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Serotype distribution, antimicrobial resistance, and molecular characterization of invasive group B Streptococcus isolates recovered from Chinese neonates

  • Author Footnotes
    1 Ping Wang, Zhuoya Ma, and Jingjing Tong contributed equally to this work.
    Ping Wang
    Footnotes
    1 Ping Wang, Zhuoya Ma, and Jingjing Tong contributed equally to this work.
    Affiliations
    Key Laboratory of Major Diseases in Children and National Key Discipline of Pediatrics (Capital Medical University), Ministry of Education, Beijing Pediatrics Research Institute, Beijing Children's Hospital Affiliated to Capital Medical University, Nan Lishi Road, Beijing 100045, China

    Department of Pediatrics, Beijing Obstetrics and Gynecology Hospital and Beijing Maternal and Child Health Care Hospital, Affiliated to Capital Medical University, Beijing, China
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  • Author Footnotes
    1 Ping Wang, Zhuoya Ma, and Jingjing Tong contributed equally to this work.
    Zhuoya Ma
    Footnotes
    1 Ping Wang, Zhuoya Ma, and Jingjing Tong contributed equally to this work.
    Affiliations
    Laboratory of Bacteriology and Department of Respiratory Diseases, Shenzhen Children's Hospital, Shenzhen, China
    Search for articles by this author
  • Author Footnotes
    1 Ping Wang, Zhuoya Ma, and Jingjing Tong contributed equally to this work.
    Jingjing Tong
    Footnotes
    1 Ping Wang, Zhuoya Ma, and Jingjing Tong contributed equally to this work.
    Affiliations
    Key Laboratory of Major Diseases in Children and National Key Discipline of Pediatrics (Capital Medical University), Ministry of Education, Beijing Pediatrics Research Institute, Beijing Children's Hospital Affiliated to Capital Medical University, Nan Lishi Road, Beijing 100045, China
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  • Ruizhen Zhao
    Affiliations
    Laboratory of Bacteriology and Department of Respiratory Diseases, Shenzhen Children's Hospital, Shenzhen, China
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  • Wei Shi
    Affiliations
    Key Laboratory of Major Diseases in Children and National Key Discipline of Pediatrics (Capital Medical University), Ministry of Education, Beijing Pediatrics Research Institute, Beijing Children's Hospital Affiliated to Capital Medical University, Nan Lishi Road, Beijing 100045, China
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  • Sangjie Yu
    Affiliations
    Key Laboratory of Major Diseases in Children and National Key Discipline of Pediatrics (Capital Medical University), Ministry of Education, Beijing Pediatrics Research Institute, Beijing Children's Hospital Affiliated to Capital Medical University, Nan Lishi Road, Beijing 100045, China
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  • Kaihu Yao
    Affiliations
    Key Laboratory of Major Diseases in Children and National Key Discipline of Pediatrics (Capital Medical University), Ministry of Education, Beijing Pediatrics Research Institute, Beijing Children's Hospital Affiliated to Capital Medical University, Nan Lishi Road, Beijing 100045, China
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  • Yuejie Zheng
    Correspondence
    Co-corresponding author.
    Affiliations
    Laboratory of Bacteriology and Department of Respiratory Diseases, Shenzhen Children's Hospital, Shenzhen, China
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  • Yonghong Yang
    Correspondence
    Corresponding author.
    Affiliations
    Key Laboratory of Major Diseases in Children and National Key Discipline of Pediatrics (Capital Medical University), Ministry of Education, Beijing Pediatrics Research Institute, Beijing Children's Hospital Affiliated to Capital Medical University, Nan Lishi Road, Beijing 100045, China
    Search for articles by this author
  • Author Footnotes
    1 Ping Wang, Zhuoya Ma, and Jingjing Tong contributed equally to this work.
Open AccessPublished:June 30, 2015DOI:https://doi.org/10.1016/j.ijid.2015.06.019

      Summary

      Background

      Group B Streptococcus (GBS) is an important neonatal pathogen associated with high morbidity and mortality in developed countries. However, data describing neonatal GBS disease in developing countries, particularly in Asia, are largely incomplete. The aim of this study was to determine the serotype distribution, antimicrobial resistance, and molecular characteristics of invasive GBS isolates recovered from Chinese neonates.

      Methods

      From 2008 to 2013, 40 GBS isolates were recovered from infected neonates less than 3 months of age. All isolates were identified with the CAMP test and commercially available techniques. Serotyping was performed by latex agglutination. Antibiotic susceptibility was tested with Etest strips and the disk diffusion method. Multilocus sequence typing and erythromycin resistance gene detection (ermB and mefA) were performed by PCR.

