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.

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.

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.

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.

Relatively high rates of cefotaxime- and ceftriaxone-non-susceptible isolates among group B streptococci with reduced penicillin susceptibility (PRGBS) in Japan.

OBJECTIVES: We have previously identified group B Streptococcus (GBS) clinical isolates with reduced penicillin susceptibility (PRGBS) that were non-susceptible to cefotaxime; however, the rates of cefotaxime and ceftriaxone non-susceptibility among PRGBS isolates have never been reported. Therefore, we first determined the MICs of 22 antibacterial drugs/compounds for 74 PRGBS isolates and then determined the rates of cefotaxime and ceftriaxone non-susceptibility among these isolates. METHODS: We used 74 clinical PRGBS isolates, previously collected in Japan and confirmed to harbour relevant amino acid substitutions in PBP2X. We also used 80 penicillin-susceptible GBS (PSGBS) clinical isolates as controls. The MICs of 22 antibacterial drugs/compounds for all 154 GBS isolates were determined via microdilution and/or agar dilution methods, as recommended by the CLSI. RESULTS: The rates of non-susceptibility/resistance to ampicillin, cefotaxime, ceftriaxone and levofloxacin for the 80 PSGBS isolates were 0%, 0%, 0% and 30%, respectively, but were 15% (P = 0.0003), 28% (P < 0.0001), 36% (P < 0.0001) and 93% (P < 0.0001) for the 74 PRGBS isolates, respectively. No PRGBS isolates were identified to be non-susceptible to meropenem, doripenem, vancomycin, quinupristin/dalfopristin, daptomycin or linezolid. CONCLUSIONS: We found that cefotaxime- and ceftriaxone-non-susceptible PRGBS isolates occur at relatively high rates in Japan. Importantly, this finding suggests that the range of drugs likely to be effective in treating PRGBS infections may be limited compared with those available for PSGBS infections; therefore, clinicians should exercise care when considering drug choice and efficacy for PRGBS infections.

Potential effect of selective pressure with different ß-lactam molecules on the emergence of reduced susceptibility to ß-lactams in group B Streptococci.

In this study, the selective potential of group B Streptococcus isolates with reduced penicillin susceptibility (PRGBS) in a neonate-hypervirulent sequence type (ST)17 lineage was investigated by in vitro exposure to ß-lactams. After 19 passages of stepwise penicillin exposure, PRGBS with a high penicillin minimum inhibitory concentration MIC (0.5 mg/L), greatly augmented ceftibuten MIC (>512 mg/L), and acquisition of G406D predicted to provide destabilizing effect (ΔΔG 0.099 kcal/mol) on PBP2X structure were identified. In early passages of stepwise cefotaxime exposure, PRGBS possessing G398E predicted to stabilize PBP2X (ΔΔG -0.038 kcal/mol) emerged with high MICs for cefotaxime (0.5 mg/L), ceftibuten (>512 mg/L) and penicillin (0.25 mg/L). Additionally, G398E + G329V + H438Y predicted to provide more stabilizing effect (ΔΔG -0.415 kcal/mol) were detected in mutants with higher MICs to cefotaxime (1 mg/L) and penicillin (0.5 mg/L). PRGBS mutants selected by penicillin and cefotaxime had a marked growth disadvantage compared with the parent strain. After two passages of stepwise ceftibuten exposure, the mutants exhibited increased MICs toward ceftibuten and acquisition of T555S predicted to provide stabilizing effect (ΔΔG -0.111 kcal/mol) in PBP 2X. In subsequent passages, gradual increases in ceftibuten MICs from 128 mg/L to 512 mg/L were found among selected mutants with accompanying stabilizing T555S+A354V (ΔΔG -0.257 kcal/mol) followed by stabilizing T555S + A354V + A536V (ΔΔG -0.322 kcal/mol), resulting in selection of a penicillin-susceptible group B Streptococcus lineage with reduced ceftibuten susceptibility (CTBr PSGBS). Notably, growth ability of CTBr PSGBS mutants was comparable to that of the parent strain. These findings may predict future failure of treatment for neonatal invasive infections caused by the neonate-hypervirulent PRGBS ST17 lineage.

