Original Studies
Etiology and Epidemiology of Children With Acute Otitis Media and Spontaneous Otorrhea in Suzhou, China Yunfang Ding, MD,* Qian Geng, MSc,†‡ Yunzhen Tao, MD,* Yuzun Lin,†‡ Yunzhong Wang, MD,* Steven Black, MD,§ Genming Zhao, PhD,†‡ and Tao Zhang, PhD†‡ Background: There are scare data about bacterial etiology and the antibiotic susceptibility, serotype distribution and molecular characteristics of pneumococci in children with acute otitis media (AOM) in China. Methods: A prospective study was conducted in Suzhou University Affiliated Children’s Hospital. All children under 18 years of age diagnosed as AOM and with spontaneous otorrhea were offered enrollment, and collection of middle ear fluid was then cultured for bacterial pathogens. The antibiotic susceptibility, serotypes, macrolide resistance genes and sequence types of Streptococcus pneumoniae strains were identified. Results: From January 2011 to December 2013, a total of 229 cases of AOM with spontaneous otorrhea were identified; of these, 159 (69.4%) middle ear fluid specimens were tested positive for bacterial pathogens. The leading cause was S. pneumoniae (47.2%), followed by Staphylococcus aureus (18.8%) and Haemophilus influenzae (7.4%). The antibiotic resistance rates of S. pneumoniae isolates to erythromycin were 99.1%, and the nonsusceptible rate to penicillin was 54.6%. The most common serotypes identified were 19A (45.1%) and 19F (35.4%). The coverage against PCV7 serotypes for this outcome was 56.1% and of PCV13 was 97.6%. The macrolide resistance was mainly mediated by both ermB and mefA/E genes (88.6%). The CC271 was the major clonal complex identified. Conclusions: S. pneumoniae was a leading cause for AOM in children in Suzhou, China. Antibiotics resistance rates of S. pneumoniae were high and mainly due to the spread of CC271 clonal complex. Key Words: acute otitis media, Streptococcus pneumoniae, clonal complex, children, China (Pediatr Infect Dis J 2015;34:e102–e106)
A
cute otitis media (AOM) is a common bacterial infection and often the primary reason for the antibiotic prescriptions in children.1–3 Streptococcus pneumoniae and nontypeable Haemophilus influenzae have been consistently reported to be the 2 major bacterial pathogens responsible for AOM.3–6 Compared with H. influenzae, S. pneumoniae is more likely to be associated with complicated otitis media, such as tympanic membrane rupture and spontaneous otorrhea.7,8 Among the more than 90 pneumococcal serotypes, only Accepted for publication October 30, 2014. From the *Department of laboratory, Suzhou University Affiliated Children’s Hospital, Suzhou, China; †Department of Epidemiology, School of Public Health, Fudan University, Shanghai, People’s Republic of China; ‡Key Laboratory of Public Health Safety, Ministry of Education, Shanghai; and §Center for Global Health, Cincinnati Children’s Hospital, Cincinnati, Ohio. Y.D. and Q.G. contributed equally to this article. This work was supported by National Natural Science Foundation of China [81102166], the Robert Austrian Research Award of ISPPD, SINO-US collaborative program on Emerging and Re-emerging Infectious Diseases [5U2GGH000018] and Shanghai Leading Public Health Discipline Project [12GWZX0101]. The authors have no other funding or conflicts of interest to disclose. Address for correspondence: Tao Zhang, PhD, Department of Epidemiology, School of Public Health, Fudan University, 138 Yi Xue Yuan Road, Shanghai 200032, People’s Republic of China. E-mail: [email protected]. Copyright © 2014 Wolters Kluwer Health, Inc. All rights reserved. ISSN: 0891-3668/15/3405-e102 DOI: 10.1097/INF.0000000000000617
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a few are responsible for most cases of AOM in children worldwide. The most commonly reported serotypes causing AOM worldwide are serotypes 3, 6A, 6B, 9V, 14, 19A, 19F and 23F.9 Seven-valent pneumococcal protein conjugate vaccine (PCV7) has been shown to prevent 6–7% of all cause episodes of AOM and prevent 51% of AOM episodes caused by either vaccine or cross-reactive serotypes of the pneumococcus.1,10 However, an increasing incidence of pneumococcal diseases caused by nonvaccine serotypes, especially 19A, has been documented in the postPCV7 era.11–13 Meanwhile, a rising prevalence of penicillin- and multidrug-resistant pneumococci among non-PCV7 serotypes has been reported.13 The emergence of these non-PCV7 strains appears to have occurred as a consequence of replacement of PCV7 strains and capsular switching among strains.12 To overcome the serotype replacement and reduce the additional pneumococcal diseases burden, 13-valent pneumococcal protein conjugate vaccine (PCV13) has been introduced in some countries. Emerging evidence suggests that PCV13 vaccination has reduced much of the residual invasive and noninvasive pneumococcal diseases burden (for instance, AOM incidence) globally.14–18 There are limited data on the bacterial etiology of AOM in China. In the 1980s, Wang et al.19,20 carried on a study showed that the positive rate for pneumococci in AOM children was 27.5%, and the dominant serogroups were 6, 3, 5 and 19. Since then, there have been no publications from China focusing on the epidemiology of pneumococcal AOM. Although the PCV7 was introduced in September 2008 for optional use in mainland China, the vaccine has not been widely used in most parts of China.21 In Suzhou, China, from 2011 to 2013, there were only 3–4% of the children aged < 5 years had PCV vaccine, and 60% of them got 2 or more doses (data not published). However, serotype distributions have changed, and increasing antibiotic resistant also occurred in China possibly due to the impact of antibiotic use on serotype selection.22–25 Due to the risk of treatment failures, surveillance of the microbiology and antibiotic susceptibility patterns of AOM pathogens in China is important. The national authorities have called for a long-term surveillance to monitor the changes of antibiotics susceptibility and molecular characteristics of S. pneumoniae.26,27 The aims of this study were to assess (1) the bacterial etiology of AOM and the antibiotic resistance patterns and (2) PCV serotypes coverage and molecular characteristics of S. pneumoniae isolated from middle ear fluid (MEF) in Suzhou, during 2011–2013.
MATERIALS AND METHODS Patients and Sample Collections This prospective study was conducted at Suzhou University Affiliated Children’s Hospital (SCH), the sole tertiary hospital center for children in Suzhou district. From 2011 to 2013, all the AOM patients aged < 18years with spontaneous otorrhea and visiting SCH were enrolled in the study after obtaining the informed consent from parents or guardians. The diagnosis of AOM was confirmed by an otolaryngologist. Children diagnosed with chronic otitis media, cholesteatomas and otitis externa and children with ventilating tubes were excluded. The MEF specimens were obtained by swab of spontaneous
The Pediatric Infectious Disease Journal • Volume 34, Number 5, May 2015
The Pediatric Infectious Disease Journal • Volume 34, Number 5, May 2015
ear pus drainage from ear canal by otolaryngologists and then sent to microbiology laboratory without any transport medium. The MEF specimens were immediately plated on trypticase agar containing 5% sheep blood and 5 µg/ml gentamicin and on chocolate agar, and incubated at 37°C for 48 h in a 5% CO2 atmosphere.
