Role of TEM-1 ß-Lactamase in the Predominance of Ampicillin-Sulbactam-Nonsusceptible Escherichia coli in Japan.

We investigated the epidemiology and resistance mechanisms of ampicillin-sulbactam-nonsusceptible Escherichia coli, focusing on the role of the TEM-1 ß-lactamase. We collected all nonduplicate E. coli clinical isolates at 10 Japanese hospitals during December 2014 and examined their antimicrobial susceptibility, ß-lactamases, TEM-1 transferability, TEM-1 ß-lactamase activity, outer membrane protein profile, membrane permeability, and clonal genotypes. Among the 329 isolates collected, 95 were ampicillin-sulbactam nonsusceptible. Of these ampicillin-sulbactam-nonsusceptible isolates, ß-lactamases conferring resistance to sulbactam, such as AmpC, were present in 33%. Hyperproduction of sulbactam-susceptible ß-lactamases, TEMs with a strong promoter, were rare (5%). The remaining 59 isolates (62%) had only sulbactam-susceptible ß-lactamases, including TEM-1 with a wild-type promoter (n = 28), CTX-Ms (n = 13), or both (n = 17). All 45 transconjugants from 96 donors with TEM-1 had higher ampicillin-sulbactam MICs (4 to 96 mg/liter) than the recipient (2 mg/liter). In donors with only TEM-1, TEM-1 activity correlated with the 50% inhibitory concentration of sulbactam and ampicillin-sulbactam MICs. The decreased membrane permeation of sulbactam was associated with an increased ampicillin-sulbactam MIC. The reduced permeation was partly attributable to deficient outer membrane proteins, which were observed in 57% of the ampicillin-sulbactam-nonsusceptible isolates with only TEM-1 and a wild-type promoter. Sequence type 131 (ST131) was the most common clonal type (52%). TEM-1 with a wild-type promoter primarily contributed to ampicillin-sulbactam nonsusceptibility in E. coli, with the partial support of other mechanisms, such as reduced permeation. Conjugative TEM-1 and the clonal spread of ST131 may contribute to the prevalence of Japanese ampicillin-sulbactam-nonsusceptible isolates.

Spread of Meropenem-Resistant Streptococcus pneumoniae Serotype 15A-ST63 Clone in Japan, 2012-2014.

After the introduction of pneumococcal conjugate vaccines, the incidence of pneumococcal infections due to meropenem-resistant serotype 15A-ST63 strains increased in Japan. By using whole-genome sequencing and comparing sequences with those of clones from the United Kingdom, the United States, and Canada, we clarified the traits of the serotype 15A-ST63 clone. Our analysis revealed that the meropenem-resistant serotype 15A-ST63 strains from Japan originated from meropenem-susceptible strains from Japan. Recombination site prediction analysis showed that the meropenem-resistant strain-specific recombination regions included the pbp1a and pbp2b regions. A detailed analysis of the composition of these genes indicated that resistance seems to be caused by pbp1a recombination. The pbp1a gene in meropenem-resistant isolates was identical to that in multidrug (including meropenem)-resistant serotype 19A-ST320 pneumococci, which have spread in the United States. The global spread of pneumococci of this lineage is noteworthy because serotype 15A is not included in the currently used 13-valent pneumococcal conjugate vaccine.

Characteristics of Carbapenemase-Producing Enterobacteriaceae in Wastewater Revealed by Genomic Analysis.

