Background: We previously revealed that several multiple-locus variable-number tandem-repeat analyses（MLVA）and P1 types of Mycoplasma pneumoniae（M. pneumoniae）cocirculated between 2011 and 2013 in Yamagata, Japan. However, the regional spread of M. pneumoniae infection by genotype is not reported yet. It remains unclear whether there is a difference in the spread of macrolide-resistant M. pneumoniae among genotypes. Methods: Genotypes were labeled according to 4-locus（Mpn 13, 14, 15, and 16）MLVA and P1 types. A total of 208 strains belonging to three major genotypes, i.e., type 4-5-7-2, 1; 4-5-7-3, 1; and 3-5-6-2, 2c, were analyzed by combining with the information of macrolide resistance-associated mutation and the patients’ information including residence. Results and Discussion: The three genotypes were widely distributed over more than four cities and towns in Yamagata Prefecture, cocirculating between late 2011 and early 2013, and there was little difference in the duration of their epidemics. Timing of macrolide-resistant strain appearance during the epidemic period differed between type 4-5-7-2, 1 and type 4-5-7-3, 1, and it did not appear throughout type 3-5-6-2, 2c epidemic. These genotypic differences can account for the variation in the prevalence of macrolide resistance-associated mutations in each of the studied areas

Hongo Seiji

Ikeda Tatsuya

Itagaki Tsutomu

Katsushima Fumio

Katsushima Yuriko

Matsuzaki Yoko

Mizuta Katsumi

Seto Junji

Shimotai Yoshitaka

Suzuki Yu

Yamagata University Academic Repository

－19－Regional spread of Mycoplasma pneumoniaeDOI  00.00000/00000000Regional spread of three distinct genotypes ofMycoplasma pneumoniae and different timing ofmacrolide-resistant strain appearance among genotypesbetween 2011 and 2013 in Yamagata, Japan＊Department of Microbiology, Yamagata Prefectural Institute of Public Health＊＊Department of Infectious Diseases, Yamagata University Faculty of Medicine＊＊＊Yamanobe Pediatric Clinic, Yamanobe＊＊＊＊Katsushima Pediatric Clinic, Yamagata（Accepted September 24, 2019）Yu Suzuki＊，＊＊， Junji Seto＊， Yoshitaka Shimotai＊＊， Tsutomu Itagaki＊＊＊，Yuriko Katsushima＊＊＊＊， Fumio Katsushima＊＊＊＊， Tatsuya Ikeda＊， Katsumi Mizuta＊，Seiji Hongo＊＊， Yoko Matsuzaki＊＊Background: We previously revealed that several multiple-locus variable-number tandem-repeat analyses（MLVA）and P1 types of Mycoplasma pneumoniae（M. pneumoniae）cocirculated between 2011 and 2013 in Yamagata, Japan. However, the regional spread of M. pneumoniae infection by genotype is not reported yet. It remains unclear whether there is a difference in the spread of macrolide-resistant M. pneumoniae among genotypes.Methods: Genotypes were labeled according to 4-locus（Mpn 13, 14, 15, and 16）MLVA and P1 types. A total of 208 strains belonging to three major genotypes, i.e., type 4-5-7-2, 1; 4-5-7-3, 1; and 3-5-6-2, 2c, were analyzed by combining with the information of macrolide resistance-associated mutation and the patients’ information including residence.Results and Discussion: The three genotypes were widely distributed over more than four cities and towns in Yamagata Prefecture, cocirculating between late 2011 and early 2013, and there was little difference in the duration of their epidemics. Timing of macrolide-resistant strain appearance during the epidemic period differed between type 4-5-7-2, 1 and type 4-5-7-3, 1, and it did not appear throughout type 3-5-6-2, 2c epidemic. These genotypic differences can account for the variation in the prevalence of macrolide resistance-associated mutations in each of the studied areas.Key words: Mycoplasma pneumoniae, molecular typing, P1 type, MLVA, macrolide resistanceIntroductionMycoplasma pneumoniae（M. pneumoniae）is a common cause of upper and lower respiratory tract infections, particularly in children and young adults１），２）. This pathogen is responsible for up to 20% of all cases of community-acquired