S. pneumoniae Strains and Antimicrobial Susceptibility Testing S. pneumoniae cultured from MEF was identified and confirmed by typical colony morphology, alpha-hemolysis, Gram staining, Optochin (Oxoid, Basingstoke, UK) susceptibility and bile solubility. Other bacteria were identified using standard microbiological procedures. The minimal inhibiting concentrations (MICs) of antimicrobial agents, including erythromycin, penicillin G, co-trimoxazole, vancomycin, cefotaxime, clindamycin, tetracycline, amoxicillin, chloramphenicol and levofloxacin were measured using the E-test methodology (AB BioDisk, Stockholm, Sweden). S. pneumoniae ATCC49619 was used as a quality control strain in antimicrobial susceptibility tests. The interpretations of MIC breakpoints and test results were made according to the recommended method of the Clinical and Laboratory Standards Institute criteria.28 Isolates not susceptible to at least 3 antibiotic families were defined as multidrug-resistant (MDR)S. pneumoniae. All strains were stored at –80°C on porous beads.
Molecular Characteristics of S. pneumoniae Strains Chromosomal DNA was extracted from subculture of S. pneumoniae isolates by lysozyme and silicon substrate column adsorption method using TIANamp Bacteria DNA Kit (TIANGEN BIOTECH, Beijing, China) according to the manufacturer’s instructions. The serotypes of pneumococcal isolates were identified by a multiplex polymerase chain reaction (PCR) method as described in previous studies.25 Nineteen kinds of serotypes were determined by 5 sequential multiplex PCR reactions: reaction 1 includes serotype 6A/B, 9V, 15B/C, 18C, 19F; reaction 2 includes serotype 3, 14, 19A, 23F, 23A; reaction 3 includes serotype1, 4, 5; reaction 4 includes serotype 7F, 15A, 22F; and reaction 5 includes serotype 20, 33F, 34. The serotypes not included in the multiplex PCR reactions were defined as nontyped serotypes. Sequence types (STs) of S. pneumoniae isolates were determined using multilocus sequence typing (MLST) technique.29 eBURSTV3 software (http://eburst.mlst.net) was used to explore the relationships among isolates and to group STs sharing 6 identical alleles of 7 loci into a clonal complex. The macrolide resistance genes ermB and mefA/E were amplified by PCR methods for all erythromycin-resistant isolates.30 A positive reference control strain was included in all PCR experiments. The PCR products were run on a 2% agarose gel electrophoresis and were visualized by ethidium bromide staining.
Statistical Analysis All the statistical analyses were performed by using the SPSS statistical package version 16.0 (SPSS, Chicago, IL). The Chi-square test was performed for comparing the proportions. All tests were 2-tailed, and P values less than 0.05 were considered statistically significant.
RESULTS Microbiology of AOM From January 2011 to December 2013, there were 229 children being diagnosed as AOM with perforation and otorrhea in SCH. Pathogenic bacteria were positive in 159 (69.4%) of 229 specimens. A single bacterium was isolated in 141 (61.6%) © 2014 Wolters Kluwer Health, Inc. All rights reserved.
Acute Otorrhea
specimens, and mixed bacteria in 18 (7.8%) specimens. S. pneumoniae was the most common bacteria (108 strains, 47.2% of all MEF specimens), followed by S. aureus (43, 18.8%) and H. influenzae (17, 7.4%) (Table 1). Of all the 229 AOM patients, 132 (57.6%) were boys and 201 (87.8%) were younger than 5 years of age. The positive rate of S. pneumoniae decreased with the age (χ2 = 9.22, P = 0.02). Compared to other seasons of the year, S. pneumoniae was more frequently cultured positive from AOM patients in winter (57.5% vs. 42.3%, χ2=4.63, P = 0.031). No significant difference was detected between females and males, as well as among the 3 study years (2011–2013) (P > 0.05) (Table2).
The Antimicrobial Susceptibility of S. pneumoniae Virtually all (99.1%) of S. pneumoniae strains were resistant to erythromycin and clindamycin. The susceptibility of S. pneumoniae to commonly applied antibiotics was still moderate (Table 3). TABLE 1. Microbiology of Middle Ear Fluid From Children With Acute Otitis Media and Spontaneous Otorrhea During 2011–2013 Pathogen
No. of strains (%)
No growth Any growth Single S. pneumoniae S. aureus H. influenzae S. pyogenes Others Mixed S. pneumoniae + S. aureus S. pneumoniae + H. influenzae S. aureus + H. influenzae S. aureus + M. catarrhalis S. pyogenes + S. maltophilia S. aureus+ Enterobacter cloacae S. aureus + other Neisseria S. pneumoniae + S. aureus + S. pyogenes
70 (30.6) 159 (69.4) 141 (61.6) 95 (41.4) 29 (12.7) 13 (5.7) 2 (0.9) 2 (0.9) 18 (7.8) 9 (3.9) 3 (1.3) 1 (0.4) 1 (0.4) 1 (0.4) 1 (0.4) 1 (0.4) 1 (0.4)
Others included S. intermedius (N = 1) and S. epidermidis (N = 1).
TABLE 2. The Population Profile of Acute Otitis Media (AOM) Pediatric Patients S. pneumoniae positive
AOM Characteristics Sex Male Female Age 0.25 >0.25 >2 >2 4 2 4 ≤4 ≤1 ≤2
≥1 >0.25 ≥16 4 >4 >4 >4 ≤4 ≤1 ≤2
≤0.25 to ≥1 ≤0.25 to >0.25 ≤2 to ≥16 ≤0.5 to ≥16 ≤0.06 to ≥8 ≤0.06 to >4 ≤0.031 to >4 ≤2 to 16 ≤1 to >1 ≤0.5 to >2
107 (99.1) 107 (97.2) 105 (97.2) 89 (82.4) 45 (41.7) 37 (34.3) 11 (10.2) 7 (6.5) 2 (1.9) 1 (0.9)
0 (0.0) 0 (0.0) 0 (0.0) 12 (11.1) 25 (23.2) 31 (28.7) 48 (44.4) 0 (0.0) 0 (0.0) 0 (0.0)
1 (0.9) 1 (0.9) 3 (2.8) 7 (6.5) 38 (35.2) 40 (37.0) 49 (45.4) 101 (93.5) 106 (98.1) 108 (99.1)
99.1 99.1 97.2 93.5 64.8 63.0 54.6 6.5 1.9 0.9
Nonsusceptible isolates included the resistant and intermediate isolates. The percentages are calculated from a total of 108 S. pneumoniae isolates.
The nonsusceptible rates to amoxicillin and cefotaxime increased with age in children younger than 5 years (χ2AMXtrend = 11.25, PAMX = 0.001; χ2CTXtrend = 6.73, PCTXtrend = 0.009). And the nonsusceptrend tible rate to penicillin in group aged 1–2 years (72.2%) was highest among all the AOM cases. There was an increasing trend of nonsusceptibility to amoxicillin during the study period (52.3% in 2011, 71.4% in 2012 and 75.9% in 2013, χ2trend = 4.66, Ptrend = 0.031). All the 108 (100.0%) S. pneumoniae strains were MDR S. pneumoniae. The most common MDR patterns were erythromycin/penicillin/cotrimoxazole/clindamycin/chloramphenicol/amoxicillin/cefotaxime and erythromycin/co-trimoxazole/clindamycin/tetracycline, with a proportion of 41.7% and 13.0%, respectively.