Wastewater is considered a major source of antibiotic-resistant bacteria released into the environment. Here, we characterized carbapenemase-producing Enterobacteriaceae (CPE) in wastewater by whole-genome analysis. Wastewater samples (n = 40) were collected from municipal wastewater treatment plants and hospital wastewater in Japan and Taiwan. Samples were screened for CPE using selective media, and the obtained isolates were sequenced using an Illumina MiSeq. The isolates (n = 45) included the following microorganisms: Klebsiella quasipneumoniae (n = 12), Escherichia coli (n = 10), Enterobacter cloacae complex (n = 10), Klebsiella pneumoniae (n = 8), Klebsiella variicola (n = 2), Raoultella ornithinolytica (n = 1), Citrobacter freundii (n = 1), and Citrobacter amalonaticus (n = 1). Among the 45 isolates, 38 harbored at least one carbapenemase-encoding gene. Of these, the blaGES (blaGES-5, blaGES-6, and blaGES-24) genes were found in 29 isolates. The genes were situated in novel class 1 integrons, but the integron structures were different between the Japanese (In1439 with blaGES-24 and In1440 with blaGES-5) and Taiwanese (In1441 with blaGES-5 and In1442 with blaGES-6) isolates. Other carbapenemase-encoding genes (blaVIM-1, blaNDM-5, blaIMP-8, blaIMP-19, and blaKPC-2) were found in one to three isolates. Notably, class 1 integrons previously reported among clinical isolates obtained in the same regions as the present study, namely, In477 with blaIMP-19 and In73 with blaIMP-8, were found among the Japanese and Taiwanese isolates, respectively. The results indicate that CPE with various carbapenemase-encoding genes in different genetic contexts were present in biologically treated wastewater, highlighting the need to monitor for antibiotic resistance in wastewater.

Molecular Analysis of a blaIMP-1-Harboring Class 3 Integron in Multidrug-Resistant Pseudomonas fulva.

A multidrug-resistant (MDR) Pseudomonas fulva strain was isolated in 2006 from a urine sample. The isolate harbored the blaIMP-1 gene, which was located in a chromosomal Tn402-like class 3 integron as a gene cassette array of aacA31-fosE-blaIMP-1 Two mutations in gyrA and one mutation in parC were detected in quinolone-resistance-determining regions (QRDRs). We report a full-length, novel, blaIMP-1-carrying class 3 integron. This integron, together with mutations in QRDRs, could have influenced the MDR phenotype.

Real-time quantitative PCR analysis of endoscopic biopsies for diagnosing CMV gastrointestinal disease in non-HIV immunocompromised patients: a diagnostic accuracy study.

Cytomegalovirus gastrointestinal diseases (CMV-GIDs) are end-organ diseases of the gastrointestinal (GI) tract caused by CMV in immunocompromised patients. We aimed to evaluate the performance of quantitative polymerase chain reaction (qPCR) on endoscopic biopsies. We retrospectively reviewed the qPCR data on endoscopic biopsies in nonhuman immunodeficiency virus (HIV) immunocompromised patients between January 2009 and May 2015. The performance of the qPCR for CMV-GID was evaluated with the sensitivity, specificity, and area under the receiver operating characteristic curve (AUROC). A total of 195 patients were included, and 28 patients with confirmed CMV-GID were identified. The AUROC of the qPCR was 0.935 (95% confidence interval [CI], 0.885 to 0.985), the sensitivity was 89.3% (95% CI, 71.8 to 97.7%), and the specificity was 85.6% (95% CI, 79.4 to 97.6%) with a cutoff value of 180 copies/µg DNA. The proportion of patients with inflammatory bowel disease in the histopathology-negative, PCR-positive group was smaller than that in the histopathology-positive group (10.7 vs 35.0%, p = 0.026), but other characteristics were not significantly different. The use of qPCR on endoscopic biopsies demonstrated good diagnostic performance for detecting CMV in non-HIV immunocompromised patients. It may increase the diagnostic yield when combined with a conventional histopathology.

Evaluation of a surface plasmon resonance imaging-based multiplex O-antigen serogrouping for Escherichia coli using eleven major serotypes of Shiga -toxin-producing E. coli.