      Results

      Four serotypes were identified. Serotype III (85%) was the most prevalent, followed by Ia (7.5%), Ib (5%), and V (2.5%). All isolates were sensitive to penicillin, ceftriaxone, and levofloxacin. However, resistance to erythromycin (92.5%), clindamycin (87.5%), and tetracycline (100%) was observed. Among erythromycin-resistant isolates, 73.0% carried the ermB gene alone, 5.4% carried the mefA gene alone, and 21.6% expressed both ermB and mefA genes. A total of seven sequence types (STs) were identified; the most prevalent was ST17, accounting for 80% of all isolates. Further, serotype III isolates contained ST17 (94.2%), ST19 (2.9%), and ST650 (2.9%).

      Conclusion

      Serotype distribution, antimicrobial susceptibility, and sequence type characterization in Asia and in other global regions may contribute to improve the prevention and treatment of neonatal GBS infections.

      Keywords

      Key points
      Group B Streptococcus isolates recovered from neonates at two Chinese hospitals were predominantly serotypes III and Ia.
      All isolates were sensitive to penicillin; however, a large proportion exhibited multidrug resistance to erythromycin, clindamycin, and tetracycline.

      1. Introduction

      Group B Streptococcus (GBS), also known as Streptococcus agalactiae, is a Gram-positive bacterium that is a leading causative pathogen of severe and invasive neonatal infections such as meningitis and sepsis, which are often associated with high morbidity and mortality. In neonates, a GBS infection occurs as one of two distinct types. Early-onset disease (EOD) is characterized by the onset of symptoms during the first week of life (0–6 days), while late-onset disease (LOD) is characterized by the onset of symptoms up to 3 months of age (7–89 days).
      • Edmond K.M.
      • Kortsalioudaki C.
      • Scott S.
      • Schrag S.J.
      • Zaidi A.K.
      • Cousens S.
      • Heath P.T.
      Group B streptococcal disease in infants aged younger than 3 months: systematic review and meta-analysis.
      An invasive GBS infection is defined as GBS isolated from a normally sterile site such as blood or cerebrospinal fluid (CSF), or, less commonly, joint, pleural, or pericardial fluid.
      • Yu H.W.
      • Lin H.C.
      • Yang P.H.
      • Hsu C.H.
      • Hsieh W.S.
      • Tsao L.Y.
      • et al.
      Group B streptococcal infection in Taiwan: maternal colonization and neonatal infection.
      Serotype distribution is critical to understanding the epidemiology of GBS infections. Currently, a total of 10 distinct GBS serotypes (Ia, Ib, and II–IX) have been characterized according to the capsular polysaccharide (CPS), one of the major known virulence factors underlying invasive GBS disease.
      • Jannati E.
      • Roshani M.
      • Arzanlou M.
      • Habibzadeh S.
      • Rahimi G.
      • Shapuri R.
      Capsular serotype and antibiotic resistance of group B streptococci isolated from pregnant women in Ardabil, Iran.
      The most common serotypes, Ia, Ib, II, III, and V, account for more than 85% of serotypes in global regions that have reported serotype data, including the Americas (96%), Europe (93%), and the Western Pacific (89%).
      • Melin P.
      • Efstratiou A.
      Group B streptococcal epidemiology and vaccine needs in developed countries.
      A substantial proportion of EOD isolates and the majority of LOD isolates are serotype III; this serotype is largely responsible for invasive GBS disease.
      • Teatero S.
      • McGeer A.
      • Low D.E.
      • Li A.
      • Demczuk W.
      • Martin I.
      • Fittipaldi N.
      Characterization of invasive group B Streptococcus from the Greater Toronto Area.
      Recently, the incidence of neonatal GBS EOD in Australia, New Zealand, and the USA has decreased putatively because of the implementation of intrapartum antibiotic prophylaxis (IAP).
      • Clifford V.
      • Garland S.M.
      • Grimwood K.
      Prevention of neonatal group B Streptococcus disease in the 21st century.
      • Baker C.J.
      • Byington C.L.
      • Polin R.A.
      Policy statement—Recommendations for the prevention of perinatal group B streptococcal (GBS) disease.
      In developing countries such as China, however, neonatal GBS disease remains problematic and has not been addressed adequately. Only a few cases have been reported.
      Widespread IAP may be disadvantageous to both mother and child as it can cause anaphylaxis and/or contribute to the development of antibiotic-resistant strains. To address these concerns, vaccination of pregnant women remains an attractive prospect for the prevention and treatment of GBS infections. To provide a basis for the application of effective clinical antimicrobial agents and the development of a GBS vaccine, the serotype distribution, antibiotic susceptibility profiles, and molecular features of invasive GBS isolates recovered from a cohort of neonates in mainland China were examined.