The Serotype Distribution of S. pneumoniae Among the 108 S. pneumoniae strains isolated from AOM patients, 79 (73.1%) strains were successfully revived and serotyped. Except for 2 strains nontyped, all the strains were typed into 7 serotypes (Fig. 1).Three specimens contained 2 serotypes: 2 having both 19A and 19F serotypes, and 1 specimen had both 19A and 6B serotypes isolated. The most prevalent serotypes were 19F (n = 37, 45.1%) and 19A (n = 29, 35.4%), accounting for 80.5% of all pneumococcal strains. The coverage rate of PCV-7, PCV-10 and
PCV-13 serotypes were 56.1%, 56.1% and 97.6%, respectively. The isolation rate of serotypes 19F rose from 32.0% in 2011 to 60.9% in 2013 (44.1% in 2012, χ2trend = 3.96, Ptrend = 0.046). While the isolation rate of serotype 19A decreased from 40.0% in 2011 to 30.4% in 2013 (35.3% in 2012, χ2trend = 0.48, Ptrend = 0.787).
MLST and Detection of Macrolide Resistance Genes The MLST resolved 79 S. pneumoniae strains into 20 STs. Four STs (ST8907, ST9246, ST9387 and ST9398), and 3 alleles (gki397, gdh401 and ddl597) were newly assigned. The most predominate STs were ST271 (n = 29, 36.7%) and ST320 (n = 25, 31.6%), which were mainly associated with serotypes 19A and 19F (Table 4). The eBURSTv3 revealed a single CC271 and 12 singletons containing 63 and 16 strains, respectively. A comparison of the strains with the international Pneumococcal Molecular Epidemiology Network revealed that strains with allelic profile that were identical to those of 4 clones (Taiwan19F-14, Taiwan23F-15, Spain6B-2 and Netherland3-31) constituted 83.9% of the strains. Taiwan19F-14 clone was the major international MDR clone in the current study. Taiwan19F-14 (ST271) clone was derived from Taiwan19F-14 (ST236) by the aroE gene mutation, usually with no change of capsular serotype. Serotype 19A ST320 clone was derived from serotype19F ST271 by ddl gene mutation. Up to 88.6% (N = 70) of strains expressed both ermB and mefA/E genes, and only 11.4% expressed ermB gene only. All Taiwan19F-14 clone strains were positive for both ermB and mefA/E genes, while only 43.8% (N = 7) of other strains were positive for 2 genes, which was significantly different (χ2 = 39.99, P < 0.001). The nonsusceptible rates of Taiwan19F-14 clones to amoxicillin, penicillin and cefotaxime were 82.5%, 71.4% and 65.1%, respectively. Statistical differences were recorded in nonsusceptible rate to penicillin and amoxicillin between Taiwan19F-14 clones and other strains (Fig. 2). In addition, Taiwan19F-14 clones showed a higher level to penicillin (MIC50 = 4 µg/ml) and amoxicillin (MIC50 = 4 µg/ml) resistance with the comparison to other strains (Table 4).
DISCUSSION FIGURE 1. The serotypes identified in 79 S. pneumoniae strains isolated from middle ear drainages of children with acute otitis media.Given to 3 co-colonization isolates, the denominator of PCV coverage rate and serotype proportions was 82. PCV7 including serotype 4, 6B, 9V, 14, 18C, 19F, 23F. Comparing to PCV7, PCV10 has 3 additional serotypes (1, 5, 7F) and PCV13 has 6 additional serotypes (1, 3, 5, 6A, 7F,19A). NT indicates nontyped serotypes.
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In this prospective study, we found that S. pneumoniae was the dominant etiologic agent causing AOM, being isolated in 47.2% of the MEF of AOM patients. A significant proportion of the pneumococcal isolates were resistant to erythromycin, clindamycin, tetracycline, co-trimoxazole and penicillin, and MDR was very common. Contrariwise, many western countries have detected a reduction and much lower rates of antibiotics resistance recently.31,32 The major reasons for this discrepancy could partially © 2014 Wolters Kluwer Health, Inc. All rights reserved.
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TABLE 4. Molecular Characteristics of 79 S. pneumoniae Strains Isolated From Middle Ear Drainages of Children With Acute Otitis Media MIC50 (range) μg/ml Clonal complex
No.
Taiwan19F-14 CC ST271 ST320 ST236(Taiwan19F-14) ST1464 ST7123 ST6993 ST2267 ST1937 Singletons ST90 (Spain6B-2) ST180 (Netherland3-31) ST4660 ST143 ST673 ST505 ST242 (Taiwan23F-15) ST8907 ST876 ST9246 ST9387 ST9398
63 29 25 4 1 1 1 1 1 16 2 2 1 1 1 2 1 1 2 1 1 1
Serotypes (no.) 19F (27), 19A/19F (2) 19A (24), 19A/6B (1) 19F (4) 19F (1) 19F (1) 19F (1) 19F (1) 19F (1) 6B (2) 3 (2) 9V (1) 14 (1) 3 (1) 3 (2) 23F (1) 23F (1) 14 (2) NT 19A (1) NT (1)
Penicillin G
Cefotaxime
Amoxicillin
4 (≤0.031 to ≥8) 2 (≤0.05 to >4) 4 (≤2 to ≥8) 4 (≤0.031 to ≥8) 4 (≤0.05 to >4) >4 (≤2 to ≥8) 4 (2 to ≥8) 2 (0.5 to >4) 4(≤2 to ≥8) 2 (2 to 4) 2 (1 to 2) ≤2(1 to ≤2) 4 2 >4 2 1 ≤2 2 1 ≤2 4 2 >4 4 2 4 2 (≤0.031 to >4) 2 (≤0.06 to >4) ≤2 (≤0.06 to ≥8) 2 (2) 1 (1) ≤2 (≤2) ≤0.031 (≤0.031 to ≤0.06) ≤0.5 (≤0.5 to ≥4) 0.25 (0.25 to ≤2) 0.063 ≤0.5 ≤2 2 1 1 0.0031 ≤0.5 ≤2 ≤0.031 (≤0.031 to 0.5) ≤0.5 (≤0.5) ≤2 (≤2) 1 1 1 4 2 4 4(4 to >4) 2 (2 to 4) ≤2 (≤2) ≤0.06 ≤0.06 ≤0.06 4 >4 ≥8 0.12 ≤0.06 ≤0.06
FIGURE 2. Comparison of nonsusceptible rates for β-lactam antibiotics between the S. pneumoniae Taiwan19F-14 clonal complex (CC) strains and non-Taiwan19F-14CC strains isolated from middle ear drainages of children with acute otitis media. R indicates resistant; I, intermediate. due to the relatively low PCV7 coverage and antibiotics abuse in China. Although we did not have the data to show how many children were previously treated with antibiotics before they visited to SCH, based on our experience, many of the children could have been treated with antibiotics before they were diagnosed as AOM. Nasopharyngeal colonization precedes and is the critical step in pathogenesis of AOM. Ascension of bacteria up the Eustachian tube from nasopharyngeal to the middle ear space, usually with a concurrent viral infection, allows establishment of bacterial infection in a closed space. However, several studies showed that nasopharyngeal cultures cannot distinguish which colonizing otogathogen actually causes AOM.4,33 In our recent publication, the nonsusceptible rates of colonizing pneumococcal strains to penicillin, cefotaxime and amoxicillin were significantly lower than that of AOM isolates in this study.25 The proportion of serotypes 19F and © 2014 Wolters Kluwer Health, Inc. All rights reserved.
