Junji Seto

Sequence and phylogenetic analyses of Saffold cardiovirus from children with exudative tonsillitis in Yamagata, Japan.

(Received 15 February 2010 ; accepted 23 May 2010 ) To the Editor, Saffold cardiovirus (SAFV) of the genus Cardiovirus and family Picornaviridae was recently recovered from faecal specimens of an infant with fever of unknown origin [1]. SAFV has also been detected in children with diseases such as gastroenteritis, respiratory tract infection, and non-polio acute fl accid paralysis [2 – 5]. However, the epidemiology and pathogenicity of SAFV is not exactly known. In this study, we detected SAFV in children with exudative tonsillitis and conducted sequence and phylogenetic analyses. We obtained nasopharyngeal swabs from 37 patients with typically exudative tonsillitis between August and December 2009. Informed consent was obtained from the parents of all subjects for the donation of the nasopharyngeal samples used in this analysis. Initially, we sought to isolate or detect pathogens from these samples using cell culture methods, quick immunochromatography (as used to detect Streptococcus), and polymerase chain reaction (PCR; as used to detect Epstein – Barr virus [6]). To isolate various viruses, we used 7 different cell lines (Vero E6, HEp-2, HEL, MDCK, GMK, HMV-II, and RD18S cells) [7,8]. These cells may be sensitive to the various agents of exudative tonsillitis – parainfl uenza viruses, infl uenza viruses, herpes simplex viruses, adenovirus, and respiratory syncytial virus [7,8]. However, these pathogens were not isolated or detected from the samples provided. Next, we attempted to detect SAFV using a nested reverse transcriptase PCR (RT-PCR). We extracted RNA from the samples and amplifi ed the VP1 coding region of SAFV by nested RT-PCR. Primer sets were newly designed by Primer Express ® version 1.5 software (Applied Biosystems LLC, Foster City, CA, USA) [9]. Primer sequences were as follows: 5 ′ -HAA RCA RGR YTG GAR YTT YNT NAT GTT-3 ′ (primer 315F) and 5 ′ DGG BCK DGG RCA RWA VAC YCT CAT-3′ (primer 738R) as outer primers, and 5 ′ -AAR CAR GRY TGG ARY TTY DTH ATG TTY TC-3′ (primer 316F) and 5 ′ -RTT RKK RAA RTY NGM RDA NCY RTT RAA CCA-3 ′ (primer 621R) as inner primers. Reverse transcription was performed for 10 min at 30 ° C, 45 min at 37 ° C, and 5 min at 95 ° C using random hexamers (TAKARA BIO Inc., Otsu, Japan). First and nested PCR conditions were as follows: 5 min at 94 ° C, followed by 40 cycles at 94 ° C for 30 s, 50 ° C for 30 s, and 72 ° C for 1 min, ending with elongation for an additional 10 min at 72 ° C. To prevent carryover contamination of nested-PCR, we took general precautions as previously described [10,11]. As a result, we obtained amplicons from 9 children who showed typical exudative tonsillitis symptoms, including fever ( 38 ° C). They lived in Yamagata Prefecture, Japan and were aged between 2 and 7 y (mean standard deviation, 3.8 1.6 y). Fever lasted for 1 to 3 days (1.8 0.7 days). Amplicons were sequenced and aligned (453 bp) [3,5,12]. Next, we performed phylogenetic LETTER TO THE EDITOR

Community outbreak of macrolide-resistant Mycoplasma pneumoniae in Yamagata, Japan in 2009.

Background: We detected a community outbreak of macrolide-resistant Mycoplasma pneumoniae infection that occurred predominantly among students at 2 schools in Yamagata, Japan. Methods: Throat swab specimens were collected from patients who were clinically suspected to have M. pneumoniae infection after testing negative for influenza virus by a nasopharyngeal swab rapid antigen test. We performed cultures for M. pneumoniae, and all isolates were sequenced for the presence of a mutation of the 23S rRNA gene. Results: Of 96 specimens collected between July 2009 and January 2010, 83 were from students attending junior high school A and primary schools B, C and D. A total of 47 M. pneumoniae isolates were obtained; among them, 25, 15 and 4 were isolated from students attending schools A, B and D, respectively, and M. pneumoniae could not be isolated from students who attended school C. An A2063T mutation in domain V of the 23S rRNA gene, which is associated with macrolide resistance, was identified in 39 (83.0%) isolates. The rates of macrolide resistance at schools A, B and D were 96.0%, 86.7% and 0%, respectively. The minimum inhibitory concentrations for isolates with an A2063T transversion showed high resistance to clarithromycin (minimum inhibitory concentration, 16–64 mg/L), and clarithromycin prescribed initially was clinically ineffective. Conclusions: This school-based cluster of macrolide-resistant M. pneumoniae infections, which was identified in 2 geographically close schools, indicates that the transmission principally occurred by close contact between students at school. Monitoring the spread of macrolide-resistant M. pneumoniae and clinical guidelines for the appropriate medication against such infections would be needed to control outbreaks of M. pneumoniae.

