Clinical isolates of Streptococcus mitis/oralis-related species with reduced carbapenem susceptibility, harboring amino acid substitutions in penicillin-binding proteins in Japan.

Streptococcus mitis/oralis group isolates with reduced carbapenem susceptibility have been reported, but its isolation rate in Japan is unknown. We collected 356 clinical α-hemolytic streptococcal isolates and identified 142 of them as S. mitis/oralis using partial sodA sequencing. The rate of meropenem non-susceptibility was 17.6% (25/142). All 25 carbapenem-non-susceptible isolates harbored amino acid substitutions in/near the conserved motifs in PBP1A, PBP2B, and PBP2X. Carbapenem non-susceptibility is common among S. mitis/oralis group isolates in Japan.

Hydroxyhexylitaconic acids as potent IMP-type metallo-ß-lactamase inhibitors for controlling carbapenem resistance in Enterobacterales.

Metallo-ß-lactamases (MBLs) represent one of the main causes of carbapenem resistance in the order Enterobacterales. To combat MBL-producing carbapenem-resistant Enterobacterales, the development of MBL inhibitors can restore carbapenem efficacy for such resistant bacteria. Microbial natural products are a promising source of attractive seed compounds for the development of antimicrobial agents. Here, we report that hydroxyhexylitaconic acids (HHIAs) produced by a member of the genus Aspergillus can suppress carbapenem resistance conferred by MBLs, particularly IMP (imipenemase)-type MBLs. HHIAs were found to be competitive inhibitors with micromolar orders of magnitude against IMP-1 and showed weak inhibitory activity toward VIM-2, while no inhibitory activity against NDM-1 was observed despite the high dosage. The elongated methylene chains of HHIAs seem to play a crucial role in exerting inhibitory activity because itaconic acid, a structural analog without long methylene chains, did not show inhibitory activity against IMP-1. The addition of HHIAs restored meropenem and imipenem efficacy to satisfactory clinical levels against IMP-type MBL-producing Escherichia coli and Klebsiella pneumoniae clinical isolates. Unlike EDTA and Aspergillomarasmine A, HHIAs did not cause the loss of zinc ions from the active site, resulting in the structural instability of MBLs. X-ray crystallography and in silico docking simulation analyses revealed that two neighboring carboxylates of HHIAs coordinated with two zinc ions in the active sites of VIM-2 and IMP-1, which formed a key interaction observed in MBL inhibitors. Our results indicated that HHIAs are promising for initiating the design of potent inhibitors of IMP-type MBLs.IMPORTANCEThe number and type of metallo-ß-lactamase (MΒL) are increasing over time. Carbapenem resistance conferred by MΒL is a significant threat to our antibiotic regimen, and the development of MΒL inhibitors is urgently required to restore carbapenem efficacy. Microbial natural products have served as important sources for developing antimicrobial agents targeting pathogenic bacteria since the discovery of antibiotics in the mid-20th century. MΒL inhibitors derived from microbial natural products are still rare compared to those derived from chemical compound libraries. Hydroxyhexylitaconic acids (HHIAs) produced by members of the genus Aspergillus have potent inhibitory activity against clinically relevant IMP-type MBL. HHIAs may be good lead compounds for the development of MBL inhibitors applicable for controlling carbapenem resistance in IMP-type MBL-producing Enterobacterales.

ß-Lactam Susceptibility of Streptococcus dysgalactiae subsp. equisimilis.

All clinical isolates of Streptococcus dysgalactiae subsp. equisimilis (SDSE) are considered susceptible to ß-lactams, the first-line drugs used for SDSE infections. However, penicillin-non-susceptible SDSE has been reported from Denmark. In this study, we attempted to detect ß-lactam-non-susceptible clinical isolates of SDSE in Japan. One hundred and fifty clinical isolates of S. dysgalactiae were collected in 2018, and species identification was performed using Rapid ID Strep API. The minimum inhibitory concentrations (MIC) of six ß-lactams (penicillin G, oxacillin, ceftizoxime, ceftibuten, cefoxitin, and cefaclor) were determined for 85 clinical isolates of SDSE using the agar dilution method standardized by the Clinical Laboratory Standards Institute. For the 85 isolates identified as SDSE, the MIC ranges of penicillin G, oxacillin, ceftizoxime, ceftibuten, cefoxitin, and cefaclor were 0.007-0.06, 0.03-0.12, 0.015-0.06, 0.25-2, 0.12-2, and 0.06-0.5 µg/mL, respectively. None of the clinical isolates were non-susceptible to penicillin G, indicating that all 85 clinical isolates of SDSE were susceptible to ß-lactams. Our findings indicate that almost all clinical isolates of SDSE in several prefectures of Japan remain susceptible to ß-lactams. Nevertheless, there remains a need for continuous and careful monitoring of drug susceptibility among clinical isolates of SDSE in Japan.