pneumonia（CAP）３）and 30% of pediatric cases of CAP４），５）. M. pneumoniae infections can be treated using macrolides（MLs）as first-line antibiotics６）－８）. However, since the year 2000, ML-resistant M. pneumoniae harboring a point mutation in its genome region coding the 23S rRNA domain V （ML resistance-associated mutation）has increased worldwide, contributing to increasing global public ABSTRACTYamagata Med J （ISSN 0288-030X）2020；38（1）：19-24 1 .15 22/0 4781－20－Suzuki，Seto，Shimotai，Itagaki，Katsushima，Katsushima，Ikeda，Mizuta，Hongo，Matsuzakihealth concerns６），９）.Molecular typing of M. pneumoniae , including 4-locus（Mpn13, 14, 15, and 16）multiple-locus variable-number tandem-repeat analysis（MLVA）and P1 gene typing based on polymorphisms in the P1 gene encoding M. pneumoniae P1 adhesion protein, is an effective tool to understand the epidemiology of M. pneumoniae infection. Using these typing methods, we reported polyclonal spread of multiple genotypes of M. pneumoniae in schools10）. In addition, we revealed that several P1 and MLVA types of M. pneumoniae had cocirculated between 2011 and 2013 in Yamagata Prefecture, Japan11），12）. However, the regional spread of M. pneumoniae infection by genotype is not reported yet. It remains unclear whether there is difference in the spread of ML-resistant M. pneumoniae among genotypes. In this study, we present detailed geographical and temporal distribution of M. pneumoniae isolates between 2011 and 2013 in Yamagata Prefecture.MethodsStudy design:Our previous study conducted between 2004 and 2014 revealed that three major MLVA types of M. pneumoniae, including type 4-5-7-2, type 4-5-7-3, and type 3-5-6-2, cocirculated between 2011 and 2013 in Yamagata, Japan11）. To elucidate the regional spread of M. pneumoniae infection by genotype, we performed a retrospective study of M. pneumoniae infection cases between 2011 and 2013 by combining the molecular typing results with the patients’ information. The profile of molecular typing and the ML susceptibility of the M. pneumoniae strains were obtained from our previous study11）. The genotypes of M. pneumoniae were defined using a combination of MLVA and P1 types and labeled in the order of MLVA type and P1 type. A total of 208 strains belonging to three major genotypes, i.e., type 4-5-7-2, 1; 4-5-7-3, 1; and 3-5-6-2, 2c, were analyzed. The patients’ information including residence, specimen collection date, age, sex, and clinical diagnosis was collected from the medical records obtained from the Yamanobe Pediatric Clinic and Katsushima Pediatric Clinic in Yamagata, Japan. This study was approved by the Yamagata University Faculty of Medicine （H27-17）and Yamagata Prefectural Institute of Public Health（YPIPHEC 16-04）ethics committees.Statistical analysis:We examined the differences among the three genotypes. Kruskal–Wallis test was used for age, and Fisher’s exact test was used for sex, diagnosis, and prevalence of the ML resistance-associated mutation. When significant differences（P value < 0.05）were detected among the three genotypes, multiple comparisons were performed. Wilcoxon rank sum test with the Benjamini–Hochberg procedure was used for age, and Fisher’s exact test with the Holm method was used for prevalence of the ML resistance-associated mutation. Statistical tests were performed using R version 3.4.1（The R Foundation, Vienna, Austria）.Results and DiscussionWe analyzed 208 strains belonging to three major genotypes, including 74 strains of type 4-5-7-2, 1, 74 strains of type 4-5-7-3, 1, and 60 strains of type 3-5-6-2, 2c. Table 1 shows the patients’ information and ML resistance-associated mutation prevalence among the three major genotypes. Statistical analysis revealed that patients infected with type 4-5-7-2, 1 were significantly younger than the patients infected with type 3-5-6-2, 2c. In addition, the prevalence of ML resistance-associated mutations in