      2. Methods

      2.1 Bacterial strains and serotype identification

      Forty non-redundant, invasive GBS isolates collected from 2008 to 2013 were analyzed in this study. These included 36 isolates recovered from neonates at Shenzhen Children's Hospital (Shenzhen, China) and four isolates recovered from neonates at Beijing Obstetrics and Gynecology Hospital (Beijing, China). Bacteria were cultured on sheep blood plates and confirmed by CAMP test and a commercially available streptococcal grouping kit according to the methods described by Wang et al.
      • Wang P.
      • Tong J.J.
      • Ma X.H.
      • Song F.L.
      • Fan L.
      • Guo C.M.
      • et al.
      Serotypes, antibiotic susceptibilities, and multi-locus sequence type profiles of Streptococcus agalactiae isolates circulating in Beijing, China.
      Serotype identification was performed with a latex agglutination kit (Statens Serum Institute, Denmark) as described previously.
      • Yao K.H.
      • Poulsen K.
      • Maione D.
      • Rinaudo C.D.
      • Baldassarri L.
      • Telford J.L.
      • et al.
      Capsular gene typing of Streptococcus agalactiae compared to serotyping by latex agglutination.
      This study protocol was in accordance with the ethical standards of the responsible regional committee on human experimentation and the Declaration of Helsinki of 1975 (revised in 1983). It was approved by the ethics committees at Shenzhen Children's Hospital and Beijing Obstetrics and Gynecology Hospital. Informed written consent was obtained from the care providers of all neonates enrolled in this study.

      2.2 Antimicrobial susceptibility tests

      The minimum inhibitory concentrations (MICs) of penicillin, ceftriaxone, erythromycin, clindamycin, clarithromycin, levofloxacin, and azithromycin were determined using Etest strips (AB Biodisk, Sweden). Susceptibilities to telithromycin and tetracycline were determined by disk diffusion method (Oxoid). The breakpoints adopted were in accordance with the 2012 criteria set by the Clinical and Laboratory Standards Institute (CLSI). The breakpoint for telithromycin was determined by comparison with the quality control culture Streptococcus pneumoniae ATCC 49619. GBS isolates were considered multidrug-resistant if they were not susceptible to three or more classes of antimicrobials tested in this study.
      All antibiotic susceptibility data were analyzed using WHONET 5.6 software, as recommended by the World Health Organization (WHO).

      2.3 Detection of erythromycin resistance genes

      Chromosomal DNA was extracted from overnight cultures of GBS isolates grown on sheep blood agar plates containing 5% defibrinated sheep blood using a DNA Mini Kit (SBS Genetech, China). PCR was used to detect the erythromycin resistance genes ermB and mefA in all erythromycin-resistant GBS isolates using primers and reaction conditions described previously.
      • Zhou L.
      • Yu S.J.
      • Gao W.
      • Yao K.H.
      • Shen A.D.
      • Yang Y.H.
      Serotype distribution and antibiotic resistance of 140 pneumococcal isolates from pediatric patients with upper respiratory infections in Beijing, 2010.
      A total of 5 μl each PCR product was analyzed by electrophoresis on a 1.5% agarose gel, stained with gold view (SBS, Beijing, China), and visualized by UV transillumination.

      2.4 Multilocus sequence typing (MLST)

      MLST analyses were based on the amplification and sequencing of internal fragments within seven housekeeping genes (adhP, pheS, atr, glnA, sdhA, glcK, and tkt). Primer pairs corresponding to these genes were designed as described previously.
      • Manning S.D.
      • Lewis M.A.
      • Springman A.C.
      • Lehotzky E.
      • Whittam T.S.
      • Davies H.D.
      Genotypic diversity and serotype distribution of group B Streptococcus isolated from women before and after delivery.
      Sequencing results were submitted to the GBS MLST website (http://pubmlst.org/sagalactiae/) to allow for allelic assignment and to determine sequence type (ST).

      3. Results

      The collection for this study comprised 40 GBS isolates recovered from 40 different neonatal patients with invasive infections. Of these, 10 neonates were diagnosed with EOD and were symptomatic within the first week after birth (0–6 days), while 30 neonates were diagnosed with LOD and were symptomatic between 7 and 89 days after birth. The majority of these GBS infection diagnoses were sepsis, pneumonia, and meningitis.