Carbapenem-Nonsusceptible Haemophilus influenzae with Penicillin-Binding Protein 3 Containing an Amino Acid Insertion.

The prevalence of ß-lactamase-negative ampicillin-resistant (BLNAR) Haemophilus influenzae has become a clinical concern. In BLNAR isolates, amino acid substitutions in penicillin-binding protein 3 (PBP3) are relevant to the ß-lactam resistance. Carbapenem-nonsusceptible H. influenzae isolates have been rarely reported. Through antimicrobial susceptibility testing, nucleotide sequence analysis of ftsI, encoding PBP3, and the utilization of a collection of H. influenzae clinical isolates in our laboratory, we obtained a carbapenem-nonsusceptible clinical isolate (NUBL1772) that possesses an altered PBP3 containing V525_N526insM. The aim of this study was to reveal the effect of altered PBP3 containing V525_N526insM on reduced carbapenem susceptibility. After generating recombinant strains with altered ftsI, we performed antimicrobial susceptibility testing and competitive binding assays with fluorescent penicillin (Bocillin FL) and carbapenems. Elevated carbapenem MICs were found for the recombinant strain harboring the entire ftsI gene of NUBL1772. The recombinant PBP3 of NUBL1772 also exhibited reduced binding to carbapenems. These results demonstrate that altered PBP3 containing V525_N526insM influences the reduced carbapenem susceptibility. The revertant mutant lacking the V525_N526insM exhibited lower MICs for carbapenems than NUBL1772, suggesting that this insertion affects reduced carbapenem susceptibility. The MICs of ß-lactams for NUBL1772 were higher than those for the recombinant possessing ftsI of NUBL1772. NUBL1772 harbored AcrR with early termination, resulting in low-level transcription of acrB and high efflux pump activity. These findings suggest that the disruption of AcrR also contributes to the reduced carbapenem susceptibility found in NUBL1772. Our results provide the first evidence that the altered PBP3 containing V525_N526insM is responsible for the reduced susceptibility to carbapenems in H. influenzae.

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.

Specific blaCTX-M-8/IncI1 Plasmid Transfer among Genetically Diverse Escherichia coli Isolates between Humans and Chickens.

We investigated the genetic backbones of 14 blaCTX-M-8-positive Escherichia coli isolates recovered from human stool samples and chicken meat. All isolates carried IncI1 plasmids with blaCTX-M-8 (blaCTX-M-8/IncI1), and most (9/14) belonged to a specific genetic lineage, namely, plasmid sequence type 113 (pST113). The genetic contexts of the nine blaCTX-M-8/IncI1 pST113 plasmids were similar, regardless of the source. These results suggest the probable local transfer of blaCTX-M-8/IncI1 between humans and chickens with genetically diverse E. coli.

Structural Insights into the TLA-3 Extended-Spectrum ß-Lactamase and Its Inhibition by Avibactam and OP0595.

The development of effective inhibitors that block extended-spectrum ß-lactamases (ESBLs) and restore the action of ß-lactams represents an effective strategy against ESBL-producing Enterobacteriaceae We evaluated the inhibitory effects of the diazabicyclooctanes avibactam and OP0595 against TLA-3, an ESBL that we identified previously. Avibactam and OP0595 inhibited TLA-3 with apparent inhibitor constants (Kiapp) of 1.71 ± 0.10 and 1.49 ± 0.05 µM, respectively, and could restore susceptibility to cephalosporins in the TLA-3-producing Escherichia coli strain. The value of the second-order acylation rate constant (k2/K, where k2 is the acylation rate constant and K is the equilibrium constant) of avibactam [(3.25 ± 0.03) × 103 M-1 · s-1] was closer to that of class C and D ß-lactamases (k2/K, <104 M-1 · s-1) than that of class A ß-lactamases (k2/K, >104 M-1 · s-1). In addition, we determined the structure of TLA-3 and that of TLA-3 complexed with avibactam or OP0595 at resolutions of 1.6, 1.6, and 2.0 Å, respectively. TLA-3 contains an inverted Ω loop and an extended loop between the ß5 and ß6 strands (insertion after Ser237), which appear only in PER-type class A ß-lactamases. These structures might favor the accommodation of cephalosporins harboring bulky R1 side chains. TLA-3 presented a high catalytic efficiency (kcat/Km ) against cephalosporins, including cephalothin, cefuroxime, and cefotaxime. Avibactam and OP0595 bound covalently to TLA-3 via the Ser70 residue and made contacts with residues Ser130, Thr235, and Ser237, which are conserved in ESBLs. Additionally, the sulfate group of the inhibitors formed polar contacts with amino acid residues in a positively charged pocket of TLA-3. Our findings provide a structural template for designing improved diazabicyclooctane-based inhibitors that are effective against ESBL-producing Enterobacteriaceae.

