論文の公開元へImproved disk diffusion method for simple detection of group B streptococci with reduced penicillin susceptibility (PRGBS)
概要
Highlights (85 characters, 3 to 5 bullet points)
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PRGBS have a multidrug-resistance tendency and tend to be misclassified as PSGBS.
We improved the disk diffusion method for detecting PRGBS.
Using 73 PRGBS and 81 PSGBS, we determined more rational cutoff values.
Oxacillin, ceftizoxime, and ceftibuten disks showed high sensitivity and specificity.
This method can be performed without other special and/or expensive equipment.
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Improved disk diffusion method for simple detection of group B streptococci with reduced
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penicillin susceptibility (PRGBS)
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Rikuko Gotoa, Wanchun Jina, 1, Jun-ichi Wachinoa, 2, Yoshichika Arakawaa, 2, Kouji Kimuraa*
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aDepartment
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Tsurumai-cho, Showa-ku, Nagoya, Aichi 466-8550, Japan
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1Present
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Moriyama-ku, Nagoya, Aichi 463-8521, Japan
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2Present
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of Bacteriology, Nagoya University Graduate School of Medicine, 65
address: College of Pharmacy, Kinjo Gakuin University, 2-1723 Omori,
address: Laboratory of Microbiology, Department of Medical Technology, Faculty
of Medical Sciences, Shubun University, 6 Nikkou-cho, Ichinomiya, Aichi 491-0938, Japan.
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Running title: Improved PRGBS detection method
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Abstract: 50 words, Main text: 991 words.
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*Address
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Bacteriology, Nagoya University Graduate School of Medicine, 65 Tsurumai-cho, Showa-ku,
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Nagoya, Aichi 466-8550, Japan, TEL: +81-52-744-2106, FAX: +81-52-744-2107.
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correspondence to Kouji Kimura, koujikim@med.nagoya-u.ac.jp, Department of
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ABSTRACT (50 words)
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We used 73 group B Streptococcus with reduced penicillin susceptibility (PRGBS)
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isolates and determined more rational cutoff values of previously developed disk diffusion
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method for detecting PRGBS using oxacillin, ceftizoxime, and ceftibuten disks. Using the
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novel cutoff values, the three disks showed high sensitivity and specificity, which were above
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90.0%.
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KEYWORDS Streptococcus agalactiae, reduced penicillin susceptibility, detection method,
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disk diffusion method, multidrug resistance
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Streptococcus agalactiae (Group B Streptococcus, GBS) is the leading cause of neonatal
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sepsis and bacterial meningitis, and is a causative pathogen of invasive diseases in the elderly
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and those suffering from underlying medical conditions, such as diabetes [1–5]. β-Lactams
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are the first-line agents for the intrapartum antibiotic prophylaxis and treatment of GBS
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infections; all clinical isolates of GBS were previously considered to be uniformly
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susceptible to β-lactams [5]. However, we reported clinical isolates of GBS with reduced
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penicillin susceptibility (PRGBS) harboring amino acid substitutions, including V405A
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and/or Q557E, in penicillin-binding protein (PBP) 2X [6, 7]. After our first report in 2008 (6),
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clinical isolates of PRGBS that were confirmed to harbor amino acid substitutions in PBPs
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were also reported from other groups in Japan [8], USA [9, 10], Canada [11, 12], Germany
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[13], Mozambique [14], China [15], Hong Kong [16], and South Korea [17]. Besides reduced
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penicillin susceptibility, PRGBS clinical isolates tend to be non-susceptible/resistant to
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fluoroquinolones and macrolides, and exhibit multidrug-resistance tendency [18]. Therefore,
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drug choice against infections caused by such multidrug-resistant PRGBS is narrower than
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that caused by penicillin-susceptible GBS (PSGBS), although clinical outcomes of high dose
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penicillins administration against PRGBS infections are unknown. Consequently, it is
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necessary to detect such multidrug-resistant PRGBS accurately and rapidly to support better
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drug choice against the infections.
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The MICs of penicillin G for PRGBS (0.25-2 μg/ml) are near the “susceptible” breakpoints
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(≤0.12 μg/ml) set by the Clinical and Laboratory Standards Institute (CLSI) [19], therefore,
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misclassifications of PRGBS to PSGBS tend to happen [20]. To overcome this, we had
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previously developed a simple disk diffusion method for detecting PRGBS using oxacillin,
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ceftizoxime, and ceftibuten disks [21]. However, at that time, it was not long since we first
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reported PRGBS, the number of PRGBS isolates used in that study was limited (16 strains of
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PRGBS and 34 strains of PSGBS), and the cutoff values between PRGBS and PSGBS were
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proposed tentatively. In this study, the number of PRGBS isolates we collected is larger than
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the number of PRGBS isolates we possessed at that time. It was reported that the MICs of
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three antibiotics, cefoxitin [22], cefaclor [23], and cefotiam [8], for GBS with reduced
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β-lactam susceptibility (GBS-RBS) including PRGBS tended to be elevated; therefore, we
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examined whether or not the three antibiotics disks: cefoxitin, cefaclor, and cefotiam, are
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useful for detecting PRGBS in addition to oxacillin, ceftizoxime, and ceftibuten disks, and
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tried to determine more rational cutoff values for each disk using a larger number of PRGBS
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isolates.
