrile homeless individuals.

A nationwide population-based surveillance of invasive Haemophilus influenzae diseases in children after the introduction of the Haemophilus influenzae type b vaccine in Japan.

BACKGROUND: Haemophilus influenzae type b (Hib) vaccine was introduced as a voluntary vaccine in December 2008 and was included in the national routine immunization program in April 2013 in Japan. Currently, no nationwide data are available to evaluate the effectiveness of Hib vaccine in Japan. METHODS: To evaluate the effectiveness of Hib vaccine in Japan, nationwide active population-based surveillance of culture-proven invasive infections caused by H. influenzae in children was performed in 2008-2017 in 10 prefectures in Japan (covering approximately 23% of the total Japanese population). Clinical data were recorded on a standardized case report form. Capsular type and antimicrobial susceptibility of the H. influenzae isolates were examined. The incidence rate ratio (IRR) and its confidence interval (CI) were calculated to compare data from 5 years before and that from after the introduction of the national routine Hib vaccine immunization program. RESULTS: During the 10-year study period, 566 invasive H. influenzae disease cases including 336 meningitis cases were identified. The average number of invasive H. influenzae disease cases among children <5 years of age during 2013-2017 decreased by 93% (IRR: 0.07, 95%CI 0.05-0.10, p < 0.001) compared with those occurring during 2008-2012. Hib strains have not been isolated from invasive H. influenzae disease cases since 2014; however, non-typeable H. influenzae and H. influenzae type f isolates have been noted as causes of invasive H. influenzae diseases among children <5 years in the post-Hib vaccine era. CONCLUSIONS: After the governmental subsidization of the Hib vaccine, invasive Hib disease cases decreased dramatically in the study population, as per our surveillance. Continuous surveillance is necessary to monitor the effectiveness of Hib vaccine and for detecting any emerging invasive capsular types.

Clinical and Bacteriologic Analysis of Nontypeable Haemophilus influenzae Strains Isolated from Children with Invasive Diseases in Japan from 2008 to 2015.

Haemophilus influenzae type b (Hib) conjugate vaccines have led to dramatic reductions in Hib disease among young children worldwide. Nontypeable H. influenzae (NTHi) is now the major cause of invasive H. influenzae infections. We investigated the clinical characteristics of invasive NTHi diseases among children in Japan, to clarify the pathogenicity of isolated NTHi strains. The mortality rate was 10.7%, with deaths occurring mainly among children with underlying comorbidities. Biotypes II and III were the most common, and most strains (64.3%) had multiple amino acid substitutions at the Asp-350, Ser-357, Ser-385, and/or Met-377 sites of penicillin-binding protein 3. Two strains were ß-lactamase positive and ampicillin-clavulanate resistant. Biofilm indices varied widely, and IS1016 was detected in 10.7% of the strains tested. Moreover, there was wide variation in the characteristics of invasive NTHi strains. NTHi strains, showing great genetic diversity, are responsible for most invasive H. influenzae infections in children in the postvaccine era. Continuous monitoring of NTHi strains responsible for invasive diseases in children is important to detect changes in the epidemiology of invasive H. influenzae infections in the postvaccine era.

Succinyl-CoA Synthetase: New Antigen Candidate of Bartonella bacilliformis.