Structural Insights into Recognition of Hydrolyzed Carbapenems and Inhibitors by Subclass B3 Metallo-ß-Lactamase SMB-1.

Metallo-ß-lactamases (MBLs) confer resistance to carbapenems, and their increasing global prevalence is a growing clinical concern. To elucidate the mechanisms by which these enzymes recognize and hydrolyze carbapenems, we solved 1.4 to 1.6 Å crystal structures of SMB-1 (Serratia metallo-ß-lactamase 1), a subclass B3 MBL, bound to hydrolyzed carbapenems (doripenem, meropenem, and imipenem). In these structures, SMB-1 interacts mainly with the carbapenem core structure via elements in the active site, including a zinc ion (Zn-2), Q157[113] (where the position in the SMB-1 sequence is in brackets after the BBL number), S221[175], and T223[177]. There is less contact with the carbapenem R2 side chains, strongly indicating that SMB-1 primarily recognizes the carbapenem core structure. This is the first report describing how a subclass B3 MBL recognizes carbapenems. We also solved the crystal structure of SMB-1 in complex with the approved drugs captopril, an inhibitor of the angiotensin-converting enzyme, and 2-mercaptoethanesulfonate, a chemoprotectant. These drugs are inhibitors of SMB-1 with Ki values of 8.9 and 184 µM, respectively. Like carbapenems, these inhibitors interact with Q157[113] and T223[177] and their thiol groups coordinate the zinc ions in the active site. Taken together, the data indicate that Q157[113], S221[175], T223[177], and the two zinc ions in the active site are key targets in the design of SMB-1 inhibitors with enhanced affinity. The structural data provide a solid foundation for the development of effective inhibitors that would overcome the carbapenem resistance of MBL-producing multidrug-resistant microbes.

Human collagen XV is a prominent histopathological component of sinusoidal capillarization in hepatocellular carcinogenesis.

BACKGROUND: Increased expression of collagen XV has been reported in hepatocellular carcinogenesis in mice. The aim of this study was to confirm the previous murine findings in human hepatocellular carcinoma (HCC) specimens, along with the histopathological distribution of collagen XV in tumoral tissues. METHODS: Sixty-three primary HCC specimens were examined. Immunostaining of collagen XV and quantitative reverse transcriptional PCR of COL15A1, which encodes collagen XV, were performed. RESULTS: Positive staining of collagen XV was observed in all tumoral regions, regardless of differentiation level or pathological type of HCC, along the sinusoid-like endothelium, whereas collagen XV was not expressed in any non-tumoral region. The intensity score of collagen XV immunostaining and the mRNA value of COL15A1 were significantly correlated. COL15A1 expression in tumors was 3.24-fold higher than in non-tumoral regions. Multivariate analysis showed that COL15A1 expression was significantly higher in the absence of hepatitis virus and moderately differentiated HCC. CONCLUSIONS: COL15A1 mRNA was up-regulated in HCC and collagen XV was expressed along the sinusoid-like endothelium of HCC but not in non-tumoral regions, which implies that collagen XV contributes to the capillarization of HCC.

Molecular epidemiological characteristics of Klebsiella pneumoniae associated with bacteremia among patients with pneumonia.