The early detection of Shiga toxin-producing Escherichia coli (STEC) is important for early diagnosis and preventing the spread of STEC. Although the confirmatory test for STEC should be based on the detection of Shiga toxin using molecular analysis, isolation permits additional characterization of STEC using a variety of methods, including O:H serotyping. The conventional slide agglutination O-antigen serogrouping used in many clinical laboratories is laborious and time-consuming. Surface plasmon resonance (SPR)-based immunosensors are commonly used to investigate a large variety of bio-interactions such as antibody/antigen, peptide/antibody, DNA/DNA, and antibody/bacteria interactions. SPR imaging (SPRi) is characterized by multiplexing capabilities for rapidly screening (approximately 100 to several hundred sensorgrams in parallel) molecules. SPRi-based O-antigen serogrouping method for STEC was recently developed by detecting the interactions between O-antigen-specific antibodies and bacterial cells themselves. The aim of this study was to evaluate its performance for E. coli serogrouping using clinical STEC isolates by comparing the results of slide agglutination tests. We tested a total of 188 isolates, including O26, O45, O91, O103, O111, O115, O121, O128, O145, O157, and O159. The overall sensitivity of SPRi-based O-antigen serogrouping was 98.9%. Only two O157 isolates were misidentified as nontypeable and O121. The detection limits of all serotypes were distributed between 1.1 × 106 and 17.6 × 106 CFU/ml. Pulsed-field gel electrophoresis (PFGE) revealed the heterogeneity of the examined isolates. In conclusion, SPRi is a useful method for the O-antigen serogrouping of STEC isolates, but the further evaluation of non-O157 minor serogroups is needed.

Occurrence of Clinically Important Lineages, Including the Sequence Type 131 C1-M27 Subclone, among Extended-Spectrum-ß-Lactamase-Producing Escherichia coli in Wastewater.

Contamination of environmental waters by extended-spectrum-ß-lactamase (ESBL)-producing Escherichia coli (ESBLEC) is of great concern. Wastewater treatment plants (WWTPs) and hospitals release large amounts of ESBLEC into the environment. In the present study, we isolated ESBLEC strains from wastewater collected from a WWTP and a hospital in Japan and performed whole-genome sequencing to characterize these strains. Genomic analysis of 54 strains (32 from the WWTP and 22 from hospital wastewater) revealed the occurrence of clinically important clonal groups with extraintestinal pathogenic E. coli status in the WWTP and hospital wastewater. Fine-scale phylogenetic analysis was performed to further characterize 15 sequence type 131 (ST131) complex strains (11 from the WWTP and 4 from hospital wastewater). These ST131 complex strains were comprised of the following different subgroups: clade A (n = 2), C1-M27 (n = 8), and C1 (non-C1-M27) (n = 1) for strains from the WWTP and clade A (n = 2), C1-M27 (n = 1), and C1 (non-C1-M27) (n = 1) for strains from hospital wastewater. The results indicate that ESBLEC strains belonging to clinically important lineages, including the C1-M27 clade, may disseminate into the environment through wastewater, highlighting the need to monitor for antibiotic resistance in wastewater.

Rapid Identification of Different Escherichia coli Sequence Type 131 Clades.

Escherichia coli sequence type 131 (ST131) is a pandemic clonal lineage that is responsible for the global increase in fluoroquinolone resistance and extended-spectrum-ß-lactamase (ESBL) producers. The members of ST131 clade C, especially subclades C2 and C1-M27, are associated with ESBLs. We developed a multiplex conventional PCR assay with the ability to detect all ST131 clades (A, B, and C), as well as C subclades (C1-M27, C1-nM27 [C1-non-M27], and C2). To validate the assay, we used 80 ST131 global isolates that had been fully sequenced. We then used the assay to define the prevalence of each clade in two Japanese collections consisting of 460 ESBL-producing E. coli ST131 (2001-12) and 329 E. coli isolates from extraintestinal sites (ExPEC) (2014). The assay correctly identified the different clades in all 80 global isolates: clades A (n = 12), B (n = 12), and C, including subclades C1-M27 (n = 16), C1-nM27 (n = 20), C2 (n = 17), and other C (n = 3). The assay also detected all 565 ST131 isolates in both collections without any false positives. Isolates from clades A (n = 54), B (n = 23), and C (n = 483) corresponded to the O serotypes and the fimH types of O16-H41, O25b-H22, and O25b-H30, respectively. Of the 483 clade C isolates, C1-M27 was the most common subclade (36%), followed by C1-nM27 (32%) and C2 (15%). The C1-M27 subclade with blaCTX-M-27 became especially prominent after 2009. Our novel multiplex PCR assay revealed the predominance of the C1-M27 subclade in recent Japanese ESBL-producing E. coli isolates and is a promising tool for epidemiological studies of ST131.