      3.1 Serotype distribution

      A total of four serotypes were identified in this study. Overall, the predominant serotype was III, accounting for 85% of all isolates, followed by Ia (7.5%), Ib (5%), and V (2.5%). A significantly greater proportion of serotype III isolates was observed in neonates with LOD (94%) when compared to those with EOD (60%). The other identified serotypes (Ia, Ib, and V) were more likely to cause EOD. Three different serotypes were identified in the 36 isolates recovered at Shenzhen Children's Hospital: 33 isolates were serotype III, two isolates were serotype Ib, and one isolate was serotype Ia. The distribution of isolates recovered from Beijing Obstetrics and Gynecology Hospital was as follows: two isolates were serotype Ia, one isolate was serotype III, and one isolate was serotype V.

      3.2 Antimicrobial susceptibility

      The antimicrobial susceptibility and MIC results for nine antimicrobial agents tested against all 40 GBS isolates are presented in Table 1. All isolates were susceptible to penicillin, ceftriaxone, levofloxacin, and telithromycin; and all were resistant to tetracycline. In contrast, 95%, 90%, 97.5%, and 100% of the 40 GBS isolates were non-susceptible to erythromycin, clindamycin, clarithromycin, and azithromycin, respectively. Approximately 90% (36/40) were multidrug-resistant, and these isolates exhibited co-resistance to erythromycin, clindamycin, and tetracycline.
      Table 1Antimicrobial susceptibility and MICs of group B Streptococcus isolates
      AntimicrobialsSusceptibilityMIC
      MIC50, minimum inhibitory concentration at which 50% of isolates were inhibited; MIC90, minimum inhibitory concentration at which 90% of isolates were inhibited; MIC range, range of minimum inhibitory concentration.
      S (%)I (%)R (%)MIC50 (mg/l)MIC90 (mg/l)Range (mg/l)
      Penicillin100000.0640.0940.032–0.125
      Ceftriaxone100000.1250.190.047–0.5
      Erythromycin52.592.5≥256≥2560.125–≥256
      Clindamycin102.587.564≥2560.094–≥256
      Clarithromycin2.5592.5≥256≥2560.25–≥256
      Levofloxacin1000011.50.5–2
      Azithromycin02.597.5≥256≥2561–≥256
      Telithromycin100%00-
      No data available for the disk diffusion test.
      -
      No data available for the disk diffusion test.
      -
      No data available for the disk diffusion test.
      Tetracycline00100-
      No data available for the disk diffusion test.
      -
      No data available for the disk diffusion test.
      -
      No data available for the disk diffusion test.
      MIC, minimum inhibitory concentration; S, susceptible; I, intermediate; R, resistant.
      a MIC50, minimum inhibitory concentration at which 50% of isolates were inhibited; MIC90, minimum inhibitory concentration at which 90% of isolates were inhibited; MIC range, range of minimum inhibitory concentration.
      b No data available for the disk diffusion test.

      3.3 Detection of macrolide resistance genes

      A total of 37 isolates were resistant to erythromycin. Of these, 27 (73.0%) harbored the ermB gene alone, two (5.4%) isolates harbored the mefA gene alone, and eight (21.6%) isolates harbored both ermB and mefA.

      3.4 MLST

      MLST analyses revealed seven different STs among the 40 GBS isolates. ST17 was the predominant ST; this accounted for 80% of all isolates. ST650 represents a new ST identified in this study. Relationships between STs and GBS serotypes of the 40 isolates characterized in this study are presented in Table 2. A correlation between serotype III and ST17 was evident, as ST17 accounted for 94.1% of all serotype III isolates. The other two serotype III isolates belonged to ST19 and ST650.
      Table 2Relationships between sequence type and serotype in group B Streptococcus isolates
      Sequence typeSerotype
      III (%)Ia (%)Ib (%)V (%)Total (%)
      ST173200032 (80)
      ST2302002 (5)
      ST1200202 (5)
      ST2401001 (2.5)
      ST1910001 (2.5)
      ST100011 (2.5)
      ST65010001 (2.5)
      Total (%)34 (85)3 (7.5)2 (5)1 (2.5)40 (100)
      ST, sequence type.