Difference in the Inhibitory Effect of Thiol Compounds and Demetallation Rates from the Zn(II) Active Site of Metallo-ß-lactamases (IMP-1 and IMP-6) Associated with a Single Amino Acid Substitution.

Gram-negative bacteria producing metallo-ß-lactamases (MBLs) have become a considerable threat to public health. MBLs including the IMP, VIM, and NDM types are Zn(II) enzymes that hydrolyze the ß-lactam ring present in a broad range of antibiotics, such as N-benzylpenicillin, meropenem, and imipenem. Among IMPs, IMP-1 and IMP-6 differ in a single amino acid substitution at position 262, where serine in IMP-1 is replaced by glycine in IMP-6, conferring a change in substrate specificity. To investigate how this mutation influences enzyme function, we examined lactamase inhibition by thiol compounds. Ethyl 3-mercaptopropionate acted as a competitive inhibitor of IMP-1, but a noncompetitive inhibitor of IMP-6. A comparison of the crystal structures previously reported for IMP-1 (PDB code: 5EV6) and IMP-6 (PDB code: 6LVJ) revealed a hydrogen bond between the side chain of Ser262 and Cys221 in IMP-1 but the absence of hydrogen bond in IMP-6, which affects the Zn2 coordination sphere in its active site. We investigated the demetallation rates of IMP-1 and IMP-6 in the presence of chelating agent ethylenediaminetetraacetic acid (EDTA) and found that the demetallation reactions had fast and slow phases with a first-order rate constant (kfast = 1.76 h-1, kslow = 0.108 h-1 for IMP-1, and kfast = 14.0 h-1 and kslow = 1.66 h-1 for IMP-6). The difference in the flexibility of the Zn2 coordination sphere between IMP-1 and IMP-6 may influence the demetallation rate, the catalytic efficiency against ß-lactam antibiotics, and the inhibitory effect of thiol compounds.

Bird's-eye MApping of plasmids (BeMAp) for visualization and comparison of genomic structures of different plasmids by mapping antimicrobial resistance genes on spreadsheets.

Effective classification and visualization of multiple antimicrobial resistance plasmids can be challenging, and few tools to analyze similarities among plasmids depending on the location of genes are available. We created a new plasmid mapping program called Bird's-eye MApping of plasmids (BeMAp) to map antimicrobial resistance genes across multiple plasmids onto a spreadsheet and visualize their similarities based on gene types, locations, alignments, and organization. We analyzed plasmids containing various antimicrobial resistance genes, together with genes coding for IMP-type metallo-ß-lactamases. Moreover, the mapping of plasmids with antimicrobial resistance genes and Incompatibility (Inc) groups showed that clustered plasmids with a similar organization of antimicrobial resistance genes were not always classified into the same Inc groups, indicating that the program displays multiple plasmids regardless of the Inc group classification. Our results showed that this calculation protocol and mapping strategy could provide a valuable tool for the practical and convenient visualization and comparison of the genomic structure of multiple plasmids in parallel.

Improved disk diffusion method for simple detection of group B streptococci with reduced penicillin susceptibility (PRGBS).

We used 73 group B Streptococcus with reduced penicillin susceptibility (PRGBS) isolates and determined more rational cutoff values of previously developed disk diffusion method for detecting PRGBS using oxacillin, ceftizoxime, and ceftibuten disks. Using the novel cutoff values, the three disks showed high sensitivity and specificity, which were above 90.0%.

Molecular Epidemiology of Enterobacter cloacae Complex Isolates with Reduced Carbapenem Susceptibility Recovered by Blood Culture.