ermB/mef genes (no.) ermB + mefA/E (63) ermB + mefA/E (29) ermB + mefA/E (25) ermB + mefA/E (4) ermB + mefA/E (1) ermB + mefA/E (1) ermB + mefA/E (1) ermB + mefA/E (1) ermB + mefA/E (1) ermB (9), ermB + mefA/E (7) ermB + mefA/E (2) ermB + mefA/E (1), ermB (1) ermB + mefA/E (1) ermB (1) ermB (1) ermB (1), ermB + mefA/E (1) ermB (1) ermB (1) ermB (1), ermB + mefA/E (1) ermB (1) ermB + mefA/E (1) ermB (1)
19A in colonizing pneumococcal strains is much lower than that of these AOM strains from the same hospital as well.25 Our results indicate that the characteristics of pneumococci isolated from nasopharyngeal and draining purulent AOM were different, the colonizing strains are not representative of the AOM isolates and it is necessary to continue survey the serotype, antibiotics susceptibility and ST of AOM and invasive pneumococci. Taiwan19F-14 clone was the major international MDR clone in the current study. The nonsusceptible rates of Taiwan19F-14 clones to amoxicillin, penicillin and cefotaxime were much higher than other clones in MEF or others colonizing straining in our previous study.25 All of Taiwan19F-14 clone strains harbored ermB and mefA/E genes. The dramatically high proportion of isolates with both ermB and mefA/E genes was associated with the remarkably high MDR rate in Suzhou. This result indicated a predominant role of Taiwan19F-14 clones in spread of antibiotics resistance in Suzhou. Taiwan19F-14 (ST271) clone was derived from Taiwan19F-14 (ST236) by the aroE gene mutation, usually with no change of capsular serotype. Serotype 19A ST320 clone was derived from serotype19F ST271 by ddl gene mutation and capsular serotype switch and became more virulent by other loci changes.11 Revealed by eBURSTv3 analysis, ST320, ST271 and ST236 were grouped into CC271. Compared with its ancestral clone ST236, serotype 19A ST320 and serotype 19F ST271 strains have higher MIC50 of penicillin G, cefotaxime and amoxicillin. Genetic evolution of pneumococcal clones from Taiwan19F-14 (ST236) to serotype19A ST320 has made this pneumococcus better able to colonize on the nasopharynx.11 In addition, serotype 19A ST320 seems to be more virulent clinically than its ancestral Taiwan19F-14 (ST236) clones.34 It is likely that the implementation of PCV7 program throughout the world, high antimicrobial nonsusceptibility and so on have driven the appearance and spread of the serotype 19A ST320 clone.35 Recently, Hu et al36 used a decision-analytic model to estimate the public health impact of nationwide vaccination of infants with PCV7 in China. They predicted that more than 16.2 million cases of pneumococcal disease and 70,9411 deaths could be prevented in China over the initial 10-year period following the www.pidj.com | e105
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introduction of the PCV7. Thus, we believed that the introduction of PCV13 would be a promising preventive strategy to control the AOM and the increasing trend of clone spread in China. The limitations of this study include the following: (1) the PCV7 vaccination histories of the study children were not available in this study; (2) all of the children were enrolled from a single local hospital, and the number of the children studied was relatively small; and (3) the children with AOM but without spontaneous otorrhea have not been included in the study. Our study results should be interpreted in light of potential selection bias. In conclusion, S. pneumoniae was a leading cause for AOM in children in Suzhou. Antibiotic resistance rates of S. pneumoniae were high and mainly due to the spread of CC271 clonal complex, especially ST320 and ST271. The introduction of PCV13 would be a potential cost–benefit strategy to control AOM.
ACKNOWLEDGEMENTS We are indebted to the parents and children who participated in this study for their time and generosity. We also would like to thank staff from Suzhou University Affiliated Children’s Hospital for their assistance in facilitating the whole procedure. REFERENCES 1. Eskola J, Kilpi T, Palmu A, et al.; Finnish Otitis Media Study Group. Efficacy of a pneumococcal conjugate vaccine against acute otitis media. N Engl J Med. 2001;344:403–409. 2. Auinger P, Lanphear BP, Kalkwarf HJ, et al. Trends in otitis media among children in the United States. Pediatrics. 2003;112(3,pt 1):514–520. 3. Arguedas A, Dagan R, Soley C, et al. Microbiology of otitis media in Costa Rican children, 1999 through 2001. Pediatr Infect Dis J. 2003;22:1063–1068. 4. Casey JR, Kaur R, Friedel VC, et al. Acute otitis media otopathogens during 2008 to 2010 in Rochester, New York. Pediatr Infect Dis J. 2013;32:805–809. 5. Parra MM, Aguilar GM, Echaniz-Aviles G, et al. Bacterial etiology and serotypes of acute otitis media in Mexican children. Vaccine. 2011;29:5544–5549. 6. Pumarola F, Marès J, Losada I, et al. Microbiology of bacteria causing recurrent acute otitis media (AOM) and AOM treatment failure in young children in Spain: shifting pathogens in the post-pneumococcal conjugate vaccination era. Int J Pediatr Otorhinolaryngol. 2013;77:1231–1236. 7. Palmu AA, Herva E, Savolainen H, Karma P, Makela PH, Kilpi TM. Association of clinical signs and symptoms with bacterial findings in acute otitis media. Clin Infect Dis 2004;38:234–242. 8. Caeymaex L, Varon E, Levy C, et al. Characteristics and outcomes of acute otitis media in children carrying Streptococcus pneumoniae or Haemophilus influenzae in their nasopharynx as a single otopathogen after introduction of the heptavalent pneumococcal conjugate vaccine. Pediatr Infect Dis J. 2014;33:533–536. 9. Rodgers GL, Arguedas A, Cohen R, et al. Global serotype distribution among Streptococcus pneumoniae isolates causing otitis media in children: potential implications for pneumococcal conjugate vaccines. Vaccine. 2009;27:3802–3810. 10. Fireman B, Black SB, Shinefield HR, et al. Impact of the pneumococcal conjugate vaccine on otitis media. Pediatr Infect Dis J. 2003;22:10–16. 11. Hsieh YC, Lin TL, Chang KY, et al. Expansion and evolution of Streptococcus pneumoniae serotype 19A ST320 clone as compared to its ancestral clone, Taiwan19F-14 (ST236). J Infect Dis. 2013;208:203–210. 12. Croucher NJ, Harris SR, Fraser C, et al. Rapid pneumococcal evolution in response to clinical interventions. Science. 2011;331:430–434. 13. Pichichero ME, Casey JR. Emergence of a multiresistant serotype 19A pneumococcal strain not included in the 7-valent conjugate vaccine as an otopathogen in children. JAMA. 2007;298:1772–1778. 14. Simonsen L, Taylor RJ, Schuck-Paim C, et al. Effect of 13-valent pneumococcal conjugate vaccine on admissions to hospital 2 years after its