Some important virulence factors have been elucidated in Klebsiella pneumoniae infections. We investigated the relationship between virulence factors and multilocus sequence types (STs) and assessed the risk factors for bacteremia in patients with pneumonia due to K. pneumoniae. From April 2004 through April 2012, a total of 120 K. pneumoniae isolates from patients with pneumonia (23 with bacteremia and 97 without bacteremia) were collected from 10 medical institutions in Japan. Additionally, 10 strains of K. pneumoniae serotype K2 that were isolated >30 years ago were included in this study. These isolates were characterized using multilocus sequence typing (MLST), and the characteristics of their virulence factors, such as hypermucoviscosity phenotype and RmpA and aerobactin production between patients with and without bacteremia, were examined. MLST analysis was performed on the 120 isolates from patients with pneumonia, and some sequence type groups were defined as genetic lineages (GLs). GL65 was more prevalent among patients with bacteremia (21.7%) than in those without bacteremia (7.2%). The majority of the strains with serotype K2 were classified into GL14 or GL65, and rmpA and the gene for aerobactin were present in all GL65-K2 strains but absent in all GL14-K2 strains. In a multivariate analysis, the independent risk factors for bacteremia included GL65 (adjusted odds ratio [AOR], 9.46; 95% confidence interval [CI], 1.81 to 49.31), as well as neoplastic disease (AOR, 9.94; 95% CI, 2.61 to 37.92), immunosuppression (AOR, 17.85; 95% CI, 1.49 to 214.17), and hypoalbuminemia (AOR, 4.76; 95% CI, 1.29 to 17.61). GL65 was more prevalent among patients with bacteremia and was associated with the virulence factors of K. pneumoniae.

Classification of group B streptococci with reduced ß-lactam susceptibility (GBS-RBS) based on the amino acid substitutions in PBPs.

All clinical isolates of group B Streptococcus (GBS; Streptococcus agalactiae) are considered uniformly susceptible to ß-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 ß-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.

New plasmid-mediated aminoglycoside 6'-N-acetyltransferase, AAC(6')-Ian, and ESBL, TLA-3, from a Serratia marcescens clinical isolate.

OBJECTIVES: Enterobacteriaceae clinical isolates showing amikacin resistance (MIC 64 to >256 mg/L) in the absence of 16S rRNA methyltransferase (MTase) genes were found. The aim of this study was to clarify the molecular mechanisms underlying amikacin resistance in Enterobacteriaceae clinical isolates that do not produce 16S rRNA MTases. METHODS: PCR was performed to detect already-known amikacin resistance determinants. Cloning experiments and sequence analyses were performed to characterize unknown amikacin resistance determinants. Transfer of amikacin resistance determinants was performed by conjugation and transformation. The complete nucleotide sequence of the plasmids was determined by next-generation sequencing technology. Amikacin resistance enzymes were purified with a column chromatography system. The enzymatic function of the purified protein was investigated by thin-layer chromatography (TLC) and HPLC. RESULTS: Among the 14 isolates, 9 were found to carry already-known amikacin resistance determinants such as aac(6')-Ia and aac(6')-Ib. Genetic analyses revealed the presence of a new amikacin acetyltransferase gene, named aac(6')-Ian, located on a 169 829 bp transferable plasmid (p11663) of the Serratia marcescens strain NUBL-11663, one of the five strains negative for known aac(6') genes by PCR. Plasmid p11663 also carried a novel ESBL gene, named blaTLA-3. HPLC and TLC analyses demonstrated that AAC(6')-Ian catalysed the transfer of an acetyl group from acetyl coenzyme A onto an amine at the 6'-position of various aminoglycosides. CONCLUSIONS: We identified aac(6')-Ian as a novel amikacin resistance determinant together with a new ESBL gene, blaTLA-3, on a transferable plasmid of a S. marcescens clinical isolate.

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.

Penicillin-susceptible group B streptococcal clinical isolates with reduced cephalosporin susceptibility.

We characterized penicillin-susceptible group B streptococcal (PSGBS) clinical isolates exhibiting no growth inhibition zone around a ceftibuten disk (CTB(r) PSGBS). The CTB(r) PSGBS isolates, for which augmented MICs of cefaclor and ceftizoxime were found, shared a T394A substitution in penicillin-binding protein 2X (PBP 2X) and a T567I substitution in PBP 2B, together with an additional G429S substitution in PBP 2X or a T145A substitution in PBP 1A, although the T145A substitution in the transglycosidase domain of PBP 1A would have no effect on the level of resistance to ceftibuten.

Characterization of multidrug-resistant group B streptococci with reduced penicillin susceptibility forming small non-Beta-hemolytic colonies on sheep blood agar plates.

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.