The Enterobacter cloacae complex (ECC) is one of the most common causes of bacteremia and leads to poor clinical outcomes. The aim of this study was to clarify the antimicrobial susceptibility profiles and genetic backgrounds of non-carbapenemase-producing reduced-carbapenem-susceptible (RCS) ECC blood isolates in Japan using agar dilution antimicrobial susceptibility testing, whole-genome sequencing, and quantitative polymerase chain reaction for ampC, ompC, and ompF transcripts. Forty-two ECC blood isolates were categorized into RCS and carbapenem-susceptible groups based on the minimum inhibitory concentration of imipenem. The RCS ECC blood isolates belonged to distinct species and sequence types and produced varying class C ß-lactamases. The E. roggenkampii, E. asburiae, and E. bugandensis isolates belonged only to the RCS group. Some E. hormaechei ssp. steigerwaltii isolates from the RCS group exhibited AmpC overexpression caused by amino acid substitutions in AmpD and AmpR along with ompF downregulation. These findings suggest that non-carbapenemase-producing RCS ECC blood isolates are genetically diverse.

Dissecting the clonality of I1 plasmids using ORF-based binarized structure network analysis of plasmids (OSNAp).

OBJECTIVES: We aimed to elucidate the relationship among blaCTX-M-carrying plasmids and their transmission between humans and domestic animals. METHODS: Phylogenetic relationship of 90 I1 plasmids harboring blaCTX-M genes encoding extended-spectrum ß-lactamase (ESBL) was analyzed using the ORF-based binarized structure network analysis of plasmids (OSNAp). RESULTS: The majority of plasmids carrying blaCTX-M-1 or blaCTX-M-8 belonged to a single lineage, respectively, and were primarily associated with domestic animals especially chickens. On the other hand, plasmids carrying blaCTX-M-14 or blaCTX-M-15, identified from both humans and domestic animals, were distributed in two or more lineages. CONCLUSION: OSNAp has revealed the phylogenetic relationships and diversity of plasmids carrying blaCTX-M more distinctly than pMLST. The findings suggest that circulation of I1 plasmids between humans and animals may contribute to their diversity.

Isolation of Group A Streptococci with Reduced In Vitro ß-Lactam Susceptibility Harboring Amino Acid Substitutions in Penicillin-Binding Proteins in Japan.

Streptococcus pyogenes (group A Streptococcus [GAS]) has long been regarded as being susceptible to ß-lactams. However, amino acid substitutions in penicillin-binding protein 2X (PBP2X) conferring reduced in vitro ß-lactam susceptibility have been indicated since 2019 in the United States and Iceland. Here, we report the first isolation of Streptococcus pyogenes possessing the PBP2X substitution conferring reduced in vitro ß-lactam susceptibility in Asia; however, the MICs were below the susceptible breakpoint of the CLSI.

Practical Agar-Based Disk Diffusion Tests Using Sulfamoyl Heteroarylcarboxylic Acids for Identification of Subclass B1 Metallo-ß-Lactamase-Producing Enterobacterales.

The worldwide distribution of carbapenemase-producing Enterobacterales (CPE) is a serious public health concern as they exhibit carbapenem resistance, thus limiting the choice of antimicrobials for treating CPE infections. Combination treatment with a ß-lactam and one of the newly approved ß-lactamase inhibitors, such as avibactam, relebactam, or vaborbactam, provides a valuable tool to cope with CPE; however, these inhibitors are active only against serine-type carbapenemases and not against metallo-ß-lactamases (MßLs). Therefore, it is important to readily differentiate carbapenemases produced by CPE by using simple and reliable methods in order to choose an appropriate treatment. Here, we developed three practical agar-based disk diffusion tests (double-disk synergy test [DDST], disk potentiation test, and modified carbapenem inactivation method [mCIM]) to discriminate the production of subclass B1 MßLs, such as IMP-, NDM-, and VIM-type MßLs, from the other carbapenemases, especially serine-type carbapenemases. This was accomplished using B1 MßL-specific sulfamoyl heteroarylcarboxylic acid inhibitors, 2,5-dimethyl-4-sulfamoylfuran-3-carboxylic acid (SFC) and 2,5-diethyl-1-methyl-4-sulfamoylpyrrole-3-carboxylic acid (SPC), originally developed by us. The DDST and mCIM using SFC and SPC revealed high sensitivity (95.3%) and specificity (100%) in detecting B1 MßL-producing Enterobacterales. In the disk potentiation test, the sensitivities using SFC and SPC were 89.1% and 93.8%, respectively, whereas the specificities for both were 100%. These methods are simple and inexpensive and have a high accuracy rate. These methods would therefore be of immense assistance in the specific detection and discrimination of B1 MßL-producing Enterobacterales in clinical microbiology laboratories and would lead to better prevention against infection with such multidrug-resistant bacteria in clinical settings.