Population structure of Japanese extraintestinal pathogenic Escherichia coli and its relationship with antimicrobial resistance.

Objectives: To define the population structure of extraintestinal pathogenic Escherichia coli (ExPEC) in Japan and its relationship with antimicrobial resistance and the major resistance mechanisms for fluoroquinolones and ß-lactams, we designed a multicentre prospective study. Methods: A total of 329 ExPEC isolates were collected at 10 Japanese acute-care hospitals during December 2014. We defined the clonal groups of ExPEC by fumC and fimH sequencing (CH typing). Antimicrobial susceptibility testing of 18 agents and the detection of mutations in quinolone resistance-determining regions (QRDRs) and ß-lactamases were performed. Results: Among the study isolates, 103 CH types were found, and CH40-30 (25%) and another 10 CH types (35% in total) constituted the major ExPEC population. Ciprofloxacin non-susceptibility, ESBLs and MDR phenotypes were found in 34%, 22% and 33%, respectively. CH40-30, corresponding to the C/H30 clade of the global pandemic ST131 clone, was associated with four QRDR mutations (100%) and bla CTX-M (60%) and was the most frequent type in 15 antimicrobial-non-susceptible populations (dominating 39%-75% of each population, the highest prevalence for ciprofloxacin), the ESBL producers (70%) and the MDR isolates (59%). Isolates that were non-susceptible to nalidixic acid and low-level resistant to ciprofloxacin with one or two QRDR mutations represented 16% of the study isolates and were distributed among the eight major and non-major CH types. Conclusions: More than half of the ExPEC population in Japan consisted of 11 major clones. Of these clones, the CH40-30-ST131-C/H30 clone was the predominant antimicrobial-resistant population. The presence of major clones with low-level ciprofloxacin resistance supports the potential future success of a non-ST131 fluoroquinolone-resistant clone.

Whole-Genome Analysis of Antimicrobial-Resistant and Extraintestinal Pathogenic Escherichia coli in River Water.

Contamination of surface waters by antimicrobial-resistant bacteria and pathogenic bacteria is a great concern. In this study, 531 Escherichia coli isolates obtained from the Yamato River in Japan were evaluated phenotypically for resistance to 25 antimicrobials. Seventy-six isolates (14.3%) were multidrug resistant (MDR), 66 (12.4%) were nonsusceptible to one or two classes of agents, and 389 (73.3%) were susceptible. We performed whole-genome sequencing of selected strains by using Illumina technology. In total, the genome sequences of 155 strains were analyzed for antibiotic resistance determinants and phylogenetic characteristics. More than 50 different resistance determinants, including acquired resistance genes and chromosomal resistance mutations, were detected. Among the sequenced MDR strains (n = 66), sequence type 155 (ST155) complex (n = 9), ST10 complex (n = 9), and ST69 complex (n = 7) were prevalent. Among extraintestinal pathogenic E. coli (ExPEC) strains (n = 58), clinically important clonal groups, namely, ST95 complex (n = 18), ST127 complex (n = 8), ST12 complex (n = 6), ST14 complex (n = 6), and ST131 complex (n = 6), were prevalent, demonstrating the clonal distribution of environmental ExPEC strains. Typing of the fimH (type 1 fimbrial adhesin) gene revealed that ST131 complex strains carried fimH22 or fimH41, and no strains belonging to the fimH30 subgroup were detected. Fine-scale phylogenetic analysis and virulence gene content analysis of strains belonging to the ST95 complex (one of the major clonal ExPEC groups causing community-onset infections) revealed no significant differences between environmental and clinical strains. The results indicate contamination of surface waters by E. coli strains belonging to clinically important clonal groups.IMPORTANCE The prevalence of antimicrobial-resistant and pathogenic E. coli strains in surface waters is a concern because surface waters are used as sources for drinking water, irrigation, and recreational purposes. In this study, MDR and ExPEC strains in river water were characterized by genomic sequencing and analysis. We detected more than 50 resistance determinants and identified clonal groups specific to MDR and ExPEC strains. This study showed contamination of surface waters by E. coli strains belonging to clinically important clonal groups. Overall, this study advances our understanding of environmental MDR and ExPEC strains.

