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Jun Hirai, Takeshi Kinjo, Shusaku Haranaga, Jiro Fujita Department of Infectious, Respiratory and Digestive Medicine, Graduate School of Medicine, University of the Ryukyus, Okinawa, JapanCorrespondence: Jun Hirai Email j.eichi419@gmail.comAbstract: Streptococcus agalactiae, also known as Group B Streptococcus (GBS), is the leading cause of bacteremia and meningitis in neonates; however, it also causes meningitis in adults, although much less frequently. After the detection of penicillin-non-susceptible GBS (PRGBS) for the first time in 2008 by Japanese researchers, clinical PRGBS isolates have been reported worldwide. These isolates need to be given due attention for being non-susceptible to multiple drugs. Herein, we present the first clinical report of meningitis caused by PRGBS. A 41-year-old Japanese male receiving an immunosuppressant visited hospital complaining of fever. Although he did not have meningitis-related symptoms or physical findings, determination of the cause of fever by Gram-staining of the spinal fluid revealed gram-positive cocci in pairs and chains. Initially, he was hospitalized on the diagnosis of cerebral meningitis caused by Streptococcus pneumoniae. However, culture of the spinal fluid revealed the β-hemolytic colonies on blood agar. Biochemical testing and mass spectrometry revealed the isolated organism as GBS (serotype Ib). The minimum inhibitory concentration (MIC) of penicillin G for the isolated strain was 0.5 μg/mL, which is greater than the MIC criteria for “susceptibility” to penicillin G for beta-hemolytic streptococci according to Clinical and Laboratory Standards Institute standards. The isolated strain was also resistant to macrolide (MIC ≥ 8 μg/mL) and fluoroquinolone (MIC ≥ 8 μg/mL). The patient recovered without neurologic sequelae upon treatment with ceftriaxone, vancomycin, and corticosteroids for 4 days, and subsequently with ampicillin for 17 days. The rate of isolation of PRGBS in the clinics has gradually increased, particularly in Japan. Although PRGBS isolated in the present case was susceptible to ampicillin and cephalosporins, strains not susceptible to ampicillin, cefotaxime, and ceftriaxone have already been isolated, indicating the prospects for limited range of effective antibiotics against PRGBS infections, including meningitis, in the near future.Keywords: adult, meningitis, Streptococcus agalactiae, PRGBS, serotype Ib, corticosteroids

Highlights • Dramatic changes in serotype prevalence noted among PRGBS in a Japanese hospital. • Over a 35-month period, 77 non-duplicate PRGBS were recovered from 73 patients. • Serotype III was predominant, followed by Ib then Ia, although they belonged to ST1. • Serotypes III and Ia isolates shared G329V, G398A, V405A and G429D substitutions in PBP2X. • Higher degree of genomic similarity was found between serotypes III and Ia strains. ABSTRACT Over a 35-month period, group B Streptococcus isolates with reduced penicillin susceptibility (PRGBS) were detected from elderly patients at a regional hospital in Japan, accompanying population-level transition of PRGBS serotypes. The genetic relatedness of 77 non-duplicate PRGBS from 73 patients was analysed. Serotype III PRGBS predominated (16 serotype III/1 serotype Ib) in the first 9 months (period I), then 3 serotype Ib isolates appeared transiently for the next 3 months (period II), which was replaced predominantly by serotype Ia (20 serotype Ia/1 serotype III/1 non-typeable) for 9 months (period III). In the last 14 months (period IV), besides 25 serotype Ia isolates, 10 serotype III were also identified. Serotypes III and Ia isolates, belonging to ST1, shared G329V, G398A, V405A and G429D substitutions in penicillin-binding protein 2X. Of three strains subjected to whole-genome sequencing, serotype III strain SU12 (period I) had a higher degree of genomic similarity with serotype Ia strain SU97 (period III) than serotype Ib strain SU67 (period II) based on average nucleotide identity and single nucleotide polymorphisms. Analysis of the cps gene clusters and the upstream and downstream flanking sequences revealed that disruption of the hyaluronidase gene located upstream of cpsY by insertion of IS 1548 was found in strain SU12, whereas ΔIS Sag8 was inserted between tRNA-Arg and rpsA genes located downstream of cpsL in strain SU97. Interestingly, most serotype III PRGBS re-emerging in period IV had this tRNA-Arg–ΔIS Sag8 – rpsA region. Capsular switching and nosocomial transmission may possibly contribute to population-level serotype replacement among ST1 PRGBS isolates. [ABSTRACT FROM AUTHOR]

We evaluated the feasibility and efficacy of a commercially available methicillin-resistant Staphylococcus aureus (MRSA)-selective agar, chromID ™ MRSA, to detect group B streptococci with reduced penicillin susceptibility (PRGBS) in this study. The results showed 72.4% (21/29) sensitivity and 98.4% (60/61) specificity to detect PRGBS using this method. [ABSTRACT FROM AUTHOR]

Penicillins remain first-line agents for treatment of group B Streptococcus ( Streptococcus agalactiae ; GBS) infections; however, several reports have confirmed the existence of GBS with reduced penicillin susceptibility (PRGBS). Because no selective agar plates for detection of PRGBS are available to date, in this investigation, we developed the selective agar plate for detection of PRGBS. We used 19 genetically well-confirmed PRGBS isolates and 38 penicillin-susceptible GBS isolates identified in Japan. For preparation of trial PRGBS-selective agar plates, we added 1 of antimicrobial agents (among oxacillin, ceftizoxime, and ceftibuten) to a well-established GBS-selective agar plate. Among 12 trial PRGBS-selective agar plates, Muller–Hinton agar containing 128 μg/mL ceftibuten with 5% sheep blood, 8 μg/mL gentamicin, and 12 μg/mL nalidixic acid was the most appropriate selective agar for PRGBS, showing 100% sensitivity and 81.6% specificity. In cases of potential nosocomial spread of PRGBS, the selective agar plate could be useful and reliable. [ABSTRACT FROM AUTHOR]

