Production of metallo-β-lactamases (MBLs), which hydrolyze carbapenems, is a cause of carbapenem resistance in Enterobacteriaceae Development of effective inhibitors for MBLs is one approach to restore carbapenem efficacy in carbapenem-resistant Enterobacteriaceae (CRE). We report here that sulfamoyl heteroarylcarboxylic acids (SHCs) can competitively inhibit the globally spreading and clinically relevant MBLs (i.e., IMP-, NDM-, and VIM-type MBLs) at nanomolar to micromolar orders of magnitude. Addition of SHCs restored meropenem efficacy against 17/19 IMP-type and 7/14 NDM-type MBL-producing Enterobacteriaceae to satisfactory clinical levels. SHCs were also effective against IMP-type MBL-producing Acinetobacter spp. and engineered Escherichia coli strains overproducing individual minor MBLs (i.e., TMB-2, SPM-1, DIM-1, SIM-1, and KHM-1). However, SHCs were less effective against MBL-producing Pseudomonas aeruginosa Combination therapy with meropenem and SHCs successfully cured mice infected with IMP-1-producing E. coli and dually NDM-1/VIM-1-producing Klebsiella pneumoniae clinical isolates. X-ray crystallographic analyses revealed the inhibition mode of SHCs against MBLs; the sulfamoyl group of SHCs coordinated to two zinc ions, and the carboxylate group coordinated to one zinc ion and bound to positively charged amino acids Lys224/Arg228 conserved in MBLs. Preclinical testing revealed that the SHCs showed low toxicity in cell lines and mice and high stability in human liver microsomes. Our results indicate that SHCs are promising lead compounds for inhibitors of MBLs to combat MBL-producing CRE.IMPORTANCE Carbapenem antibiotics are the last resort for control of severe infectious diseases, bloodstream infections, and pneumonia caused by Gram-negative bacteria, including Enterobacteriaceae However, carbapenem-resistant Enterobacteriaceae (CRE) strains have spread globally and are a critical concern in clinical settings because CRE infections are recognized as a leading cause of increased mortality among hospitalized patients. Most CRE produce certain kinds of serine carbapenemases (e.g., KPC- and GES-type β-lactamases) or metallo-β-lactamases (MBLs), which can hydrolyze carbapenems. Although effective MBL inhibitors are expected to restore carbapenem efficacy against MBL-producing CRE, no MBL inhibitor is currently clinically available. Here, we synthesized 2,5-diethyl-1-methyl-4-sulfamoylpyrrole-3-carboxylic acid (SPC), which is a potent inhibitor of MBLs. SPC is a remarkable lead compound for clinically useful MBL inhibitors and can potentially provide a considerable benefit to patients receiving treatment for lethal infectious diseases caused by MBL-producing CRE.

We characterized 12 clinical isolates of Klebsiella oxytoca with the extended-spectrum β-lactamase (ESBL) phenotype (high minimum inhibitory concentration [MIC] values of ceftriaxone) recovered over 9 months at a university hospital in Japan. To determine the clonality of the isolates, we used pulsed-field gel electrophoresis (PFGE), multi-locus sequence typing (MLST), and PCR analyses to detect blaRBI, which encodes the β-lactamase RbiA, OXY-2-4 with overproduce-type promoter. Moreover, we performed the isoelectric focusing (IEF) of β-lactamases, and the determination of the MICs of β-lactams including piperacillin/tazobactam for 12 clinical isolates and E. coli HB101 with pKOB23, which contains blaRBI, by the agar dilution method. Finally, we performed the initial screening and phenotypic confirmatory tests for ESBLs. Each of the 12 clinical isolates had an identical PFGE pulsotype and MLST sequence type (ST9). All 12 clinical isolates harbored identical blaRBI. The IEF revealed that the clinical isolate produced only one β-lactamase. E. coli HB101 (pKOB23) and all 12 isolates demonstrated equally resistance to piperacillin/tazobactam (MICs, >128 μg/ml). The phenotypic confirmatory test after the initial screening test for ESBLs can discriminate β-lactamase RbiA-producing K. oxytoca from β-lactamase CTX-M-producing K. oxytoca. Twelve clinical isolates of K. oxytoca, which were recovered from an outbreak at one university hospital, had identical genotypes and produced β-lactamase RbiA that conferred resistance to piperacillin/tazobactam. In order to detect K. oxytoca isolates that produce RbiA to promote research concerning β-lactamase RbiA-producing K. oxytoca, the phenotypic confirmatory test after the initial screening test for ESBLs would be useful.