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introduction in the USA: a time series analysis. Lancet Respir Med. 2014;2:387–394. 15. Kaplan SL, Barson WJ, Lin PL, et al. Early trends for invasive pneumococcal infections in children after the introduction of the 13-valent pneumococcal conjugate vaccine. Pediatr Infect Dis J. 2013;32:203–207. 16. Marom T, Tan A, Wilkinson GS, et al. Trends in otitis media-related health care use in the United States, 2001-2011. JAMA Pediatr. 2014;168:68–75. 17. Azzari C, Martinón-Torres F, Schmitt HJ, et al. Evolving role of 13-valent pneumococcal conjugate vaccine in clinical practice. Pediatr Infect Dis J. 2014;33:858–864. 18. Strutton DR, Farkouh RA, Earnshaw SR, et al. Cost-effectiveness of 13-valent pneumococcal conjugate vaccine: Germany, Greece, and The Netherlands. J Infect. 2012;64:54–67. 19. Wang R, Wang H, Zhu Xg, et al. Study of pneumococcal acute otitis media in infants. Chinese J Otorhinolaryngol. 1985;20:132–134. 20. Wang R, Ding S, Wang X, et al. Study of pneumococcal acute otitis media and the serotype distribution. Chinese J Otorhinolaryngol. 1989;24:84–86. 21. Yu H, Yang W, Varma JK. To save children’s lives, China should adopt an initiative to speed introduction of pneumonia vaccines. Health Aff. 2012;31:2545–2553. 22. Kim SH, Song JH, Chung DR, et al.; ANSORP Study Group. Changing trends in antimicrobial resistance and serotypes of Streptococcus pneumoniae isolates in Asian countries: an Asian Network for Surveillance of Resistant Pathogens (ANSORP) study. Antimicrob Agents Chemother. 2012;56:1418–1426. 23. Yao KH, Wang LB, Zhao GM, et al. Pneumococcal serotype distribution and antimicrobial resistance in Chinese children hospitalized for pneumonia. Vaccine. 2011;29:2296–2301. 24. Xue L, Yao K, Xie G, et al. Serotype distribution and antimicrobial resistance of Streptococcus pneumoniae isolates that cause invasive disease among Chinese children. Clin Infect Dis. 2010;50:741–744. 25. Geng Q, Zhang T, Ding Y, et al. Molecular characterization and antimicrobial susceptibility of Streptococcus pneumoniae isolated from children hospitalized with respiratory infections in Suzhou, China. PLoS One. 2014;9:e93752. 26. Zhou L, Yu SJ, Gao W, et al. Serotype distribution and antibiotic resistance of 140 pneumococcal isolates from pediatric patients with upper respiratory infections in Beijing, 2010. Vaccine. 2011;29:7704–7710. 27. Ma X, Zhao R, Ma Z, et al. Serotype distribution and antimicrobial resistance of Streptococcus pneumoniae isolates causing invasive diseases from Shenzhen Children’s Hospital. PLoS One. 2013;8:e67507. 28. CLSI. Performance Standards for Antimicrobial Susceptibility Testing: 21st Informational Supplement. M100–S21. Clinical and Laboratory Standards Institute, Wayne, PA; 2011. 29. Enright MC, Spratt BG. A multilocus sequence typing scheme for Streptococcus pneumoniae: identification of clones associated with serious invasive disease. Microbiology. 1998;144(pt 11):3049–3060. 30. Rong C. Study on Resistance of Streptococcus pneumoniae and Etiological Distribution in Hospitalized Pediatric Patients with Respiratory Infections. Fudan University, Shanghai, People’s Republic of China; 2010. 31. Henderson KL, Muller-Pebody B, Blackburn RM, et al. Reduction in erythromycin resistance in invasive pneumococci from young children in England and Wales. J Antimicrob Chemother. 2010;65:369–370. 32. Siira L, Jalava J, Kaijalainen T, Ollgren J, Lyytikäinen O, Virolainen A. Antimicrobial resistance in relation to sero- and genotypes among invasive Streptococcus pneumoniae in Finland, 2007–2011. Microb Drug Resist. 2014;20:124–130. 33. Eldan M, Leibovitz E, Piglansky L, et al. Predictive value of pneumococcal nasopharyngeal cultures for the assessment of nonresponsive acute otitis media in children. Pediatr Infect Dis J. 2000;19:298–303. 34. Hsieh YC, Wang CW, Lai SH, et al. Necrotizing pneumococcal pneumonia with bronchopleural fistula among children in Taiwan. Pediatr Infect Dis J. 2011;30:740–744. 35. Tyrrell GJ. The changing epidemiology of Streptococcus pneumoniae serotype 19A clonal complexes. J Infect Dis. 2011;203:1345–1347. 36. Hu S, Shi Q, Chen CI, et al. Estimated public health impact of nationwide vaccination of infants with 7-valent pneumococcal conjugate vaccine (PCV7) in China. Int J Infect Dis. 2014;26:116–122.
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Etiology and Epidemiology of Children With Acute Otitis Media and Spontaneous Otorrhea in Suzhou, China Yunfang Ding, MD,* Qian Geng, MSc,†‡ Yunzhen Tao, MD,* Yuzun Lin,†‡ Yunzhong Wang, MD,* Steven Black, MD,§ Genming Zhao, PhD,†‡ and Tao Zhang, PhD†‡ Background: There are scare data about bacterial etiology and the antibiotic susceptibility, serotype distribution and molecular characteristics of pneumococci in children with acute otitis media (AOM) in China. Methods: A prospective study was conducted in Suzhou University Affiliated Children’s Hospital. All children under 18 years of age diagnosed as AOM and with spontaneous otorrhea were offered enrollment, and collection of middle ear fluid was then cultured for bacterial pathogens. The antibiotic susceptibility, serotypes, macrolide resistance genes and sequence types of Streptococcus pneumoniae strains were identified. Results: From January 2011 to December 2013, a total of 229 cases of AOM with spontaneous otorrhea were identified; of these, 159 (69.4%) middle ear fluid specimens were tested positive for bacterial pathogens. The leading cause was S. pneumoniae (47.2%), followed by Staphylococcus aureus (18.8%) and Haemophilus influenzae (7.4%). The antibiotic resistance rates of S. pneumoniae isolates to erythromycin were 99.1%, and the nonsusceptible rate to penicillin was 54.6%. The most common serotypes identified were 19A (45.1%) and 19F (35.4%). The coverage against PCV7 serotypes for this outcome was 56.1% and of PCV13 was 97.6%. The macrolide resistance was mainly mediated by both ermB and mefA/E genes (88.6%). The CC271 was the major clonal complex identified. Conclusions: S. pneumoniae was a leading cause for AOM in children in Suzhou, China. Antibiotics resistance rates of S. pneumoniae were high and mainly due to the spread of CC271 clonal complex. Key Words: acute otitis media, Streptococcus pneumoniae, clonal complex, children, China (Pediatr Infect Dis J 2015;34:e102–e106)
A
cute otitis media (AOM) is a common bacterial infection and often the primary reason for the antibiotic prescriptions in children.1–3 Streptococcus pneumoniae and nontypeable Haemophilus influenzae have been consistently reported to be the 2 major bacterial pathogens responsible for AOM.3–6 Compared with H. influenzae, S. pneumoniae is more likely to be associated with complicated otitis media, such as tympanic membrane rupture and spontaneous otorrhea.7,8 Among the more than 90 pneumococcal serotypes, only Accepted for publication October 30, 2014. From the *Department of laboratory, Suzhou University Affiliated Children’s Hospital, Suzhou, China; †Department of Epidemiology, School of Public Health, Fudan University, Shanghai, People’s Republic of China; ‡Key Laboratory of Public Health Safety, Ministry of Education, Shanghai; and §Center for Global Health, Cincinnati Children’s Hospital, Cincinnati, Ohio. Y.D. and Q.G. contributed equally to this article. This work was supported by National Natural Science Foundation of China [81102166], the Robert Austrian Research Award of ISPPD, SINO-US collaborative program on Emerging and Re-emerging Infectious Diseases [5U2GGH000018] and Shanghai Leading Public Health Discipline Project [12GWZX0101]. The authors have no other funding or conflicts of interest to disclose. Address for correspondence: Tao Zhang, PhD, Department of Epidemiology, School of Public Health, Fudan University, 138 Yi Xue Yuan Road, Shanghai 200032, People’s Republic of China. E-mail: [email protected]. Copyright © 2014 Wolters Kluwer Health, Inc. All rights reserved. ISSN: 0891-3668/15/3405-e102 DOI: 10.1097/INF.0000000000000617