Defining the Genome Features of Escherichia albertii, an Emerging Enteropathogen Closely Related to Escherichia coli

Escherichia albertii is a recently recognized close relative of Escherichia coli. This emerging enteropathogen possesses a type III secretion system (T3SS) encoded by the locus of enterocyte effacement, similar to enteropathogenic and enterohemorrhagic E. coli (EPEC and EHEC). Shiga toxin-producing strains have also been identified. The genomic features of E. albertii, particularly differences from other Escherichia species, have not yet been well clarified. Here, we sequenced the genome of 29 E. albertii strains (3 complete and 26 draft sequences) isolated from multiple sources and performed intraspecies and intragenus genomic comparisons. The sizes of the E. albertii genomes range from 4.5 to 5.1 Mb, smaller than those of E. coli strains. Intraspecies genomic comparisons identified five phylogroups of E. albertii. Intragenus genomic comparison revealed that the possible core genome of E. albertii comprises 3,250 genes, whereas that of the genus Escherichia comprises 1,345 genes. Our analysis further revealed several unique or notable genetic features of E. albertii, including those responsible for known biochemical features and virulence factors and a possibly active second T3SS known as ETT2 (E. coli T3SS 2) that is inactivated in E. coli. Although this organism has been observed to be nonmotile in vitro, genes for flagellar biosynthesis are fully conserved; chemotaxis-related genes have been selectively deleted. Based on these results, we have developed a nested polymerase chain reaction system to directly detect E. albertii. Our data define the genomic features of E. albertii and provide a valuable basis for future studies of this important emerging enteropathogen.

Two cases of macrolide resistance in Mycoplasma pneumoniae acquired during the treatment period