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.

Segmentation of a natural scene into objects and background is a fundamental but challenging task for recognizing objects. Investigating intermediate-level visual cortical areas with a focus on local information is a crucial step towards understanding the formation of the cortical representations of figure and ground. We examined the activity of a population of macaque V4 neurons during the presentation of natural image patches and their respective variations. The natural image patches were optimized to exclude the influence of global context but included various characteristics of local stimulus. Around one fourth of the patch-responsive V4 neurons exhibited significant modulation of firing activity that was dependent on the positional relation between the figural region of the stimulus and the classical receptive field of the neuron. However, the individual neurons showed low consistency in figure-ground modulation across a variety of image patches (55-62%), indicating that individual neurons were capable of correctly signaling figure and ground only for a limited number of stimuli. We examined whether integration of the activity of multiple neurons enabled higher consistency across a variety of natural patches by training a support vector machine to classify figure and ground of the stimuli from the population firing activity. The integration of the activity of a few tens of neurons yielded discrimination accuracy much greater than that of single neurons (up to 85%), suggesting a crucial role of population coding for figure-ground discrimination in natural images.

This study aimed to investigate genomic traits underlying the antimicrobial resistance and virulence of multidrug-resistant (MDR) group B streptococci with reduced penicillin susceptibility (PRGBS) recovered from elderly patients with bloodstream infections, which remain poorly characterized. The pangenome was found to be open, with the predicted pan- and core genome sizes being 3,531 and 1,694 genes, respectively. Accessory and unique genes were enriched for the Clusters of Orthologous Groups (COG) categories L, Replication, recombination, and repair, and K, Transcription. All MDR PRGBS isolates retained a core virulence gene repertoire (bibA, fbsA/-B/-C, cspA, cfb, hylB, scpB, lmb, and the cyl operon), supporting an invasive ability similar to that of the other invasive GBS, penicillin-susceptible GBS (PSGBS), and noninvasive PRGBS isolates. The putative sequence type 1 (ST1)-specific AlpST-1 virulence gene was also retained among the serotype Ia/ST1 PRGBS isolates. In addition to tet(M) and erm(B), mef(A)-msr(D) elements or the high-level gentamicin resistance gene aac(6')-aph(2″), which are both rare in PSGBS, were detected among those MDR PRGBS isolates. In the core single-nucleotide polymorphism (SNP) phylogenetic tree, all invasive ST1 PRGBS isolates with serotypes Ia and III were placed together in a clade with a recombination rate of 3.97, which was 36 times higher than the value found for a clade formed by serotype V/ST1 PSGBS isolates derived mostly from human blood. ST1 has been the predominant sequence type among the PRGBS isolates in Japan, and serotypes Ia and III have been very rare among the ST1 PSGBS isolates. Thus, these lineages that mostly consisted of serotypes Ia/ST1 and III/ST1 PRGBS could possibly emerge through recombination within the ST1 populations. IMPORTANCE Streptococcus agalactiae, or group B Streptococcus (GBS), is recognized as the leading cause of neonatal invasive infections. However, an increasing incidence of invasive GBS infections among nonpregnant adults, particularly the elderly and those with underlying diseases, has been observed. There is a trend toward the increasing occurrence of penicillin nonsusceptibility among GBS clinical isolates, from 4.8% in 2008 to 5.8% in 2020 in Japan. Also, in the United States, the frequency of adult invasive GBS isolates suggestive of β-lactam nonsusceptibility increased from 0.7% in 2015 to 1.0% in 2016. In adults, mortality has been significantly higher among patients with bacteremia than among those without bacteremia. Our study revealed that invasive GBS with reduced penicillin susceptibility (PRGBS) isolates harbor major virulence and resistance genes known among GBS, highlighting the need for large population-based genomic surveillance studies to better understand the clinical relevance of invasive PRGBS isolates.

No abstract available