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a few are responsible for most cases of AOM in children worldwide. The most commonly reported serotypes causing AOM worldwide are serotypes 3, 6A, 6B, 9V, 14, 19A, 19F and 23F.9 Seven-valent pneumococcal protein conjugate vaccine (PCV7) has been shown to prevent 6–7% of all cause episodes of AOM and prevent 51% of AOM episodes caused by either vaccine or cross-reactive serotypes of the pneumococcus.1,10 However, an increasing incidence of pneumococcal diseases caused by nonvaccine serotypes, especially 19A, has been documented in the postPCV7 era.11–13 Meanwhile, a rising prevalence of penicillin- and multidrug-resistant pneumococci among non-PCV7 serotypes has been reported.13 The emergence of these non-PCV7 strains appears to have occurred as a consequence of replacement of PCV7 strains and capsular switching among strains.12 To overcome the serotype replacement and reduce the additional pneumococcal diseases burden, 13-valent pneumococcal protein conjugate vaccine (PCV13) has been introduced in some countries. Emerging evidence suggests that PCV13 vaccination has reduced much of the residual invasive and noninvasive pneumococcal diseases burden (for instance, AOM incidence) globally.14–18 There are limited data on the bacterial etiology of AOM in China. In the 1980s, Wang et al.19,20 carried on a study showed that the positive rate for pneumococci in AOM children was 27.5%, and the dominant serogroups were 6, 3, 5 and 19. Since then, there have been no publications from China focusing on the epidemiology of pneumococcal AOM. Although the PCV7 was introduced in September 2008 for optional use in mainland China, the vaccine has not been widely used in most parts of China.21 In Suzhou, China, from 2011 to 2013, there were only 3–4% of the children aged < 5 years had PCV vaccine, and 60% of them got 2 or more doses (data not published). However, serotype distributions have changed, and increasing antibiotic resistant also occurred in China possibly due to the impact of antibiotic use on serotype selection.22–25 Due to the risk of treatment failures, surveillance of the microbiology and antibiotic susceptibility patterns of AOM pathogens in China is important. The national authorities have called for a long-term surveillance to monitor the changes of antibiotics susceptibility and molecular characteristics of S. pneumoniae.26,27 The aims of this study were to assess (1) the bacterial etiology of AOM and the antibiotic resistance patterns and (2) PCV serotypes coverage and molecular characteristics of S. pneumoniae isolated from middle ear fluid (MEF) in Suzhou, during 2011–2013.
MATERIALS AND METHODS Patients and Sample Collections This prospective study was conducted at Suzhou University Affiliated Children’s Hospital (SCH), the sole tertiary hospital center for children in Suzhou district. From 2011 to 2013, all the AOM patients aged < 18years with spontaneous otorrhea and visiting SCH were enrolled in the study after obtaining the informed consent from parents or guardians. The diagnosis of AOM was confirmed by an otolaryngologist. Children diagnosed with chronic otitis media, cholesteatomas and otitis externa and children with ventilating tubes were excluded. The MEF specimens were obtained by swab of spontaneous
The Pediatric Infectious Disease Journal • Volume 34, Number 5, May 2015
The Pediatric Infectious Disease Journal • Volume 34, Number 5, May 2015
ear pus drainage from ear canal by otolaryngologists and then sent to microbiology laboratory without any transport medium. The MEF specimens were immediately plated on trypticase agar containing 5% sheep blood and 5 µg/ml gentamicin and on chocolate agar, and incubated at 37°C for 48 h in a 5% CO2 atmosphere.
S. pneumoniae Strains and Antimicrobial Susceptibility Testing S. pneumoniae cultured from MEF was identified and confirmed by typical colony morphology, alpha-hemolysis, Gram staining, Optochin (Oxoid, Basingstoke, UK) susceptibility and bile solubility. Other bacteria were identified using standard microbiological procedures. The minimal inhibiting concentrations (MICs) of antimicrobial agents, including erythromycin, penicillin G, co-trimoxazole, vancomycin, cefotaxime, clindamycin, tetracycline, amoxicillin, chloramphenicol and levofloxacin were measured using the E-test methodology (AB BioDisk, Stockholm, Sweden). S. pneumoniae ATCC49619 was used as a quality control strain in antimicrobial susceptibility tests. The interpretations of MIC breakpoints and test results were made according to the recommended method of the Clinical and Laboratory Standards Institute criteria.28 Isolates not susceptible to at least 3 antibiotic families were defined as multidrug-resistant (MDR)S. pneumoniae. All strains were stored at –80°C on porous beads.
Molecular Characteristics of S. pneumoniae Strains Chromosomal DNA was extracted from subculture of S. pneumoniae isolates by lysozyme and silicon substrate column adsorption method using TIANamp Bacteria DNA Kit (TIANGEN BIOTECH, Beijing, China) according to the manufacturer’s instructions. The serotypes of pneumococcal isolates were identified by a multiplex polymerase chain reaction (PCR) method as described in previous studies.25 Nineteen kinds of serotypes were determined by 5 sequential multiplex PCR reactions: reaction 1 includes serotype 6A/B, 9V, 15B/C, 18C, 19F; reaction 2 includes serotype 3, 14, 19A, 23F, 23A; reaction 3 includes serotype1, 4, 5; reaction 4 includes serotype 7F, 15A, 22F; and reaction 5 includes serotype 20, 33F, 34. The serotypes not included in the multiplex PCR reactions were defined as nontyped serotypes. Sequence types (STs) of S. pneumoniae isolates were determined using multilocus sequence typing (MLST) technique.29 eBURSTV3 software (http://eburst.mlst.net) was used to explore the relationships among isolates and to group STs sharing 6 identical alleles of 7 loci into a clonal complex. The macrolide resistance genes ermB and mefA/E were amplified by PCR methods for all erythromycin-resistant isolates.30 A positive reference control strain was included in all PCR experiments. The PCR products were run on a 2% agarose gel electrophoresis and were visualized by ethidium bromide staining.
Statistical Analysis All the statistical analyses were performed by using the SPSS statistical package version 16.0 (SPSS, Chicago, IL). The Chi-square test was performed for comparing the proportions. All tests were 2-tailed, and P values less than 0.05 were considered statistically significant.
RESULTS Microbiology of AOM From January 2011 to December 2013, there were 229 children being diagnosed as AOM with perforation and otorrhea in SCH. Pathogenic bacteria were positive in 159 (69.4%) of 229 specimens. A single bacterium was isolated in 141 (61.6%) © 2014 Wolters Kluwer Health, Inc. All rights reserved.
Acute Otorrhea
specimens, and mixed bacteria in 18 (7.8%) specimens. S. pneumoniae was the most common bacteria (108 strains, 47.2% of all MEF specimens), followed by S. aureus (43, 18.8%) and H. influenzae (17, 7.4%) (Table 1). Of all the 229 AOM patients, 132 (57.6%) were boys and 201 (87.8%) were younger than 5 years of age. The positive rate of S. pneumoniae decreased with the age (χ2 = 9.22, P = 0.02). Compared to other seasons of the year, S. pneumoniae was more frequently cultured positive from AOM patients in winter (57.5% vs. 42.3%, χ2=4.63, P = 0.031). No significant difference was detected between females and males, as well as among the 3 study years (2011–2013) (P > 0.05) (Table2).