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We defined strains with the MICs of penicillin G ≥ 0.25 μg/ml as PRGBS and strains with
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the MICs of penicillin G ≤ 0.12 μg/ml as PSGBS, as determined by the agar dilution method
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that was set by the CLSI [19]. In accordance with this definition, we used 73 PRGBS and 81
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PSGBS isolates. All 73 PRGBS isolates harbored amino acid substitutions in the PBPs.
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Disk diffusion methods standardized by the CLSI were performed using 10U penicillin G, 1
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μg oxacillin, 30 μg ceftizoxime, 30 μg ceftibuten, 30 μg cefoxitin, 30 μg cefaclor, and 30 μg
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cefotiam disks (Eiken Chemical Co. Ltd., Tokyo, Japan). We compared the MICs of penicillin
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G determined by the agar dilution method standardized by the CLSI and the diameters of the
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growth inhibitory zones around each disk of the disk diffusion method. We performed
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receiver operating characteristic (ROC) curve analysis and determined the cutoff value for
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each
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https://www.jichi.ac.jp/saitama-sct/SaitamaHP.files/statmedEN.html
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2022]). We calculated the sensitivity and specificity for the determined cutoff values.
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Comparison between the diameters of the growth inhibitory zones around each disk identified
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from the disk diffusion method and the MICs of penicillin G determined by the agar dilution
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method standardized by the CLSI are shown in Fig. 1. The selected cutoff values, sensitivity,
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and specificity of each disk diffusion method are listed in Table 1 (also see Fig. S1 and S2).
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The cutoff values of penicillin G, oxacillin, ceftizoxime, ceftibuten, cefoxitin, cefaclor, and
disk
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(EZR;
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cefotiam, were 31 mm, 18 mm (previous tentative cutoff: 16 mm), 29 mm (previous tentative
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cutoff: 28 mm), 19 mm (previous tentative cutoff: 19 mm), 24 mm, 29 mm, and 27 mm,
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respectively. Among the seven disks investigated in this study, penicillin G, cefoxitin,
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cefaclor, and cefotiam disks exhibited relatively lower sensitivity and specificity (82.2% and
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86.4%, 78.1% and 87.7%, 64.4% and 87.7%, and 84.9% and 67.9%, respectively); therefore,
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these disks are not very useful for detecting PRGBS using disk diffusion method. However,
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oxacillin, ceftizoxime, and ceftibuten disks exhibited higher sensitivity and specificity
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(93.2% and 98.8%, 90.4% and 95.1%, and 94.5% and 95.1%, respectively); consequently, the
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disk diffusion method using these disks are very useful for detecting PRGBS. Moreover, in
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the case of that we judge as PRGBS when the diameters around oxacillin or ceftibuten disks
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are below or equal to cutoff values, the sensitivity and specificity were 97.3% and 95.1%,
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respectively.
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The fact that all clinical isolates used in this study are recovered from only Japan is one of
the limitations of this study.
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Although most clinical isolates of PRGBS were recovered from respiratory specimens of
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adults, especially the elderly, PRGBS clinical isolates from neonatal invasive GBS diseases
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which were confirmed to harbor amino acid substitutions in PBP2X have been already
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reported from Mozambique [14]. Therefore, active screening for PRGBS among clinical
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isolates, including clinical isolates recovered from neonatal invasive GBS infections, using
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this method for detecting PRGBS, is required to support the determination of better drug
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choice against GBS infections and to facilitate the accumulation of clinical information as
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well as promoting researches concerning PRGBS through efficient detection of PRGBS.
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In conclusion, using the novel cutoff values, oxacillin, ceftizoxime, and ceftibuten disks
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showed high sensitivity and specificity. This improvement to our previous disk diffusion
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method for detecting PRGBS could serve as a promising method for detecting PRGBS at
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bacterial laboratories in medical institutes worldwide, without need of other special and/or
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expensive equipment.
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Acknowledgments
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We thank all members of Prof. Arakawa’s laboratory for their technical advice and beneficial
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discussions. The manuscript was edited by Editage, an English language editing company.
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This work was supported by the Research Program on Emerging/Re-emerging Infectious
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Disease Project of the Japan Agency for Medical Research and Development, AMED. The
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funding source had no involvement in study design, in the collection, in analysis and
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interpretation of data, in the writing of the report, and in the decision to submit the journal for
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publication.
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Competing interests statement
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The authors declare no competing interests.
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References
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[1] Lamagni TL, Keshishian C, Efstratiou A, Guy R, Henderson KL, Broughton K, Sheridan
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