Analysis of multidrug resistant group B streptococci with reduced penicillin susceptibility forming small, less hemolytic colonies.

Hirotsugu Banno,Jun-Ichi Wachino,Yoshichika Arakawa,Kouji Kimura,Tsuyoshi Sekizuka,Keigo Shibayama,Wanchun Jin,Keiko Yamada,Makoto Kuroda,Yosuke Tanaka,Herman Tse

doi:10.1371/journal.pone.0183453

Hirotsugu Banno, Jun-Ichi Wachino + Show 9 more

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https://doi.org/10.1371/journal.pone.0183453

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Abstract

Group B streptococci (GBS; Streptococcus agalactiae) are the leading cause of neonatal invasive diseases and are also important pathogens for elderly adults. Until now, nearly all GBS with reduced penicillin susceptibility (PRGBS) have shown β-hemolytic activity and grow on sheep blood agar. However, we have previously reported three PRGBS clinical isolates harboring a CylK deletion that form small less hemolytic colonies. In this study, we examined the causes of small, less hemolytic colony formation in these clinical isolates. Isogenic strains were sequenced to identify the mutation related to a small colony size. We identified a 276_277insG nucleic acid insertion in the thiamin pyrophosphokinase (tpk) gene, resulting in premature termination at amino acid 103 in TPK, as a candidate mutation responsible for small colony formation. The recombinant strain Δtpk, which harbored the 276_277insG insertion in the tpk gene, showed small colony formation. The recombinant strain ΔcylK, which harbored the G379T substitution in cylK, showed a reduction in hemolytic activity. The phenotypes of both recombinant strains were complemented by the expression of intact TPK or CylK, respectively. Moreover, the use of Rapid ID 32 API and VITEK MS to identify strains as GBS was evaluated clinical isolates and recombinant strains. VITEK MS, but not Rapid ID 32 API, was able to accurately identify the strains as GBS. In conclusion, we determined that mutations in tpk and cylK caused small colonies and reduced hemolytic activity, respectively, and characterized the clinical isolates in detail.

Highlights

Streptococcus agalactiae (GBS) is the leading cause of neonatal sepsis and meningitis and is responsible for high mortality and morbidity, in neonates and those suffering from underlying medical conditions, such as diabetes [1,2,3]. β-Lactams are first-line antimicrobial agents for intrapartum antibiotic prophylaxis and the treatment of Group B streptococci (GBS) infections [4, 5]
To determine if ΔcylK causes reduced hemolytic activity and/or small colony formation, we constructed ΔcylK based on BAA-611 harboring the G379T substitution in cylK gene
Group B streptococci with reduced penicillin susceptibility forming small, less hemolytic colonies was complemented with a plasmid harboring cylK and the resulting strain showed similar hemolytic activity to that of the wild type on sheep blood agar plates (S1 Fig)

Summary

Introduction

Streptococcus agalactiae (GBS) is the leading cause of neonatal sepsis and meningitis and is responsible for high mortality and morbidity, in neonates and those suffering from underlying medical conditions, such as diabetes [1,2,3]. β-Lactams are first-line antimicrobial agents for intrapartum antibiotic prophylaxis and the treatment of GBS infections [4, 5]. GBS clinical isolates with reduced penicillin susceptibility (PRGBS) have emerged via the acquisition of substitutions, including V405A and/or Q557E, in penicillinbinding protein 2X [6,7,8,9,10]. Most PRGBS clinical isolates show β-hemolytic activity and grow on sheep blood agar. We previously reported three multidrugresistant PRGBS clinical isolates (MRY11-004, MRY11-005, and NUBL-2449) that form atypical, small less-β-hemolytic colonies on sheep blood agar [14]. These clinical isolates harbor a G379T nucleic acid substitution in the cylK gene, resulting in premature termination at amino acid 127 in CylK, which is required for full hemolytic activity of GBS[14]

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