Hirotsugu Banno

High isolation rate of MDR group B streptococci with reduced penicillin susceptibility in Japan

OBJECTIVES In Japan, the isolation rate of group B Streptococcus (GBS) with reduced penicillin susceptibility (PRGBS) was 2.3% between 2005 and 2006. However, no data on this have been available since then; moreover, the isolation rate of MDR-PRGBS has never been reported. The aim of this study was to obtain recent data on the PRGBS isolation rate and to investigate, for the first time, the isolation rate of MDR-PRGBS. METHODS We collected 306 clinical GBS isolates from various regions in Japan between January 2012 and July 2013. The MICs of penicillin G, oxacillin, ceftizoxime and ceftibuten for all GBS isolates and the MICs of erythromycin and levofloxacin for PRGBS isolates were determined by the agar dilution method. The mutations in the genes involved in antibiotic resistance and antibiotic resistance genes were analysed by PCR and DNA sequencing. RESULTS We detected 45 clinical PRGBS isolates, with a PRGBS isolation rate among GBS of 14.7% (45/306). Among the PRGBS isolates, 32 (32/45, 71.1%) and 43 (43/45, 95.6%) were resistant/non-susceptible to erythromycin and levofloxacin, respectively. Furthermore, 31 PRGBS isolates, which constituted 68.9% of the PRGBS (31/45) and 10.1% of the GBS (31/306), respectively, were resistant/non-susceptible to both macrolides and fluoroquinolones, indicating multidrug resistance. CONCLUSIONS These results suggest that the number of clinical PRGBS isolates with a tendency to multidrug resistance increased rapidly between 2005-06 and 2012-13 in Japan.

Characterization of Multidrug-Resistant Group B Streptococci with Reduced Penicillin Susceptibility Forming Small Non-Beta-Hemolytic Colonies on Sheep Blood Agar Plates

ABSTRACT We isolated and characterized three multidrug-resistant clinical isolates of group B streptococci with reduced penicillin susceptibility (PRGBS) that formed small non-beta-hemolytic colonies on sheep blood agar plates but grew well on chocolate agar plates. They can be overlooked in the bacterial identification step, leading to clinical misdiagnosis and treatment failure.

In vitro reduction of antibacterial activity of tigecycline against multidrug-resistant Acinetobacter baumannii with host stress hormone norepinephrine

The host stress hormone norepinephrine (NE), also called noradrenaline, is reported to augment bacterial growth and pathogenicity, but few studies have focused on the effect of NE on the activity of antimicrobials. The aim of this study was to clarify whether NE affects antimicrobial activity against multidrug-resistant Acinetobacter baumannii (MDR-AB). Time-kill studies of tigecycline (TIG) and colistin (COL) against MDR-AB as well as assays for factors contributing to antibiotic resistance were performed using MDR-AB clinical strains both in the presence and absence of 10 µM NE. In addition, expression of three efflux pump genes (adeB, adeJ and adeG) in the presence and absence of NE was analysed by quantitative reverse transcription PCR. Viable bacterial cell counts in TIG-supplemented medium containing NE were significantly increased compared with those in medium without NE. In contrast, NE had little influence on viable bacterial cell counts in the presence of COL. NE-supplemented medium resulted in an ca. 2 log increase in growth and in bacterial cell numbers adhering on polyurethane, silicone and polyvinylchloride surfaces. Amounts of biofilm in the presence of NE were ca. 3-fold higher than without NE. Expression of the adeG gene was upregulated 4-6-fold in the presence of NE. In conclusion, NE augmented factors contributing to antibiotic resistance and markedly reduced the in vitro antibacterial activity of TIG against MDR-AB. These findings suggest that NE treatment may contribute to the failure of TIG therapy in patients with MDR-AB infections.

Ceftibuten-containing agar plate for detecting group B streptococci with reduced penicillin susceptibility (PRGBS).

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.

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

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.

High isolation rate and multidrug resistance tendency of penicillin-susceptible group B Streptococcus with reduced ceftibuten susceptibility in Japan

Group B Streptococcus (GBS) clinical isolates with reduced penicillin susceptibility (PRGBS) have emerged through acquisition of amino acid substitutions in penicillin-binding protein 2X (PBP2X). Moreover, we also reported the emergence of penicillin-susceptible GBS clinical isolates with reduced ceftibuten susceptibility (CTBr PSGBS) due to amino acid substitutions in PBPs. However, whether or not these amino acid substitutions are responsible for the reduced ceftibuten susceptibility (RCTBS) profile remains unclear. Furthermore, the rate of CTBr PSGBS isolation and their multidrug resistance tendency remain uncertain. Therefore, we collected 377 clinical GBS isolates from multiple regions in Japan between August 2013 and August 2015. These isolates were characterized by determining MICs and sequencing the pbp2x gene. The isolation rate of CTBr PSGBS was 7.2% (27/377). CTBr PSGBS isolate harbor two types of amino acid substitutions in PBP2X [(T394A type) and (I377V, G398A, Q412L, and H438H type)]. The relevance of the amino acid substitutions found to the RCTBS was confirmed with allelic exchange techniques. Allelic exchange recombinant clones acquired two types of amino acid substitutions in PBP2X showed RCTBS. Furthermore, total ratio of resistance and non-susceptibility to both macrolides and fluoroquinolones in CTBr PSGBS was 51.9% (14/27). The isolation rate of CTBr PSGBS is non-negligibly high and the CTBr PSGBS tends to exhibit resistance and non-susceptible profile to both macrolides and fluoroquinolones.

Effectual detection of group B streptococci with reduced penicillin susceptibility (PRGBS) by commercially available methicillin-resistant-Staphylococcus aureus (MRSA)-selective agar

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.