      4. Discussion

      Reported data on invasive neonatal GBS infections in Asia are sparse. To address this knowledge gap, a study that examined serotype distribution, antimicrobial resistance, and molecular characterization of invasive GBS isolates recovered from neonatal patients at two different hospitals situated in mainland China was performed.
      In this study, it was found that neonatal GBS infections were most commonly caused by serotypes III and Ia, and this finding concurs with a previous report from the USA.
      • Johri A.K.
      • Lata H.
      • Yadav P.
      • Dua M.
      • Yang Y.H.
      • Xu X.N.
      • et al.
      Epidemiology of group B Streptococcus in developing countries.
      However, the data differ from the results of a previous study comprising 1500 pregnant Chinese women that was performed in the 1990s, wherein the reported serotype distribution was 36% serotype II, 26% serotype III, and 18% serotype Ia, and neonates born from this cohort were colonized with a serotype distribution of 13.5% Ia, 9.5% II, 11.5% III, and 57% NT (non-typeable).
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      Additionally, it was found in the present study that serotype III and Ia isolates were responsible for 60% and 10% of EOD, as well as 94% and 3% of LOD, respectively. These results differ from those of a previous report from France in which serotypes III and Ia were recovered in 54% and 24% of EOD, and 82% and 7% of LOD, respectively.
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      It is hypothesized that serotypes responsible for GBS infection differ globally, and that these serotypes may vary over time. Therefore, active investigation of serotype distribution in China and Asia, as well as other global regions, should precede vaccine development.
      A retrospective study was performed of 200 children with fatal neonatal pneumonia who died between 1953 and 2004, with 34 fatal neonatal cases without any infectious disease serving as a control group. Paraffin-embedded lung tissues were collected for total genomic DNA extraction, and PCR and Southern blot analyses were used for GBS detection and molecular serotyping. It was demonstrated that the positive GBS rate of the pneumonia group was significantly higher than that of the control group (PCR: 26% vs. 3% (p < 0 .01); Southern blot: 65% vs. 18% (p < 0.01)). The positive rate in neonates aged younger than 7 days was significantly higher than that in neonates aged older than 7 days in the pneumonia group. The isolates of 22 GBS-positive neonates were serotypeable. Of these, seven were identified as serotype Ia, six isolates were serotype III, five were serotype II, and one was serotype Ib.
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      In this study, all isolates were susceptible to penicillin and ceftriaxone. These data suggest that penicillin should be the first choice for antibiotic prophylaxis and treatment of GBS infections, as recommended previously.
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      GBS isolates with reduced penicillin susceptibility have also been reported in Japan
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      Further, all GBS isolates tested in this study were sensitive to levofloxacin; however, fluoroquinolone-resistant GBS clinical isolates, mainly belonging to serotype III/ST19, have been reported previously.
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      The incidence of erythromycin resistance was 92.5%. This is higher when compared to Taiwan (58.3%) and Western countries, with reported resistance ranging from 11.5% to 32%.
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      Capsular type and antibiotic resistance in Streptococcus agalactiae isolates from patients, ranging from newborns to the elderly, with invasive infections.
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      • Chu C.
      • Chiu C.H.
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      Last, the non-susceptible rate to clindamycin reported here is higher than that in Korea (54.0%)
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      and the USA (38.4%).
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      MLST allowed for the comparison of genetic profiles of isolates recovered from various geographic areas. In the present study, ST17 was most prevalent – as high as 80.0%. Interestingly, it has been suggested previously that ST17 isolates have unique characteristics independent of cps type that contribute to more invasive disease.
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      • Springman A.C.
      • Lehotzky E.
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      Multilocus sequence types associated with neonatal group B streptococcal sepsis and meningitis in Canada.
      In summary, the data from this study provide important epidemiological information on invasive GBS isolates recovered from neonates in China. The most prevalent serotype III /ST17 clone exhibited increased resistance to clindamycin and erythromycin. This study contributes to the basic knowledge required for successful GBS vaccine development suited for disease prevention and treatment in China, as well as the implementation of effective clinical antimicrobials.

      Acknowledgements

      The authors sincerely thank all staff who took part in this study, in particular co-workers at Shenzhen Children's Hospital, for their valuable assistance in collecting the GBS isolates.
      Funding: This work was supported by the Research Funds of Profession Quota Budget administered by the Beijing Municipal Science and Technology Commission (2015-bjsekyjs-3).
      Ethical approval: The study protocol was in accordance with the ethical standards of the responsible regional committee on human experimentation and the Declaration of Helsinki of 1975 (revised in 1983). It was approved by the ethics committees of the two hospitals, Ethics Committee of Beijing Obstetrics and Genecology Hospital and Ethics Committee of Shenzhen Children's Hospital.
      Conflict of interest: No conflict of interest for all authors.

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