type 4-5-7-2, 1 （83.8%; 62/74）was significantly higher than that in type 4-5-7-3, 1（25.7%; 19/74）and type 3-5-6-2; 2c（0%; 0/60）. However, there were no significant differences in clinical diagnosis, defined as the ratio of lower to upper respiratory tract infections, among the three genotypes, suggesting that there is no association between genotype and disease severity.Figure 1 shows the geographical information of the three genotypes of M. pneumoniae, mapped according to districts（cities and towns）. Of the 208 M. pneumoniae strains, 181（87%）were isolated in four of the studied districts: 72（34.6%）in Yamagata City, 48（23.1%）in Yamanobe Town, 42（20.2%）in Sagae City, and 19（9.1%）in Nakayama Town. In these four districts, each of the three genotypes －21－Regional spread of Mycoplasma pneumoniaewas isolated. In the remaining six districts（Oe Town, Asahi Town, Kaminoyama City, Kahoku Town, Higashine City, and Tendo City）, one to two of the three genotypes were isolated. These findings suggest that the three genotypes of M. pneumoniae were widely distributed in the studied districts. Prevalence of ML resistance-associated mutation varied within each district, with 100%（2/2）in Kahoku Town; 75%（3/4）in Kaminoyama City; 52.1%（25/48）in Yamanobe Town; 44.4%（32/72）in Yamagata City; 40.5%（17/42）in Sagae City; 7.7%（1/13）in Oe Town; 5.3%（1/19）in Nakayama Town; and 0% in Higashine City, Tendo City, and Asahi Town. The observed differences in the prevalence of this mutation according to different districts may reflect differences in its prevalence according to genotype（Table 1）.Geographical and temporal information of the three genotypes of M. pneumoniae strains is shown in Figure 2（a）, where each year has been divided into three terms. A single genotype of type 4-5-7-3, 1 was isolated during the first and second terms of 2011, whereas the three genotypes were isolated between the third term of 2011 and the first term of 2013. Each of the three genotypes was isolated for seven terms（approximately a 28-month period）in succession, which gradually spread to multiple surrounding districts. Monthly occurrence of each genotype with or without the ML resistance-associated mutation is shown in Figure 2（b）. Type 4-5-7-2, 1 was first isolated in October 2011, whereas the ML-resistant strain was prevalent from the initial period of its epidemic. Type 4-5-7-3, 1 was first isolated in January 2011; however, it did not possess the ML resistance-associated mutation until its emergence 11 months after the initial epidemic. Furthermore, type 3-5-6-2, 2c; first isolated in December 2011 and intermittently occurring until 8 (1–45) 8 (3–63) 9 (1–56) 0.042‡Sex Male 42 40 34 0.953Female 32 34 26Diagnosis 40 31 24 0.201Upper respiratorytract infection 34 43 36Positive 62 19 0 <0.001§Negative 12 55 60Lower respiratorytract infection* Genotype was represented by the combination of VNTR numbers at 4 loci asMpn13–Mpn14–Mpn15–Mpn16 and P1 type.† Kruskal–Wallis test was used for age and Fisher’s exact test was used for sex,diagnosis, and prevalence of the ML resistance-associated mutation. When significantdifferences (P value < 0.05) were detected among the three genotypes, multiplecomparisons were performed.‡ Significant differences were obtained between type 4-5-7-2, 1 and 3-5-6-2, 2c (P  =0.045) by multiple comparison (Wilcoxon rank sum test with the Benjamini–Hochberg procedure).