BACKGROUND: Bartonella bacilliformis is the causative agent of Carrion's disease, a neglected illness with mortality rates of 40-85% in the absence of treatment. The lack of a diagnostic technique to overcome misdiagnosis and treat asymptomatic carriers is of note. This study aimed to identify new B. bacilliformis antigenic candidates that could lead to a new diagnostic tool able to be implemented in endemic rural areas. METHODOLOGY/PRINCIPAL FINDINGS: Blood (n = 198) and serum (n = 177) samples were collected in northern Peru. Clinical data were recorded. Specific 16S rRNA amplification by RT-PCR, IFA and ELISA for IgM/IgG with whole cells as antigens was done. Western blot analysis and N-terminal amino acid sequencing detected seroreactive proteins. ELISAs for IgM/IgG for the antigenic candidates were performed. Of the population 33.3% reported at least one symptom compatible with Carrion's disease; 25.4% (IFA), 27.1% (ELISA-IgG), 33.9% (ELISA-IgM) and 38.9% (RT-PCR) of samples were positive. Four proteins were considered potential antigenic candidates, including two new antigenic candidates, succinyl-CoA synthetase subunit α (SCS-α) and succinyl-CoA synthetase subunit ß (SCS-ß). On Western blot both Pap31 and SCS-α interacted with IgM, while GroEL and SCS-ß interacted with IgG. The presence of specific antibodies against the antigenic candidates varied from 34.5% (IgG against SCS-α) to 97.2% (IgM against Pap31). CONCLUSIONS/SIGNIFICANCE: RT-PCR and the high levels of positivity for specific ELISAs demonstrate high levels of B. bacilliformis exposure and asymptomatic carriers among inhabitants. The new antigens identified might be used as a new rapid diagnostic tool to diagnose acute Carrion's disease and identify asymptomatic carriers.

Hydroxychloroquine-inhibited dengue virus is associated with host defense machinery.

Hydroxychloroquine (HCQ) is an antimalarial drug also used in treating autoimmune diseases. Its antiviral activity was demonstrated in restricting HIV infection in vitro; however, the clinical implications remain controversial. Infection with dengue virus (DENV) is a global public health problem, and we lack an antiviral drug for DENV. Here, we evaluated the anti-DENV potential of treatment with HCQ. Immunofluorescence assays demonstrated that HCQ could inhibit DENV serotype 1-4 infection in vitro. RT-qPCR analysis of HCQ-treated cells showed induced expression of interferon (IFN)-related antiviral proteins and certain inflammatory cytokines. Mechanistic study suggested that HCQ activated the innate immune signaling pathways of IFN-ß, AP-1, and NFκB. Knocking down mitochondrial antiviral signaling protein (MAVS), inhibiting TANK binding kinase 1 (TBK1)/inhibitor-κB kinase ɛ (IKKɛ), and blocking type I IFN receptor reduced the efficiency of HCQ against DENV-2 infection. Furthermore, HCQ significantly induced cellular production of reactive oxygen species (ROS), which was involved in the host defense system. Suppression of ROS production attenuated the innate immune activation and anti-DENV-2 effect of HCQ. In summary, HCQ triggers the host defense machinery by inducing ROS- and MAVS-mediated innate immune activation against DENV infection and may be a candidate drug for DENV infection.

Functionally distinct effects of the C-terminal regions of IKKε and TBK1 on type I IFN production.

Inhibitor of κB kinase ε (IKKε) and TANK binding kinase 1 (TBK1), so-called non-canonical IKKs or IKK-related kinases, are involved in the cellular innate immunity by inducing type I IFNs. Two kinases commonly phosphorylate transcription factors IRF3 and IRF7 in type I IFN production pathway. In contrast to TBK1, underlying mechanisms of IKKε activation and regions required for activation of downstream molecules are poorly understood. In this study, we investigated regions of IKKε required for the activation of type I IFN promoter specially, by focusing on the C-terminal region. To show the functional significance of the IKKε C-terminal region on type I IFN production, we employed various mutant forms of IKKε and compared to corresponding region of TBK1. We identified the specific regions and residues of IKKε involved in the activation of downstream signaling. Interestingly, corresponding region and residues are not required for activation of downstream signaling by TBK1. The results highlight the importance of the C-terminal region in the functional activity of IKKε in innate immune response and also the difference in activation mechanisms between IKKε and the closely related TBK1.

PIASy mediates hypoxia-induced SIRT1 transcriptional repression and epithelial-to-mesenchymal transition in ovarian cancer cells.