The Antimicrobial Susceptibility of S. pneumoniae Virtually all (99.1%) of S. pneumoniae strains were resistant to erythromycin and clindamycin. The susceptibility of S. pneumoniae to commonly applied antibiotics was still moderate (Table 3). TABLE 1. Microbiology of Middle Ear Fluid From Children With Acute Otitis Media and Spontaneous Otorrhea During 2011–2013 Pathogen
No. of strains (%)
No growth Any growth Single S. pneumoniae S. aureus H. influenzae S. pyogenes Others Mixed S. pneumoniae + S. aureus S. pneumoniae + H. influenzae S. aureus + H. influenzae S. aureus + M. catarrhalis S. pyogenes + S. maltophilia S. aureus+ Enterobacter cloacae S. aureus + other Neisseria S. pneumoniae + S. aureus + S. pyogenes
70 (30.6) 159 (69.4) 141 (61.6) 95 (41.4) 29 (12.7) 13 (5.7) 2 (0.9) 2 (0.9) 18 (7.8) 9 (3.9) 3 (1.3) 1 (0.4) 1 (0.4) 1 (0.4) 1 (0.4) 1 (0.4) 1 (0.4)
Others included S. intermedius (N = 1) and S. epidermidis (N = 1).
TABLE 2. The Population Profile of Acute Otitis Media (AOM) Pediatric Patients S. pneumoniae positive
AOM Characteristics Sex Male Female Age 0.25 >0.25 >2 >2 4 2 4 ≤4 ≤1 ≤2
≥1 >0.25 ≥16 4 >4 >4 >4 ≤4 ≤1 ≤2
≤0.25 to ≥1 ≤0.25 to >0.25 ≤2 to ≥16 ≤0.5 to ≥16 ≤0.06 to ≥8 ≤0.06 to >4 ≤0.031 to >4 ≤2 to 16 ≤1 to >1 ≤0.5 to >2
107 (99.1) 107 (97.2) 105 (97.2) 89 (82.4) 45 (41.7) 37 (34.3) 11 (10.2) 7 (6.5) 2 (1.9) 1 (0.9)
0 (0.0) 0 (0.0) 0 (0.0) 12 (11.1) 25 (23.2) 31 (28.7) 48 (44.4) 0 (0.0) 0 (0.0) 0 (0.0)
1 (0.9) 1 (0.9) 3 (2.8) 7 (6.5) 38 (35.2) 40 (37.0) 49 (45.4) 101 (93.5) 106 (98.1) 108 (99.1)
99.1 99.1 97.2 93.5 64.8 63.0 54.6 6.5 1.9 0.9
Nonsusceptible isolates included the resistant and intermediate isolates. The percentages are calculated from a total of 108 S. pneumoniae isolates.
The nonsusceptible rates to amoxicillin and cefotaxime increased with age in children younger than 5 years (χ2AMXtrend = 11.25, PAMX = 0.001; χ2CTXtrend = 6.73, PCTXtrend = 0.009). And the nonsusceptrend tible rate to penicillin in group aged 1–2 years (72.2%) was highest among all the AOM cases. There was an increasing trend of nonsusceptibility to amoxicillin during the study period (52.3% in 2011, 71.4% in 2012 and 75.9% in 2013, χ2trend = 4.66, Ptrend = 0.031). All the 108 (100.0%) S. pneumoniae strains were MDR S. pneumoniae. The most common MDR patterns were erythromycin/penicillin/cotrimoxazole/clindamycin/chloramphenicol/amoxicillin/cefotaxime and erythromycin/co-trimoxazole/clindamycin/tetracycline, with a proportion of 41.7% and 13.0%, respectively.
The Serotype Distribution of S. pneumoniae Among the 108 S. pneumoniae strains isolated from AOM patients, 79 (73.1%) strains were successfully revived and serotyped. Except for 2 strains nontyped, all the strains were typed into 7 serotypes (Fig. 1).Three specimens contained 2 serotypes: 2 having both 19A and 19F serotypes, and 1 specimen had both 19A and 6B serotypes isolated. The most prevalent serotypes were 19F (n = 37, 45.1%) and 19A (n = 29, 35.4%), accounting for 80.5% of all pneumococcal strains. The coverage rate of PCV-7, PCV-10 and
PCV-13 serotypes were 56.1%, 56.1% and 97.6%, respectively. The isolation rate of serotypes 19F rose from 32.0% in 2011 to 60.9% in 2013 (44.1% in 2012, χ2trend = 3.96, Ptrend = 0.046). While the isolation rate of serotype 19A decreased from 40.0% in 2011 to 30.4% in 2013 (35.3% in 2012, χ2trend = 0.48, Ptrend = 0.787).
MLST and Detection of Macrolide Resistance Genes The MLST resolved 79 S. pneumoniae strains into 20 STs. Four STs (ST8907, ST9246, ST9387 and ST9398), and 3 alleles (gki397, gdh401 and ddl597) were newly assigned. The most predominate STs were ST271 (n = 29, 36.7%) and ST320 (n = 25, 31.6%), which were mainly associated with serotypes 19A and 19F (Table 4). The eBURSTv3 revealed a single CC271 and 12 singletons containing 63 and 16 strains, respectively. A comparison of the strains with the international Pneumococcal Molecular Epidemiology Network revealed that strains with allelic profile that were identical to those of 4 clones (Taiwan19F-14, Taiwan23F-15, Spain6B-2 and Netherland3-31) constituted 83.9% of the strains. Taiwan19F-14 clone was the major international MDR clone in the current study. Taiwan19F-14 (ST271) clone was derived from Taiwan19F-14 (ST236) by the aroE gene mutation, usually with no change of capsular serotype. Serotype 19A ST320 clone was derived from serotype19F ST271 by ddl gene mutation. Up to 88.6% (N = 70) of strains expressed both ermB and mefA/E genes, and only 11.4% expressed ermB gene only. All Taiwan19F-14 clone strains were positive for both ermB and mefA/E genes, while only 43.8% (N = 7) of other strains were positive for 2 genes, which was significantly different (χ2 = 39.99, P < 0.001). The nonsusceptible rates of Taiwan19F-14 clones to amoxicillin, penicillin and cefotaxime were 82.5%, 71.4% and 65.1%, respectively. Statistical differences were recorded in nonsusceptible rate to penicillin and amoxicillin between Taiwan19F-14 clones and other strains (Fig. 2). In addition, Taiwan19F-14 clones showed a higher level to penicillin (MIC50 = 4 µg/ml) and amoxicillin (MIC50 = 4 µg/ml) resistance with the comparison to other strains (Table 4).
DISCUSSION FIGURE 1. The serotypes identified in 79 S. pneumoniae strains isolated from middle ear drainages of children with acute otitis media.Given to 3 co-colonization isolates, the denominator of PCV coverage rate and serotype proportions was 82. PCV7 including serotype 4, 6B, 9V, 14, 18C, 19F, 23F. Comparing to PCV7, PCV10 has 3 additional serotypes (1, 5, 7F) and PCV13 has 6 additional serotypes (1, 3, 5, 6A, 7F,19A). NT indicates nontyped serotypes.
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In this prospective study, we found that S. pneumoniae was the dominant etiologic agent causing AOM, being isolated in 47.2% of the MEF of AOM patients. A significant proportion of the pneumococcal isolates were resistant to erythromycin, clindamycin, tetracycline, co-trimoxazole and penicillin, and MDR was very common. Contrariwise, many western countries have detected a reduction and much lower rates of antibiotics resistance recently.31,32 The major reasons for this discrepancy could partially © 2014 Wolters Kluwer Health, Inc. All rights reserved.