Sir, Mycoplasma pneumoniae is a common cause of upper and lower respiratory tract infections, especially in children and young adults. M. pneumoniae infections can be treated with macrolides, which are generally considered to be the first-choice antibiotics for children. However, the prevalence of macrolide-resistant M. pneumoniae has gradually increased worldwide and has reached a rate of 90% in China. In Japan, it has been reported that the prevalence of macrolide-resistant M. pneumoniae isolates reached 65.5% in 2010 and 89.5% in 2011. M. pneumoniae resistance to macrolides is caused by point mutations in domain V of the 23S rRNA gene that interfere with the binding of macrolides to rRNA. An A-to-G transition at position 2063 (A2063G) is the most frequently detected mutation among resistant strains, closely followed by an A-to-G transition at position 2064 (A2064G). Although resistant strains of M. pneumoniae have been generated in vitro by selection with subinhibitory concentrations of macrolides, in humans there is only one report of the acquisition of a macrolide resistance mutation during the course of infection. We report herein two cases in which macrolide-resistant M. pneumoniae isolates are thought to have emerged during treatment with a macrolide. Patient 1 is an 8-year-old child (Table 1). He visited the Yamanobe Paediatric Clinic with a 1 day history of fever of up to 39.48C and a mild cough in November 2011. His temperature was 37.48C. After taking a pharyngeal swab sample, he was treated with azithromycin (10 mg/kg/day) for 3 days. His temperature rapidly decreased 24 h later. Because of the persistent dry cough without fever, he visited the same clinic at 6 days after starting the antibiotic treatment and a second pharyngeal swab sample was taken. The patient’s guardian gave written informed consent for the publication of these data. Patient 2 is a 5-year-old child (Table 1). She was brought to the Yamanobe Paediatric Clinic in January 2012, with a 4 day history of fever up to 39.88C and coughing. On initial physical examination, her temperature was 38.78C and rales were heard during lung auscultation. Initial laboratory tests showed a leucocyte count of 9300 cells/mL and a C-reactive protein level of 0.8 mg/dL. A chest X-ray showed infiltrates in the inferior lobe of her left lung, a result compatible with pneumonia. After taking a pharyngeal swab sample, she was given clarithromycin (10 mg/kg/day) for 5 days. On day 2 of the oral antibiotic treatment, the patient was brought to the same clinic because she vomited whenever she took the medicine; the clarithromycin was stopped on day 3 because her temperature decreased. On day 6 after starting the antibiotic treatment, her temperature increased again up to 38.08C. The next day, her leucocyte count was 28300 cells/mL and her C-reactive protein level was 0.4 mg/dL. A second pharyngeal swab sample was taken and she was treated with tosufloxacin (12 mg/kg/day) for 5 days. Her condition resolved within 1 week. The patient’s guardian gave written informed consent for the publication of these data. In both cases, M. pneumoniae was isolated from the first and second samples using pleuropneumonia-like organism broth (Difco, Detroit, MI, USA). PCR amplification and sequence analysis of the 23S rRNA gene were performed on the DNA extracted from four strains of M. pneumoniae using the procedures reported by Matsuoka et al. The MICs of the prescribed antibiotics were determined using a broth microdilution method as described by Okazaki et al. No mutation in domain V of the 23S rRNA gene was detected in the M. pneumoniae isolates from the first samples of both patients, which were obtained before the initiation of macrolide treatment. However, an A2063G transition was detected in the M. pneumoniae isolate from the second sample of Patient 1 and an A2064G transition was detected in the M. pneumoniae isolate from the second sample of Patient 2. The MICs of azithromycin for the strains isolated from the first and second samples of Patient 1 were 0.000488 and 16 mg/L, respectively, and the MICs of clarithromycin for the strains isolated from Patient 2 were 0.00195 and 64 mg/L, respectively, indicating the acquisition of resistance to the initially prescribed antibiotics. The MICs of tosufloxacin for the strains isolated from Patient 2 were 0.25 and 1 mg/L, respectively. In the cases reported by Chironna et al., it could not be determined when the macrolide resistance mutation occurred. In our two cases, it is apparent that the macrolide-resistant M. pneumoniae strains emerged within 6 or 7 days after the initiation of azithromycin or clarithromycin treatment. It is likely that the emergence of macrolide resistance in Patient 2, who was treated with clarithromycin, was the result of treatment with subinhibitory concentrations due to the vomiting that occurred whenever the patient took the medicine. In Patient 1, who was treated with the proper dosage of azithromycin, the fever decreased immediately; however, macrolide-resistant M. pneumoniae still emerged. These cases suggest that care must be taken when determining the appropriate dose and treatment duration for macrolides and other first-choice antibiotics to inhibit the emergence of resistant strains.

Characteristics of Mycoplasma pneumoniae infection identified on culture in a pediatric clinic

The appropriate choice of antibiotics against Mycoplasma pneumoniae infection has become difficult, as the prevalence of macrolide‐resistant M. pneumoniae has increased.

A Geographically Widespread Outbreak Investigation and Development of a Rapid Screening Method Using Whole Genome Sequences of Enterohemorrhagic Escherichia coli O121

From 2014 to 2015, we investigated a suspected nationwide outbreak of enterohemorrhagic Escherichia coli serogroup O121. However, similar pulsed field gel electrophoresis (PFGE) profiles and the lack of epidemiological links between the isolates made detection of the outbreak difficult. To elucidate a more precise genetic distance among the isolates, whole genome sequence (WGS) analyses were implemented in the investigation. The WGS-based single nucleotide polymorphism (SNP) analysis showed that 23 out of 44 isolates formed a distinct cluster (the number of intra-cluster SNPs was ≤8). Specific genomic regions in the clustered isolates were used to develop a specific PCR analysis. The PCR analysis detected all the clustered isolates and was suitable for rapid screening during the outbreak investigation. Our results showed that WGS analyses were useful for the detection of a geographically widespread outbreak, especially for isolates showing similar PFGE profiles and for the development of a rapid and cost-effective screening method.