Epithelial-mesenchymal transition (EMT) has an essential role in organogenesis and contributes to a host of pathologies, including carcinogenesis. Hypoxia (low oxygen supply) aids tumor metastasis in part by promoting EMT in cancer cells. The underlying mechanism whereby hypoxia orchestrates EMT remains poorly defined. Here we report that SIRT1, a multifaceted player in tumorigenesis, opposed ovarian cancer metastasis in vitro and in vivo by impeding EMT. Hypoxic stress downregulated the expression of SIRT1, primarily at the transcriptional level, by reducing the occupancy of the transcriptional activator Sp1 on the proximal promoter of the SIRT1 gene in a SUMOylation-dependent manner. Further analysis revealed that the SUMO E3 ligase PIASy (also known as PIAS4) was induced by hypoxia and prevented Sp1 from binding to the SIRT1 promoter. Conversely, knockdown of PIASy by small interfering RNA (siRNA) restored Sp1 binding and SIRT1 expression in cancer cells challenged with hypobaric hypoxia, reversed cancer cell EMT, and attenuated metastasis in vivo in nude mice. Importantly, analysis of human ovarian tumor specimens indicated that PIASy expression was positively, whereas SIRT1 expression was inversely, correlated with cancer aggressiveness. In summary, our work has identified a new pathway that links downregulation of SIRT1 to hypoxia-induced EMT in ovarian cancer cells and, as such, sheds light on the development of novel anti-tumor therapeutics.

Hemin-binding proteins as potent markers for serological diagnosis of infections with Bartonella quintana.

It is difficult to distinguish infections with different Bartonella species using commercially available immunofluorescence (indirect immunofluorescent antibody [IFA]) assay kits. To identify appropriate proteins for serodiagnosis of Bartonella quintana infections, we investigated the antigenicity of B. quintana proteins using sera from homeless people with high B. quintana IgG titers in IFA assay. These sera reacted strongly to an outer membrane protein, hemin-binding protein D (HbpD). Further, serum from an endocarditis patient infected with B. quintana reacted to HbpB and HbpD. To locate the antigenic sites within the proteins, we generated deletion mutants of HbpB and HbpD. Amino acid residues 89 to 220 of HbpB and 151 to 200 of HbpD were identified as the minimum regions required for recognition by these sera. Several oligopeptides comprising parts of the minimum regions of HbpB and HbpD were synthesized, and their immunoreactivity with the above-mentioned sera was tested by enzyme-linked immunosorbent assay (ELISA). Serum from the endocarditis patient reacted similarly to synthetic peptides HbpB2 (amino acid residues 144 to 173 of HbpB) and HbpD3 (151 to 200 residues of HbpD), while sera from the other subjects reacted to HbpD3. These results indicate that synthetic peptides HbpB2 and HbpD3 might be suitable for developing serological tools for differential diagnosis of B. quintana infections from other Bartonella infections.

Dengue virus serotype 2 blocks extracellular signal-regulated kinase and nuclear factor-κB activation to downregulate cytokine production.

BACKGROUND: Dengue virus (DENV) infection is the most common mosquito-borne viral disease threatening human health around the world. Type I interferon (IFN) and cytokine production are crucial in the innate immune system. We previously reported that DENV serotype 2 (DENV-2) induced low levels of interferon regulatory factor 3 and NF-κB activation, thus leading to reduced production of IFN-ß in the early phase of infection. Here, we determined whether DENV infection not only hampers type I IFN activation but also cytokine production triggered by Toll-like receptor (TLR) signaling. METHODOLOGY/PRINCIPAL FINDINGS: We used quantitative RT-PCR and found that only low levels of IFN-ß and inflammatory cytokines such as interleukin 10 (IL-10), IL-12 and tumor necrosis factor α (TNFα) mRNA were detected in DENV-2-infected bone-marrow-derived dendritic cells. Furthermore, DENV-2 infection repressed cytokine production triggered by TLR signaling. To elucidate the molecular mechanisms underlying this suppression event, we measured NF-κB activation by p65 nuclear translocation and luciferase reporter assay and found that NF-κB activation triggered by TLR ligands was blocked by DENV-2 infection. As well, extracellular signal-regulated kinase (ERK) activity was suppressed by DENV-2 infection. CONCLUSIONS/SIGNIFICANCE: To downregulate the host innate immunity, DENV-2 by itself is a weak inducer of type I IFN and cytokines, furthermore DENV-2 can also block the TLR-triggered ERK-NF-κB activation and cytokine production.