§ Significant differences were obtained between type 4-5-7-2, 1 and 4-5-7-3, 1; 3-5-6-2, 2c and between type 4-5-7-3, 1 and 3-5-6-2, 2c, respectively (P  < 0.001) bymultiple comparison (Fisher’s exact test with the Holm method).4-5-7-2, 1(n = 74)4-5-7-3, 1(n = 74)3-5-6-2, 2c(n = 60)Genotype* of M. pneumoniae isolateP  value†Age, median (range)Macrolide resistance-associated mutationTable 1． Comparison of patient characteristics and macrolide  resistance-associated mutation prevalence among the  three major genotypes of Mycoplasma pneumoniae is  olated between 2011 and 2013 in Yamagata, Japan－22－Suzuki，Seto，Shimotai，Itagaki，Katsushima，Katsushima，Ikeda，Mizuta，Hongo，Matsuzaki010(a)Type 4-5-7-3, 1Type 4-5-7-2, 1Type 3-5-6-2, 2cNo. of isolatesSept–DecSept–Dec Sept–Dec Jan–Apr2011 2012 2013 (year)Jan–Apr May–Aug Jan–Apr May–Aug May–Aug(b): ML resistant mutation positive: ML resistant mutation negative: ML resistant mutation positive: ML resistant mutation negative: ML resistant mutation negativeType 4-5-7-2, 1Type 4-5-7-3, 1Type 3-5-6-2, 2cFigure 22011 2012 2013J   F  M  A  M  J   J  A  S  O  N  D J   F  M  A  M  J   J  A  S  O  N  D J   F  M  A  M  J   J  A  S  O  N  D(year)(month)10511051105101020010Figure２．Geographical and temporal information of the three genotypes （type 4-5-7-2, 1; type 4-5-7-3, 1; and type 3-5-6-2, 2c）of Mycoplasma pneumoniae isolated between 2011 and 2013 in Yamagata Prefecture, Japan.（a）Distribution of M. pneumoniae strains by district, where each year was divided into three terms. Maps correspond to the gray area shown in Figure 1.（b）Monthly occurrence of M. pneumoniae strains with or without the ML resistance-associated mutation.Figure 10 400kmJapan0 20km Yamagata PrefectureGenotype4-5-7-2, 1    4-5-7-3, 13-5-6-2, 2cMacrolide resistance-associated mutationPositive    NegativeYamagata City332415Tendo City1Kahoku Town1 1Kaminoyama City4Asahi Town15Oe Town112Sagae City151710Yamanobe Town241113Nakayama Town1 315Higashine City1n<10n<20n<50Scale of outer circlen<80Figure１．Geographical information of the three genotypes（type 4-5-7-2, 1; type 4-5-7-3, 1; and type 3-5-6-2, 2c）of Mycoplasma pneumoniae isolated between 2011 and 2013 in Yamagata Prefecture, Japan.Gray area of the map displays the ten districts from where M. pneumoniae strains were derived. The proportion of genotypes and prevalence of the ML resistance-associated mutation by genotype are shown in the outer ring and inner circle, respectively. The numbers in the outer ring indicate the number of M. pneumoniae genotypes detected within each district.－23－Regional spread of Mycoplasma pneumoniaeDecember 2013, did not possess the ML resistance-associated mutation throughout the entirety of its epidemic period.Prevalence of early emergence of the ML-resistant type 4-5-7-2, 1 M. pneumoniae may be due to the isolation of ML-resistant type 4-5-7-2, 1 strain in Yamagata Prefecture before 201111），12）. Type 4-5-7-3, 1 strain, which had not been isolated before 2011 in Yamagata Prefecture11），12）, was ML-susceptible during the initial period of its epidemic. Therefore, emergence of ML-resistant type 4-5-7-3, 1 strain may be associated with a selective pressure resulting from therapeutic ML use. Although type 3-5-6-2, 2c strain, as well as type 4-5-7-3, 1 strain, had low prevalence in Yamagata Prefecture prior to 201111），12）, ML-resistant type 3-5-6-2, 2c strain did not appear throughout its epidemic period. However, we previously reported that ML resistance-associated mutation emerged after ML treatment in a child infected with type 3-5-6-2, 2c M. pneumoniae as well as in children infected with type 4-5-7-3, 113）. Hence, our current findings suggest that type 3-5-6-2, 2c M. pneumoniae with a ML resistance-associated mutation has poor transmission ability compared with other genotypes. Further surveillance is needed to clarify the potential emergence of a ML-resistant type 3-5-6-2, 2c strain in this area.There are several limitations in this study. First, this study was conducted based on M. pneumoniae strains isolated from patients who visited only two pediatric clinics. The two clinics are located in Yamanobe Town and Yamagata City of Yamagata Prefecture; therefore, obtained findings may not reflect the situation of the whole prefecture. Second, we could not collect clinical information on anything except clinical diagnosis; therefore, we could not sufficiently compare severity among