Crystal Structures of Metallo-ß-Lactamase (IMP-1) and Its D120E Mutant in Complexes with Citrate and the Inhibitory Effect of the Benzyl Group in Citrate Monobenzyl Ester.

The emergence and rapid spread of carbapenem-resistant pathogens producing metallo-ß-lactamases such as IMP-1 and NDM-1 have been of great concern in the global clinical setting. The X-ray crystal structures of IMP-1 from Serratia marcescens and its single mutant, D120E, in complexes with citrate were determined at resolutions of 2.00 and 1.85 Å, respectively. Two crystal structures indicate that a single mutation at position 120 caused a structural change around Zn1, where the geometry changes from a tetrahedron in the native IMP-1 to a square pyramid in D120E. Based on these two complex structures, the authors synthesized citrate monobenzyl ester 1 to evaluate the structural requirement for the inhibitory activity against IMP-1 and compared the inhibitory activities with nonsubstituted citrate. The introduction of a benzyl group into citrate enhanced the inhibitory activity in comparison to citrate (IC50 > 5 mM).

Daptomycin Susceptibility of Group B Streptococcus.

We previously reported the emergence and high prevalence of group B streptococci (GBS) with reduced penicillin susceptibility (PRGBS) clinical isolates in Japan. PRGBS tend to be non-susceptible to macrolides and fluoroquinolones. In our previous study, we found that the minimum inhibitory concentration (MIC) of daptomycin for one clinical isolate of GBS was above the susceptible breakpoint settled by the Clinical and Laboratory Standards Institute (CLSI). This suggests the possibility of the unrecognized spread of daptomycin-non-susceptible clinical GBS isolates in Japan. This study aimed to analyze the daptomycin susceptibility in 1,046 clinical GBS isolates that were recovered after the approval of daptomycin in Japan. MICs of daptomycin for the 1,046 clinical isolates were determined by the microdilution method recommended by the CLSI. The MIC range was 0.12-1 µg/mL, and the MIC50 and MIC90 were 0.5 µg/mL and 1 µg/mL, respectively. All the GBS isolates evaluated in this study were susceptible to daptomycin. Therefore, at present, daptomycin might be considered as a new option to treat GBS infections, especially multidrug-resistant PRGBS infections.

Aminoglycoside Resistance: Updates with a Focus on Acquired 16S Ribosomal RNA Methyltransferases.

The clinical usefulness of aminoglycosides has been revisited as an effective choice against ß-lactam-resistant and fluoroquinolone-resistant gram-negative bacterial infections. Plazomicin, a next-generation aminoglycoside, was introduced for the treatment of complicated urinary tract infections and acute pyelonephritis. In contrast, bacteria have resisted aminoglycosides, including plazomicin, by producing 16S ribosomal RNA (rRNA) methyltransferases (MTases) that confer high-level and broad-range aminoglycoside resistance. Aminoglycoside-resistant 16S rRNA MTase-producing gram-negative pathogens are widespread in various settings and are becoming a grave concern. This article provides up-to-date information with a focus on aminoglycoside-resistant 16S rRNA MTases.

Sulfamoyl Heteroarylcarboxylic Acids as Promising Metallo-ß-Lactamase Inhibitors for Controlling Bacterial Carbapenem Resistance.

Production of metallo-ß-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 ß-lactamases) or metallo-ß-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.

Characterization of blaCTX-M-27/F1:A2:B20 Plasmids Harbored by Escherichia coli Sequence Type 131 Sublineage C1/H30R Isolates Spreading among Elderly Japanese in Nonacute-Care Settings.