PIASy inhibits virus-induced and interferon-stimulated transcription through distinct mechanisms.

The protein inhibitor of activated STAT (PIAS) family proteins regulates innate immune responses by controlling transcription induced by Toll-like receptor, RIG-I-like receptor signaling, and JAK/STAT pathways. Here, we show that PIASy negatively regulates type I interferon (IFN) transcription. Virus infection led to enhanced type I IFN induction in PIASy null cells, and conversely PIASy overexpression reduced IFN transcription. A mutation in the LXXLL motif of the SAP domain abolished inhibition of IFN-stimulated gene expression but did not affect virus or Toll-like receptor/RIG-I-like receptor-stimulated IFN transcription, indicating that PIASy employs distinct mechanisms to inhibit virus-induced and IFN-stimulated transcription. SUMO E3 activity was not required for PIASy inhibition of IFN transcription; however, PIASy relied on the SUMO modification mechanism to inhibit IFN transcription, because the activity of the SUMO-interacting motif was required for inhibition, and knockdown of SUMO E2 enzyme UBC9 decreased inhibitory activity of PIASy. Our results demonstrate that PIASy negatively regulates both IFN transcription and IFN-stimulated gene expression through multiple mechanisms utilizing the function of different domains.

Ebola Zaire virus blocks type I interferon production by exploiting the host SUMO modification machinery.

Ebola Zaire virus is highly pathogenic for humans, with case fatality rates approaching 90% in large outbreaks in Africa. The virus replicates in macrophages and dendritic cells (DCs), suppressing production of type I interferons (IFNs) while inducing the release of large quantities of proinflammatory cytokines. Although the viral VP35 protein has been shown to inhibit IFN responses, the mechanism by which it blocks IFN production has not been fully elucidated. We expressed VP35 from a mouse-adapted variant of Ebola Zaire virus in murine DCs by retroviral gene transfer, and tested for IFN transcription upon Newcastle Disease virus (NDV) infection and toll-like receptor signaling. We found that VP35 inhibited IFN transcription in DCs following these stimuli by disabling the activity of IRF7, a transcription factor required for IFN transcription. By yeast two-hybrid screens and coimmunoprecipitation assays, we found that VP35 interacted with IRF7, Ubc9 and PIAS1. The latter two are the host SUMO E2 enzyme and E3 ligase, respectively. VP35, while not itself a SUMO ligase, increased PIAS1-mediated SUMOylation of IRF7, and repressed Ifn transcription. In contrast, VP35 did not interfere with the activation of NF-kappaB, which is required for induction of many proinflammatory cytokines. Our findings indicate that Ebola Zaire virus exploits the cellular SUMOylation machinery for its advantage and help to explain how the virus overcomes host innate defenses, causing rapidly overwhelming infection to produce a syndrome resembling fulminant septic shock.

Ebolavirus VP35 interacts with the cytoplasmic dynein light chain 8.

The viral protein VP35 of ebolavirus (EBOV) is implicated to have diverse roles in the viral life cycle. We employed a yeast two-hybrid screen to search for VP35 binding partners and identified the cytoplasmic dynein light chain (DLC8) as a protein that interacts with VP35. Mapping analysis unraveled a consensus motif, SQTQT, within VP35 through which VP35 binds to DLC8. The disruption of DLC8 binding does not affect the ability of VP35 to inhibit type I IFN production. Given that VP35 from various EBOV species interacts with DLC8, this interaction may have a role in regulating the EBOV life cycle.