The Pediatric Infectious Disease Journal • Volume 34, Number 5, May 2015
Acute Otorrhea
TABLE 4. Molecular Characteristics of 79 S. pneumoniae Strains Isolated From Middle Ear Drainages of Children With Acute Otitis Media MIC50 (range) μg/ml Clonal complex
No.
Taiwan19F-14 CC ST271 ST320 ST236(Taiwan19F-14) ST1464 ST7123 ST6993 ST2267 ST1937 Singletons ST90 (Spain6B-2) ST180 (Netherland3-31) ST4660 ST143 ST673 ST505 ST242 (Taiwan23F-15) ST8907 ST876 ST9246 ST9387 ST9398
63 29 25 4 1 1 1 1 1 16 2 2 1 1 1 2 1 1 2 1 1 1
Serotypes (no.) 19F (27), 19A/19F (2) 19A (24), 19A/6B (1) 19F (4) 19F (1) 19F (1) 19F (1) 19F (1) 19F (1) 6B (2) 3 (2) 9V (1) 14 (1) 3 (1) 3 (2) 23F (1) 23F (1) 14 (2) NT 19A (1) NT (1)
Penicillin G
Cefotaxime
Amoxicillin
4 (≤0.031 to ≥8) 2 (≤0.05 to >4) 4 (≤2 to ≥8) 4 (≤0.031 to ≥8) 4 (≤0.05 to >4) >4 (≤2 to ≥8) 4 (2 to ≥8) 2 (0.5 to >4) 4(≤2 to ≥8) 2 (2 to 4) 2 (1 to 2) ≤2(1 to ≤2) 4 2 >4 2 1 ≤2 2 1 ≤2 4 2 >4 4 2 4 2 (≤0.031 to >4) 2 (≤0.06 to >4) ≤2 (≤0.06 to ≥8) 2 (2) 1 (1) ≤2 (≤2) ≤0.031 (≤0.031 to ≤0.06) ≤0.5 (≤0.5 to ≥4) 0.25 (0.25 to ≤2) 0.063 ≤0.5 ≤2 2 1 1 0.0031 ≤0.5 ≤2 ≤0.031 (≤0.031 to 0.5) ≤0.5 (≤0.5) ≤2 (≤2) 1 1 1 4 2 4 4(4 to >4) 2 (2 to 4) ≤2 (≤2) ≤0.06 ≤0.06 ≤0.06 4 >4 ≥8 0.12 ≤0.06 ≤0.06
FIGURE 2. Comparison of nonsusceptible rates for β-lactam antibiotics between the S. pneumoniae Taiwan19F-14 clonal complex (CC) strains and non-Taiwan19F-14CC strains isolated from middle ear drainages of children with acute otitis media. R indicates resistant; I, intermediate. due to the relatively low PCV7 coverage and antibiotics abuse in China. Although we did not have the data to show how many children were previously treated with antibiotics before they visited to SCH, based on our experience, many of the children could have been treated with antibiotics before they were diagnosed as AOM. Nasopharyngeal colonization precedes and is the critical step in pathogenesis of AOM. Ascension of bacteria up the Eustachian tube from nasopharyngeal to the middle ear space, usually with a concurrent viral infection, allows establishment of bacterial infection in a closed space. However, several studies showed that nasopharyngeal cultures cannot distinguish which colonizing otogathogen actually causes AOM.4,33 In our recent publication, the nonsusceptible rates of colonizing pneumococcal strains to penicillin, cefotaxime and amoxicillin were significantly lower than that of AOM isolates in this study.25 The proportion of serotypes 19F and © 2014 Wolters Kluwer Health, Inc. All rights reserved.
ermB/mef genes (no.) ermB + mefA/E (63) ermB + mefA/E (29) ermB + mefA/E (25) ermB + mefA/E (4) ermB + mefA/E (1) ermB + mefA/E (1) ermB + mefA/E (1) ermB + mefA/E (1) ermB + mefA/E (1) ermB (9), ermB + mefA/E (7) ermB + mefA/E (2) ermB + mefA/E (1), ermB (1) ermB + mefA/E (1) ermB (1) ermB (1) ermB (1), ermB + mefA/E (1) ermB (1) ermB (1) ermB (1), ermB + mefA/E (1) ermB (1) ermB + mefA/E (1) ermB (1)
19A in colonizing pneumococcal strains is much lower than that of these AOM strains from the same hospital as well.25 Our results indicate that the characteristics of pneumococci isolated from nasopharyngeal and draining purulent AOM were different, the colonizing strains are not representative of the AOM isolates and it is necessary to continue survey the serotype, antibiotics susceptibility and ST of AOM and invasive pneumococci. Taiwan19F-14 clone was the major international MDR clone in the current study. The nonsusceptible rates of Taiwan19F-14 clones to amoxicillin, penicillin and cefotaxime were much higher than other clones in MEF or others colonizing straining in our previous study.25 All of Taiwan19F-14 clone strains harbored ermB and mefA/E genes. The dramatically high proportion of isolates with both ermB and mefA/E genes was associated with the remarkably high MDR rate in Suzhou. This result indicated a predominant role of Taiwan19F-14 clones in spread of antibiotics resistance in Suzhou. Taiwan19F-14 (ST271) clone was derived from Taiwan19F-14 (ST236) by the aroE gene mutation, usually with no change of capsular serotype. Serotype 19A ST320 clone was derived from serotype19F ST271 by ddl gene mutation and capsular serotype switch and became more virulent by other loci changes.11 Revealed by eBURSTv3 analysis, ST320, ST271 and ST236 were grouped into CC271. Compared with its ancestral clone ST236, serotype 19A ST320 and serotype 19F ST271 strains have higher MIC50 of penicillin G, cefotaxime and amoxicillin. Genetic evolution of pneumococcal clones from Taiwan19F-14 (ST236) to serotype19A ST320 has made this pneumococcus better able to colonize on the nasopharynx.11 In addition, serotype 19A ST320 seems to be more virulent clinically than its ancestral Taiwan19F-14 (ST236) clones.34 It is likely that the implementation of PCV7 program throughout the world, high antimicrobial nonsusceptibility and so on have driven the appearance and spread of the serotype 19A ST320 clone.35 Recently, Hu et al36 used a decision-analytic model to estimate the public health impact of nationwide vaccination of infants with PCV7 in China. They predicted that more than 16.2 million cases of pneumococcal disease and 70,9411 deaths could be prevented in China over the initial 10-year period following the www.pidj.com | e105
The Pediatric Infectious Disease Journal • Volume 34, Number 5, May 2015
Ding et al
introduction of the PCV7. Thus, we believed that the introduction of PCV13 would be a promising preventive strategy to control the AOM and the increasing trend of clone spread in China. The limitations of this study include the following: (1) the PCV7 vaccination histories of the study children were not available in this study; (2) all of the children were enrolled from a single local hospital, and the number of the children studied was relatively small; and (3) the children with AOM but without spontaneous otorrhea have not been included in the study. Our study results should be interpreted in light of potential selection bias. In conclusion, S. pneumoniae was a leading cause for AOM in children in Suzhou. Antibiotic resistance rates of S. pneumoniae were high and mainly due to the spread of CC271 clonal complex, especially ST320 and ST271. The introduction of PCV13 would be a potential cost–benefit strategy to control AOM.
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