We characterized 29 blaCTX-M-27-harboring plasmids of Escherichia coli sequence type 131 (ST131) sublineage C1/H30R isolates from healthy individuals and long-term-care facility (LTCF) residents. Most (27/29) plasmids were of the FIA, FIB, and FII multireplicon type with the same plasmid multilocus sequence typing (pMLST). Several plasmids (7/23) from LTCF residents harbored only blaCTX-M-27 as the resistance gene; however, their fundamental structures were very similar to those of previously isolated blaCTX-M-27/F1:A2:B20 plasmids, suggesting their prevalence as a newly arising public health concern.

4-Amino-2-Sulfanylbenzoic Acid as a Potent Subclass B3 Metallo-ß-Lactamase-Specific Inhibitor Applicable for Distinguishing Metallo-ß-Lactamase Subclasses.

The number of cases of infection with carbapenem-resistant Enterobacteriaceae (CRE) has been increasing and has become a major clinical and public health concern. Production of metallo-ß-lactamases (MBLs) is one of the principal carbapenem resistance mechanisms in CRE. Therefore, developing MBL inhibitors is a promising strategy to overcome the problems of carbapenem resistance conferred by MBLs. To date, the development and evaluation of MBL inhibitors have focused on subclass B1 MBLs but not on B3 MBLs. In the present study, we searched for B3 MBL (specifically, SMB-1) inhibitors and found thiosalicylic acid (TSA) to be a potent inhibitor of B3 SMB-1 MBL (50% inhibitory concentration [IC50], 0.95 µM). TSA inhibited the purified SMB-1 to a considerable degree but was not active against Escherichia coli cells producing SMB-1, as the meropenem (MEM) MIC for the SMB-1 producer was only slightly reduced with TSA. We then introduced a primary amine to TSA and synthesized 4-amino-2-sulfanylbenzoic acid (ASB), which substantially reduced the MEM MICs for SMB-1 producers. X-ray crystallographic analyses revealed that ASB binds to the two zinc ions, Ser221, and Thr223 at the active site of SMB-1. These are ubiquitously conserved residues across clinically relevant B3 MBLs. ASB also significantly inhibited other B3 MBLs, including AIM-1, LMB-1, and L1. Therefore, the characterization of ASB provides a starting point for the development of optimum B3 MBL inhibitors.

Erythromycin-Susceptible but Clindamycin-Resistant Phenotype of Clinical ermB-PCR-Positive Group B Streptococci Isolates with IS1216E-Inserted ermB.

Streptococcus agalactiae (Group B Streptococcus, GBS) is a pathogen which causes neo natal sepsis, meningitis, and invasive infections in the elderly and people with medical conditions. Macrolide and lincosamide resistance rates of GBS strains have been increasing worldwide. A macrolide resistance gene, erythromycin ribosomal methylase (erm), typically confers macrolides, lincosamides, streptogramin B resistance phenotype. However, in the current study, we recovered and characterized 3 clinical ermB-PCR-positive isolates of GBS with L phenotype. The presence of ermB and lnuB (lincosamide nucleotidyltransferase) genes in all 3 clinical isolates was confirmed using PCR. The ermB gene of the clinical isolates harbored C222T (N74N), T224C (I75T), and A299G (N100S) nucleotide (amino acid) substitutions, and insertion of an IS1216E element at nucleotide position 643, resulted in the deletion of a segment spanning nucleotides 643-738 of ermB gene, which suggested the loss-of-function of ErmB protein in the 3 clinical isolates. Since these clinical isolates show positive PCR result for a drug resistance gene despite its partial deletion, these results contradict their drug resistance phenotype. These factors must be considered while performing PCR-based detection of antimicrobial drug resistance genes.

Intercellular Transfer of Chromosomal Antimicrobial Resistance Genes between Acinetobacter baumannii Strains Mediated by Prophages.