Virus infection triggers SUMOylation of IRF3 and IRF7, leading to the negative regulation of type I interferon gene expression.

Viral infection activates Toll-like receptor and RIG-I (retinoic acid-inducible gene I) signaling pathways, leading to phosphorylation of IRF3 (interferon regulatory factor 3) and IRF7 and stimulation of type I interferon (IFN) transcription, a process important for innate immunity. We show that upon vesicular stomatitis virus infection, IRF3 and IRF7 are modified not only by phosphorylation but by the small ubiquitin-related modifiers SUMO1, SUMO2, and SUMO3. SUMOylation of IRF3 and IRF7 was dependent on the activation of Toll-like receptor and RIG-I pathways but not on the IFN-stimulated pathway. However, SUMOylation of IRF3 and IRF7 was not dependent on their phosphorylation, and vice versa. We identified Lys(152) of IRF3 and Lys(406) of IRF7 to be their sole small ubiquitin-related modifier (SUMO) conjugation site. IRF3 and IRF7 mutants defective in SUMOylation led to higher levels of IFN mRNA induction after viral infection, relative to the wild type IRFs, indicating a negative role for SUMOylation in IFN transcription. Together, SUMO modification is an integral part of IRF3 and IRF7 activity that contributes to postactivation attenuation of IFN production.

Genotyping analysis of Mycoplasma pneumoniae clinical strains in Japan between 1995 and 2005: type shift phenomenon of M. pneumoniae clinical strains.

Mycoplasma pneumoniae clinical isolates obtained between 1995 and 2005 were examined to determine the prevalent genotype. One hundred and twenty-seven strains isolated from bronchitis and pneumonia patients were genotyped by a PCR-RFLP method based on nucleotide sequence polymorphisms of the p1 gene, which encodes the major adhesin protein. The typing results established that 66 of the isolates were group I strains, 45 were group II strains and 16 were group II variants. Analysis of the annual occurrence of these isolates showed a predominance of group II strains between 1995 and 2001 (n=37). No group I strain was found during this period. However, group I strains appeared in the isolates from 2002 (2/5 isolates, 40 %) and increased in specimens taken after 2003, thereby constituting a large proportion of the isolates. In 2004 and 2005, no group II strains were found among the isolates (n=49), although there were nine group II variants. Throat swabs and sputum samples obtained from patients with respiratory infections between 1997 and 2005 were also analysed by PCR-RFLP or a new nested PCR to detect the p1 gene DNA. Typing analysis of these p1 gene DNAs also showed that the group I p1 gene was not present in specimens taken before 2000, but was present and dominant in specimens taken after 2001. These results indicate that, in Japan, the prevalent type of M. pneumoniae changed from a group II strain to a group I strain around 2002.

Genetic and phenotypic characterization of Haemophilus influenzae type b isolated from children with meningitis and their family members in Vietnam.

To investigate Haemophilus influenzae type b (Hib) infection in Vietnamese children under the age of 5 years, cerebrospinal fluid (CSF) samples from patients with meningitis were screened for Hib, and isolates were subjected to evaluation of susceptibility to 12 antibiotics, biotyping, and genotyping with pulsed-field gel electrophoresis (PFGE). The major biotype was type II (68.3%), followed by type I (22.8%). Among 79 Hib isolates, 45 (57%) were beta-lactamase-producing and ampicillin-resistant (44 and 1 isolates produced TEM-1- and ROB-1-type beta-lactamases, respectively), and 34 isolates (43%) were beta-lactamase-nonproducing and ampicillin-sensitive. No beta-lactamase-nonproducing and ampicillin-resistant isolates were found. The PFGE patterns of Hib isolates were highly divergent, but most could be classified into three clusters. We also investigated Hib colonization in household contacts of patients, and found that Hib isolates from the CSF of patients and from nasopharyngeal cavities of household contacts showed the same PFGE patterns. This observation suggested that household contacts of patients are a possible reservoir of Hib.