Complete adherence to evidence-based quality-of-care indicators for Staphylococcus aureus bacteremia resulted in better prognosis.

PURPOSE: Staphylococcus aureus bacteremia (SAB) is a serious clinical condition associated with high morbidity and mortality. Recent studies have revealed that adherence to evidence-based quality-of-care indicators (QCIs) for the management of SAB could result in reduced mortality. We aimed to determine whether compliance with QCIs was associated with mortality and whether compliance with QCIs predicted the mortality of patients with SAB. METHODS: In a university hospital in Kyoto, Japan, SAB patients, who survived at least 14 days after positive blood cultures were analyzed from 2006 to 2014 to assess their compliance with QCIs and the trend in mortality. In addition, the predicted mortality, which was stratified by the number of fulfilled QCIs (QCI points), was calculated. In this study, the following five main QCI points were evaluated: (1) follow-up blood cultures; (2) early source control when applicable; (3) echocardiography; (4) the early use of appropriate antibiotics, and (5) the appropriate duration of therapy. RESULTS: We identified 477 eligible SAB cases, of which 199 were MRSA cases (41.7 %). The proportion of SAB cases in which the physicians adhered to at least four QCIs increased gradually from 47.5 % in 2006 to 79.3 % in 2014 (P = 0.001); whereas, the 30-day mortality decreased from 10.0 to 3.4 % after treatment completion. CONCLUSIONS: With an increase in the proportion of SAB cases adhering to QCIs, better prognoses were observed for patients with SAB. The QCI points reflected the 30-day mortality.

Clinical and microbiologic characteristics of cefotaxime-non-susceptible Enterobacteriaceae bacteremia: a case control study.

BACKGROUND: Cefotaxime plays an important role in the treatment of patients with bacteremia due to Enterobacteriaceae, although cefotaxime resistance is reported to be increasing in association with extended-spectrum ß-lactamase (ESBL) and AmpC ß-lactamase (AmpC). METHODS: We conducted a case-control study in a Japanese university hospital between 2011 and 2012. We assessed the risk factors and clinical outcomes of bacteremia due to cefotaxime-non-susceptible Enterobacteriaceae (CTXNS-En) and analyzed the resistance mechanisms. RESULTS: Of 316 patients with Enterobacteriaceae bacteremia, 37 patients with bacteremia caused by CTXNS-En were matched to 74 patients who had bacteremia caused by cefotaxime-susceptible Enterobacteriaceae (CTXS-En). The most common CTXNS-En was Escherichia coli (43%), followed by Enterobacter spp. (24%) and Klebsiella spp. (22%). Independent risk factors for CTXNS-En bacteremia included previous infection or colonization of CTXNS-En, cardiac disease, the presence of intravascular catheter and prior surgery within 30 days. Patients with CTXNS-En bacteremia were less likely to receive appropriate empirical therapy and to achieve a complete response at 72 h than patients with CTXS-En bacteremia. Mortality was comparable between CTXNS-En and CTXS-En patients (5 vs. 3%). CTXNS-En isolates exhibited multidrug resistance but remained highly susceptible to amikacin and meropenem. CTX-M-type ESBLs accounted for 76% of the ß-lactamase genes responsible for CTXNS E. coli and Klebsiella spp. isolates, followed by plasmid-mediated AmpC (12%). Chromosomal AmpC was responsible for 89% of CTXNS Enterobacter spp. isolates. CONCLUSIONS: CTXNS-En isolates harboring ESBL and AmpC caused delays in appropriate therapy among bacteremic patients. Risk factors and antibiograms may improve the selection of appropriate therapy for CTXNS-En bacteremia. Prevalent mechanisms of resistance in CTXNS-En were ESBL and chromosomal AmpC.