Mycobacterium tuberculosis Transmission among Elderly Persons, Yamagata Prefecture, Japan, 2009–2015

In many countries with low to moderate tuberculosis (TB) incidence, cases have shifted to elderly persons. It is unclear, however, whether these cases are associated with recent Mycobacterium tuberculosis transmissionor represent reactivation of past disease. During 2009–2015, we performed a population-based TB investigation in Yamagata Prefecture, Japan, using in-depth contact tracing and 24-loci variable-number tandem-repeat typing optimized for Beijing family M. tuberculosis strains. We analyzed 494 strains, of which 387 (78.3%) were derived from elderly patients. Recent transmission with an epidemiologic link was confirmed in 22 clusters (70 cases). In 17 (77.3%) clusters, the source patient was elderly; 11 (64.7%) of the 17 clusters occurred in a hospital or nursing home. In this setting, the increase in TB cases was associated with M. tuberculosis transmissions from elderly persons. Prevention of transmission in places where elderly persons gather will be an effective strategy for decreasing TB incidence among predominantly elderly populations.

Proposed vector candidate: Leptotrombidium palpale for Shimokoshi type Orientia tsutsugamushi

To identify the vector species for Shimokoshi type Orientia tsutsugamushi, a survey of larval trombiculid mites was conducted in Yamagata Prefecture, Japan from April to May 2012. In all, 2889 larval trombiculid mites were obtained from 21 Apodemus speciosus rodent hosts, 2600 of which were morphologically classified into eight species in three genera. After screening of O. tsutsugamushi DNA in individual larval trombiculid mites using real‐time PCR targeting the 16S ribosomal RNA gene, serotype‐specific nested PCRs targeting the 56 kDa protein gene were performed, followed by sequencing analysis. As a result, Shimokoshi type O. tsutsugamushi DNA was identified from 3 (1.9%) of 157 Leptotrombidium palpale. This is the first study to identify Shimokoshi type O. tsutsugamushi DNA in L. palpale. The results indicate that L. palpale is a possible vector for Shimokoshi type O. tsutsugamushi.

Turtle-Associated Salmonella Infections in Kanagawa, Japan.

In this paper, we examine 2 case reports for different reptile-related Salmonella enterica subspecies enterica serotypes. In case 1, a 5-year-old boy presented with gastroenteritis caused by S. enterica subspecies enterica serovar Poona. The suspected source of infection was a turtle kept at the patients home. In case 2, a 4-year-old boy presented with gastroenteritis caused by S. enterica subspecies enterica serovar Abony. The Pulsed-field gel electrophoresis analysis suggested that a tortoise kept at the patients home was the source of infection. This paper presents a review of the literature and an examination of cases regarding turtle-associated salmonellosis in Japan.

Development of macrolide resistance-associated mutations after macrolide treatment in children infected with Mycoplasma pneumoniae

Purpose. To determine the timing of the emergence of macrolide‐resistant mutations after macrolide treatment in individuals with Mycoplasma pneumoniae infections. Methodology. Between October 2011 and December 2013, serial pharyngeal swab specimens were collected before and after macrolide treatment from 21 otherwise healthy children infected with M. pneumoniae without macrolide‐resistant mutations. The copy numbers of a M. pneumoniae gene and the proportion of clones showing macrolide‐resistance mutations were determined for each specimen. Results. After macrolide treatment (10‐15 mg kg−1 day−1 clarithromycin for 5‐10 days or 10 mg kg−1 day−1 azithromycin for 3 days), fever resolved in 19 (90%) of 21 children within 1 to 2 days, and the M. pneumoniae gene copy number decreased in all but one specimen in the second set of specimens relative to the number in the corresponding initial specimens. None of the second specimens, which were collected 2‐4 days after initiation of macrolide treatment, showed mutations in the 23S rRNA gene. However, the proportion of mutant clones with A2063G and A2064G mutations in the specimens collected 7‐24 days after initiation of treatment increased to 100%. We identified a family in which three members had M. pneumoniae infections. The analysis of transmission in this household indicated that the M. pneumoniae harbouring a macrolide‐resistant mutation that developed in the index patient after macrolide treatment was not transmitted to the family members. Conclusion. A macrolide‐resistant population might develop in individual patients up to 24 days after initiation of macrolide treatment. However, the decrease in M. pneumoniae load after macrolide administration effectively reduces interpersonal transmission.