Objectives Group B Streptococcus (GBS; Streptococcus agalactiae) has been regarded as uniformly susceptible to penicillins. However, we recently reported the existence of GBS with reduced penicillin susceptibility (PRGBS), with amino acid substitutions in penicillin-binding protein (PBP) 2X. Although most PRGBS show high MICs of ceftizoxime (4–64 mg/L) and cefotaxime (0.12–1 mg/L), those for strain B1 are exceptionally high (ceftizoxime MIC ≥256 mg/L and cefotaxime MIC 2 mg/L). We previously found an amino acid substitution (G539S) neighbouring the conserved K540TG motif in PBP1A in addition to the PRGBS-specific amino acid substitution Q557E in PBP2X of B1. The aim of this study was to reveal the effect of the amino acid substitutions in PBP1A and PBP2X of B1 on the high cephalosporin resistance. Methods A ceftizoxime competition assay was performed to reveal the PBPs that are the main targets of ceftizoxime. We generated two allelic exchange mutants from β-lactam-susceptible GBS BAA-611. BAA-611 (B1PBP2X) contained the PBP2X gene derived from B1 and BAA-611 (B1PBP2X, B1PBP1A) contained both the PBP2X and the PBP1A gene derived from B1. These allelic exchange mutants and strain B1 were subjected to susceptibility testing. Results The ceftizoxime competition assay revealed that PBP1A and PBP2X were the main targets of ceftizoxime. Although the MICs of ceftizoxime and cefotaxime for BAA-611 (B1PBP2X) were 64 and 0.5 mg/L, respectively, BAA-611 (B1PBP2X, B1PBP1A) showed high cephalosporin resistance (ceftizoxime MIC ≥256 mg/L and cefotaxime MIC 2 mg/L) comparable to B1. Conclusions The high cephalosporin resistance of GBS was caused by amino acid substitutions in PBP1A and PBP2X. [ABSTRACT FROM PUBLISHER]

Objectives Recently several clinical isolates of Streptococcus agalactiae [also known as group B Streptococcus (GBS)] that have acquired reduced penicillin susceptibility (PRGBS) by amino acid substitutions in the penicillin-binding protein 2X have emerged. The frequency of fluoroquinolone (FQ)- and macrolide-resistant streptococci among PRGBS is not yet known. Methods Fifty-seven GBS [19 PRGBS and 38 penicillin-susceptible GBS (PSGBS)], isolated from different medical institutions in Japan, were studied. For GBS, the MICs of penicillin G, levofloxacin and erythromycin were determined using the agar dilution method. Nineteen PRGBS were previously confirmed as genetically diverse streptococci by PFGE. Further, the mechanisms underlying penicillin, FQ and macrolide non-susceptibility/resistance were analysed. Results The frequency of non-susceptibility to FQs among PSGBS was 18.4% (7/38), whereas that among PRGBS was 100% (19/19). The frequency of resistance to erythromycin among PSGBS was 7.9% (3/38), while that among PRGBS was 47.4% (9/19). Statistical significance was determined using Fisher's exact test between reduced penicillin susceptibility and FQ non-susceptibility (P ≤ 0.0001) and macrolide resistance (P = 0.0012). The resistance/non-susceptibility mechanisms among PRGBS were diverse, suggesting that the PRGBS examined were not clonal. Conclusions PRGBS isolates tend to show resistance to FQs and/or macrolides. Because the drug choice for treating these multidrug-resistant GBS is more limited than that for usual GBS, these strains may present future public health challenges. [ABSTRACT FROM PUBLISHER]

All clinical isolates of group B Streptococcus (GBS; Streptococcus agalactiae) are considered uniformly susceptible to β-lactams, including penicillins. However, GBS with reduced penicillin susceptibility (PRGBS) were first identified by our group in Japan and have also been reported from North America. PRGBS are non-susceptible to penicillin because of acquisition of amino acid substitutions near the conserved active-site motifs in PBP2X. In particular, V405A and Q557E are considered the key amino acid substitutions responsible for penicillin non-susceptibility. We revealed that in addition to the substitutions in PBP2X, an amino acid substitution in PBP1A confers high-level cephalosporin resistance in GBS. As the number of publications on GBS with reduced β-lactam susceptibility (GBS-RBS), especially PRGBS, and concomitantly the need for a systematic classification of GBS-RBS is increasing, we propose here a classification of GBS-RBS based on the amino acid substitutions in their PBPs. [ABSTRACT FROM AUTHOR]

All clinical isolates of group B Streptococcus (GBS; Streptococcus agalactiae) are considered uniformly susceptible to β-lactams, including penicillins. However, GBS with reduced penicillin susceptibility (PRGBS) were first identified by our group in Japan and have also been reported from North America. PRGBS are non-susceptible to penicillin because of acquisition of amino acid substitutions near the conserved active-site motifs in PBP2X. In particular, V405A and Q557E are considered the key amino acid substitutions responsible for penicillin non-susceptibility. We revealed that in addition to the substitutions in PBP2X, an amino acid substitution in PBP1A confers high-level cephalosporin resistance in GBS. As the number of publications on GBS with reduced β-lactam susceptibility (GBS-RBS), especially PRGBS, and concomitantly the need for a systematic classification of GBS-RBS is increasing, we propose here a classification of GBS-RBS based on the amino acid substitutions in their PBPs., (© The Author 2015. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)