Genetic identification and antimicrobial susceptibility of clinically isolated anaerobic bacteria: A prospective multicenter surveillance study in Japan.

This prospective multicenter surveillance study was designed to provide antimicrobial susceptibility profiles of clinical anaerobic bacteria with genetic species identification in Japan. In 2014, a total of 526 non-duplicate clinical anaerobic isolates were collected from 11 acute-care hospitals in the Kyoto and Shiga regions of Japan. Genetic identification was performed using 16S rRNA sequencing. Minimum inhibitory concentrations were determined in the central laboratory and were interpreted using the CLSI criteria. Genetic analysis provided species-level identification for 496 isolates (83 species in 40 genera) and genus-level identification for 21 isolates (13 genera). Among these 517 isolates, the most frequent anaerobes were Bacteroides spp. (n = 207), Prevotella spp. (n = 43), Clostridium spp. (n = 40), and Peptoniphilus spp. (n = 40). B. fragilis was the most common species (n = 107) and showed 91.6%-97.2% susceptibility to ß-lactam/ß-lactamase inhibitor combinations (BLBLIs; ampicillin-sulbactam, amoxicillin-clavulanate, and piperacillin-tazobactam) and carbapenems (imipenem and meropenem) as well as 100% susceptibility to metronidazole. Gram-negative anaerobes were highly susceptible to metronidazole (99.0%) followed by BLBLIs and carbapenems (>90% each). BLBLIs or carbapenems also retained activity against Gram-positive anaerobes (99.5%-100%) except Clostridioides difficile. All isolates were susceptible to combinations of metronidazole with BLBLIs or carbapenems. Thus, BLBLIs or carbapenems are first choices for empirical therapy of anaerobic infections in Japan, and these antimicrobials in combination with metronidazole should be reserved for very severe infections and targeted therapy.

[FLT3 Mutations in Acute Myeloid Leukemia].

FMS-like tyrosine kinase 3 (FLT3), a class III tyrosine kinase receptor, plays an important role in the pro- liferation, survival, and differentiation of hematopoietic stem/progenitor cells. Approximately 30% of pa- tients with cytogenetically normal acute myeloid leukemia (CN-AML) harbor FLT3 mutations. The most frequent FLT3 mutations are internal tandem duplications (ITDs) in the juxtamembrane domain. FLT3-ITD mutations cause ligand-independent dimerization of FLT3 and the constitutive activation of its downstream signaling pathways, such as PI3K/AKT, A4APK/ERK, and STAT5, leading to dysregulated cellular prolifera- tion. The relapse risk of CN-AML patients with FLT3-ITD is higher and the overall survival (OS) of such patients is shorter than those of patients with wild-type FLT3. Recently genome-wide studies with next-generation sequencing have suggested that mutational combinations of genes related to signal transduction, transcription, splicing, cancer suppressors, and epigenetics contribute to the pathogenesis of AML. These mutations including FLT3-ITD may be prognostic factors facilitating the risk stratification for CN-AML. The point mutations D835/I836 in the tyrosine kinase domain (TKD) are detected in 5-7% of AML patients. The clinical relevance of these FLT3-TKD mutations remains unclear. FLT3-TKD mutations are detected even in patients treated with FLT3 inhibitors as secondary mutations, suggesting that the mutations are as- sociated with the resistance. Therefore, the detection of these mutations might provide us with the opportunity to consider appropriate treatment for patients. The molecular abnormalities in AML patients give us insights into the pathology of AML and clinically significant information required for the diagnosis, prognosis, and treatment decisions. [Review].