The spread of antimicrobial resistance genes (ARGs) among Gram-negative pathogens, including Acinetobacter baumannii, is primarily mediated by transferable plasmids; however, ARGs are frequently integrated into its chromosome. How ARG gets horizontally incorporated into the chromosome of A. baumannii, and whether it functions as a cause for further spread of ARG, remains unknown. Here, we demonstrated intercellular prophage-mediated transfer of chromosomal ARGs without direct cell-cell interaction in A. baumannii We prepared ARG-harboring extracellular DNA (eDNA) components from the culture supernatant of a multidrug-resistant (MDR) A. baumannii NU-60 strain and exposed an antimicrobial-susceptible (AS) A. baumannii ATCC 17978 strain to the eDNA components. The antimicrobial-resistant (AR) A. baumannii ATCC 17978 derivatives appeared to acquire various ARGs, originating from dispersed loci of the MDR A. baumannii chromosome, along with their surrounding regions, by homologous recombination, with the ARGs including armA (aminoglycoside resistance), blaTEM-1 (ß-lactam resistance), tet(B) (tetracycline resistance), and gyrA-81L (nalidixic acid resistance) genes. Notably, the eDNAs conferring antimicrobial resistance were enveloped in specific capsid proteins consisting of phage particles, thereby protecting the eDNAs from detergent and DNase treatments. The phages containing ARGs were likely released into the extracellular space from MDR A. baumannii, thereby transducing ARGs into AS A. baumannii, resulting in the acquisition of AR properties by the recipient. We concluded that the generalized transduction, in which phages were capable of carrying random pieces of A. baumannii genomic DNAs, enabled efficacious intercellular transfer of chromosomal ARGs between A. baumannii strains without direct cell-cell interaction.

Isolation of group B Streptococcus with reduced ß-lactam susceptibility from pregnant women.

ß-Lactam antibiotics are first-line agents for the treatment and prevention of group B Streptococcus (GBS) infections. We previously reported clinical GBS isolates with reduced ß-lactam susceptibility (GBS-RBS) and characterized them as harbouring amino acid substitutions in penicillin-binding proteins (PBPs). However, to our knowledge, GBS-RBS clinical isolates have never previously been isolated from pregnant women worldwide. We obtained 477 clinical GBS isolates from vaginal/rectal swabs of 4530 pregnant women in Japan. We determined the MICs of seven ß-lactams for all 477 clinical isolates. Five clinical isolates showed reduced ceftibuten susceptibility. For these isolates, we performed sequencing analysis of pbp genes. None of the 477 isolates were non-susceptible to penicillin G, ampicillin, and meropenem. For five isolates, the MICs of ceftibuten were relatively high (64-128 µg/ml). Each of these isolates possessed a single amino acid substitution in PBP2X, and some of the substitutions had been previously found in GBS with reduced penicillin susceptibility. This is the first report of the isolation of clinical GBS-RBS isolates harbouring amino acid substitutions in PBP2X that confer reduced ceftibuten susceptibility from pregnant women.

Spread of seb-Positive Methicillin-Resistant Staphylococcus aureus SCCmec Type II-ST764 Among Elderly Japanese in Nonacute Care Settings.

We investigated the prevalence and molecular epidemiology of methicillin-resistant Staphylococcus aureus (MRSA) among 356 residents of nine long-term care facilities (LTCFs) in Japan during 2015 and 2017. In total, 800 specimens were tested and 39 MRSA isolates were recovered from 31 (8.71%) residents. PCR-based open reading frame typing (POT) and pulsed-field gel electrophoresis typing were performed for the 39 MRSA isolates; five of them showing identical pulsotypes, and POT scores were excluded in further analysis. Staphylococcal cassette chromosome mec (SCCmec) typing, multilocus sequence typing, and toxin gene detection were performed for one representative MRSA isolate per resident. Among the 34 unrelated MRSA isolates, 15 (44.1%) and 19 (55.9%) were of SCCmec types II and IV, respectively, and belonged to seven sequence types (STs). Among the 15 SCCmec II isolates, 11 (73.3%), 3, and 1 belonged to ST764 (clonal complex [CC]5), ST5 (CC5), and ST630 (CC8), respectively. Among the 19 SCCmec IV isolates, 13 (68.4%), 3, 2, and 1 belonged to ST1 (CC1), ST474 (CC1), ST8 (CC8), and ST380 (CC8), respectively. Among the 14 CC5 lineage-SCCmec II isolates, one ST5 isolate and 7 of the 11 ST764 isolates (63.6%) carried seb gene, and 14 (87.5%) of 16 CC1 lineage-SCCmec IV isolates had sea gene (p < 0.05). The results indicate that the seb-positive SCCmec type II-ST764 clone has spread in Japanese LTCF environments. As LTCF residents have multiple comorbidities and increased susceptibility to infections, it is necessary to monitor MRSA colonization in LTCFs through periodic screening to prevent dissemination.