patients who were infected with different genotypes of M. pneumoniae. Our study was restricted to a local area and retrospectively performed; however, no similar studies that described regional difference in the prevalence of ML resistance-associated mutation by combining the molecular typing results is reported from other areas in Japan.In summary, this study reveals that the three distinct genotypes of M. pneumoniae geographically and temporally cocirculate in Yamagata Prefecture, Japan and there is little difference in the duration of their epidemics. However, the timing of emergence of the ML-resistant strain differs for each genotype. These genotypic distinctions may account for the observed differences in the prevalence of ML resistance-associated mutation in each of the studied areas. Provision of early information about the genotype of  circulating in each region will help clinicians choose specific antibiotics against  infection. References１． Atkinson TP, Balish MF, Waites KB: Epidemiology, clinical manifestations, pathogenesis and laboratory detection of Mycoplasma pneumoniae infections. FEMS Microbiol Rev 2008; 32: 956-973２． Waites KB, Talkington DF: Mycoplasma pneumoniae and its role as a human pathogen. Clin Microbiol Rev 2004; 17: 697-728３． Foy HM: Infections caused by Mycoplasma pneumoniae and possible carrier state in different populations of patients. Clin Infect Dis 1993; 17: S37-46４． Block S, Hedrick J, Hammerschlag MR, Cassell GH, Craft JC: Mycoplasma pneumoniae and Chlamydia pneumoniae  in pediatr ic community-  acquired pneumonia: comparative efficacy and safety of clarithromycin vs. erythromycin ethylsuccinate. Pediatr Infect Dis J 1995; 14: 471-477５． Korppi M, Heiskanen-Kosma T, Kleemola M: Incidence of community-acquired pneumonia in children caused by Mycoplasma pneumoniae: serological results of a prospective, population-based study in primary health care. Respirology 2004; 9: 109-114６． Yamazaki T, Kenri T: Epidemiology of Mycoplasma pneumoniae Infections in Japan and Therapeutic Strategies for Macrolide-Resistant M. pneumoniae. Front Microbiol 2016; 7: 693７． Sasaki T, Kenri T, Okazaki N, Iseki M, Yamashita R, Shintani M, et al. : Epidemiological study of Mycoplasma pneumoniae infections in Japan based on PCR-restriction fragment length polymorphism of the P1 cytadhesin gene. J Clin Microbiol 1996; 34: 447-449８． Dégrange S, Cazanave C, Charron A, Renaudin H, Bébéar C, Bébéar CM: Development of multiple-locus variable-number tandem-repeat analysis for molecular typing of Mycoplasma pneumoniae. J Clin Microbiol 2009; 47: 914-923－24－Suzuki，Seto，Shimotai，Itagaki，Katsushima，Katsushima，Ikeda，Mizuta，Hongo，Matsuzaki９． Pereyre S, Goret J, Bébéar C: Mycoplasma pneumoniae: current knowledge on macrolide resistance and treatment. Front Microbiol 2016; 7: 97410. Suzuki Y, Seto J, Shimotai Y, Itagaki T, Katsushima Y, Katsushima F, et al. : Polyclonal spread of multiple genotypes of Mycoplasma pneumoniae in semi-closed settings in Yamagata, Japan. J Med Microbiol 2019; 68: 785-79011. Suzuki Y, Seto J, Shimotai Y, Itagaki T, Katsushima Y, Katsushima F, et al. : Multiple-Locus Variable-Number Tandem-Repeat Analysis of Mycoplasma pneumoniae Isolates between 2004 and 2014 in Yamagata, Japan: Change in Molecular Characteristics during an 11-year Period. Jpn J Infect Dis 2017; 70: 642-64612． Suzuki Y, Seto J, Itagaki T, Aoki T, Abiko C, Matsuzaki Y: Gene mutations associated with macrolide-resistance p1 gene typing of Mycoplasma pneumoniae isolated in Yamagata, Japan, between 2004 and 2013. Kansenshogaku Zasshi 2015; 89: 16-22 （Article in Japanese）13． Suzuki Y, Shimotai Y, Itagaki T, Seto J, Ikeda T, Yahagi K, et al. : Development of macrolide resistance-associated mutations after macrolide treatment in children infected with Mycoplasma pneumoniae. J Med Microbiol 2017; 66: 1531-1538

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