Global Escherichia coli Sequence Type 131 Clade with blaCTX-M-27 Gene.

The Escherichia coli sequence type (ST) 131 C2/H30Rx clade with the blaCTX-M-15 gene had been most responsible for the global dissemination of extended-spectrum ß-lactamase (ESBL)-producing E. coli. ST131 C1/H30R with blaCTX-M-27 emerged among ESBL-producing E. coli in Japan during the late 2000s. To investigate the possible expansion of a single clade, we performed whole-genome sequencing for 43 Japan and 10 global ST131 isolates with blaCTX-M-27 (n = 16), blaCTX-M-14 (n = 16), blaCTX-M-15 (n = 13), and others (n = 8). We also included 8 ST131 genomes available in public databases. Core genome-based analysis of 61 isolates showed that ST131 with blaCTX-M-27 from 5 countries formed a distinct cluster within the C1/H30R clade, named C1-M27 clade. Accessory genome analysis identified a unique prophage-like region, supporting C1-M27 as a distinct clade. Our findings indicate that the increase of ESBL-producing E. coli in Japan is due mainly to emergence of the C1-M27 clade.

Interspecies Dissemination of a Mobilizable Plasmid Harboring blaIMP-19 and the Possibility of Horizontal Gene Transfer in a Single Patient.

Carbapenemase-producing Gram-negative bacilli have been a global concern over the past 2 decades because these organisms can cause severe infections with high mortality rates. Carbapenemase genes are often carried by mobile genetic elements, and resistance plasmids can be transferred through conjugation. We conducted whole-genome sequencing (WGS) to demonstrate that the same plasmid harboring a metallo-ß-lactamase gene was detected in two different species isolated from a single patient. Metallo-ß-lactamase-producing Achromobacter xylosoxidans (KUN4507), non-metallo-ß-lactamase-producing Klebsiella pneumoniae (KUN4843), and metallo-ß-lactamase-producing K. pneumoniae (KUN5033) were sequentially isolated from a single patient and then analyzed in this study. Antimicrobial susceptibility testing, molecular typing (pulsed-field gel electrophoresis and multilocus sequence typing), and conjugation analyses were performed by conventional methods. Phylogenetic and molecular clock analysis of K. pneumoniae isolates were performed with WGS, and the nucleotide sequences of plasmids detected from these isolates were determined using WGS. Conventional molecular typing revealed that KUN4843 and KUN5033 were identical, whereas the phylogenetic tree analysis revealed a slight difference. These two isolates were separated from the most recent common ancestor 0.74 years before they were isolated. The same resistance plasmid harboring blaIMP-19 was detected in metallo-ß-lactamase-producing A. xylosoxidans and K. pneumoniae Although this plasmid was not self-transferable, the conjugation of this plasmid from A. xylosoxidans to non-metallo-ß-lactamase-producing K. pneumoniae was successfully performed. The susceptibility patterns for metallo-ß-lactamase-producing K. pneumoniae and the transconjugant were similar. These findings supported the possibility of the horizontal transfer of plasmid-borne blaIMP-19 from A. xylosoxidans to K. pneumoniae in a single patient.

Development of a Surface Plasmon Resonance-Based Immunosensor for Detection of 10 Major O-Antigens on Shiga Toxin-Producing Escherichia coli, with a Gel Displacement Technique To Remove Bound Bacteria.