Epidemiological studies on Bartonella quintana infections among homeless people in Tokyo, Japan.

In an epidemiological investigation of trench fever in Japan, we compared the seroprevalence of Bartonella quintana in homeless people and in the general population. In homeless rescue outreach programs held in Tokyo from May 2001 to March 2003, 151 blood samples were taken from non-hospitalized homeless people. The prevalence of IgM and IgG antibodies against B. quintana in these people was compared with that in 200 healthy blood donors using a commercially available indirect fluorescent antibody test. Although IgG titers of > or = 1:128 were found in 57% (86/151) of homeless people and 51% (101/200) of blood donors, high titers of > or = to 1:1,024 were encountered only in homeless people (11%, 16/151). Attempts to isolate B. quintana from the blood of homeless people were unsuccessful, but polymerase chain reaction based detection, using Bartonella genus specific primers, demonstrated the presence of B. quintana DNA in the blood of 10 homeless people. Our data suggest that urban trench fever is endemic among the Japanese homeless population.

Clinical evaluation of macrolide-resistant Mycoplasma pneumoniae.

Macrolide-resistant Mycoplasma pneumoniae (MR M. pneumoniae) has been isolated from clinical specimens in Japan since 2000. A comparative study was carried out to determine whether or not macrolides are effective in treating patients infected with MR M. pneumoniae. The clinical courses of 11 patients with MR M. pneumoniae infection (MR patients) treated with macrolides were compared with those of 26 patients with macrolide-susceptible M. pneumoniae infection (MS patients). The total febrile days and the number of febrile days during macrolide administration were longer in the MR patients than in the MS patients (median of 8 days versus median of 5 days [P = 0.019] and 3 days versus 1 day [P = 0.002], respectively). In addition, the MR patients were more likely than the MS patients to have had a change of the initially prescribed macrolide to another antimicrobial agent (63.6% versus 3.8%; odds ratio, 43.8; P < 0.001), which might reflect the pediatrician's judgment that the initially prescribed macrolide was not sufficiently effective in these patients. Despite the fact that the febrile period was prolonged in MR patients given macrolides, the fever resolved even when the initial prescription was not changed. These results show that macrolides are certainly less effective in MR patients.

Characterization and molecular analysis of macrolide-resistant Mycoplasma pneumoniae clinical isolates obtained in Japan.

In recent years, Mycoplasma pneumoniae strains that are clinically resistant to macrolide antibiotics have occasionally been encountered in Japan. Of 76 strains of M. pneumoniae isolated in three different areas in Japan during 2000 to 2003, 13 strains were erythromycin (ERY) resistant. Of these 13 strains, 12 were highly ERY resistant (MIC, > or =256 microg/ml) and 1 was weakly resistant (MIC, 8 microg/ml). Nucleotide sequencing of domains II and V of 23S rRNA and ribosomal proteins L4 and L22, which are associated with ERY resistance, showed that 10 strains had an A-to-G transition at position 2063 (corresponding to 2058 in Escherichia coli numbering), 1 strain showed A-to-C transversion at position 2063, 1 strain showed an A-to-G transition at position 2064, and the weakly ERY-resistant strain showed C-to-G transversion at position 2617 (corresponding to 2611 in E. coli numbering) of domain V. Domain II and ribosomal proteins L4 and L22 were not involved in the ERY resistance of these clinical M. pneumoniae strains. In addition, by using our established restriction fragment length polymorphism technique to detect point mutations of PCR products for domain V of the 23S rRNA gene of M. pneumoniae, we found that 23 (24%) of 94 PCR-positive oral samples taken from children with respiratory infections showed A2063G mutation. These results suggest that ERY-resistant M. pneumoniae infection is not unusual in Japan.