A surface plasmon resonance-based immunosensor (SPR-immunosensor) was developed for the detection of Shiga toxin-producing Escherichia coli (STEC) belonging to the O-antigen groups O26, O91, O103, O111, O115, O121, O128, O145, O157, and O159. The polyclonal antibodies (PoAbs) generated against each of the STEC O-antigen types in rabbits were purified and were immobilized on the sensor chip at 0.5 mg/mL. The limit of detection for STEC O157 by the SPR-immunosensor was found to be 6.3 × 10(4) cells for 75 s. Each of the examined 10 O-antigens on the STECs was detected by the corresponding PoAb with almost no reaction to the other PoAbs. The detected STECs were sufficiently removed from the PoAbs using gelatin or agarose gel without deactivation of the PoAbs, enabling repeatable use of the sensor chip. The developed SPR-immunosensor can be applied for the detection of multiple STEC O-antigens. Furthermore, the new antigen removal technique using the gel displacement approach can be utilized with various immunosensors to improve the detection of pathogens in clinical and public health settings.

Bundled strategies against infection after liver transplantation: Lessons from multidrug-resistant Pseudomonas aeruginosa.

Infection is a life-threatening complication after liver transplantation (LT). A recent outbreak of multidrug-resistant Pseudomonas aeruginosa triggered changes in our infection control measures. This study investigated the usefulness of our bundled interventions against postoperative infection after LT. This before-and-after analysis enrolled 130 patients who underwent living donor or deceased donor LT between January 2011 and October 2014. We initiated 3 measures after January 2013: (1) we required LT candidates to be able to walk independently; (2) we increased the hand hygiene compliance rate and contact precautions; and (3) we introduced procalcitonin (PCT) measurement for a more precise determination of empirical antimicrobial treatment. We compared factors affecting the emergence of drug-resistant microorganisms, such as the duration of antimicrobial and carbapenem therapy and hospital stay, and outcomes such as bacteremia and death from infection between before (n = 77) and after (n = 53) the LT suspension period. The utility of PCT measurement was also evaluated. Patients' backgrounds were not significantly different before and after the protocol revision. Incidence of bacteremia (44% versus 25%; P = 0.02), detection rate of multiple bacteria (18% versus 4%; P = 0.01), and deaths from infections (12% versus 2%; P = 0.04) significantly decreased after the protocol revision. Duration of antibiotic (42.3 versus 25.1 days; P = 0.002) and carbapenem administration (15.1 versus 5.2 days; P < 0.001) and the length of postoperative hospital stay (85.4 versus 63.5 days; P = 0.048) also decreased after the protocol revision. PCT mean values were significantly higher in the bacteremia group (10.10 ng/mL), compared with the uneventful group (0.65 ng/mL; P = 0.002) and rejection group (2.30 ng/mL; P = 0.02). One-year overall survival after LT significantly increased in the latter period (71% versus 94%; P = 0.001). In conclusion, the bundled interventions were useful in preventing infections and lengthening overall survival after LT.

Epidemiology of invasive fungal infections after liver transplantation and the risk factors of late-onset invasive aspergillosis.

Invasive fungal infection (IFI) in liver transplant recipients is associated with poor outcomes. Targeted antifungal prophylaxis is recommended for high-risk populations; however, the epidemiology of IFI has changed, and the risk criteria remain unclear. In addition, the risk factors for late-onset invasive aspergillosis (IA) have not been fully characterized. We examined 279 recipients over 16 years of age to uncover their IFI epidemiology, clinical characteristics and outcomes. In addition, a case-control study was performed to identify the risk factors of late-onset IA. Of the 279 recipients, 96.1% underwent living donor liver transplantation. Antifungal prophylaxis was administered to 80.6% of the recipients. IFI occurred in 15 patients, among which 8 cases were early-onset (≤90 days after liver transplantation) and 7 cases were late-onset (>90 days after liver transplantation). Five of the late-onset cases were invasive pulmonary aspergillosis, and 2 were fungemia cases. The mortality rate of late-onset IA was 80.0%. According to a multivariate analysis, steroid use before liver transplantation, bloodstream infection within 90 days after liver transplantation and reoperation within 90 days after liver transplantation were significant risk factors for late-onset IA after liver transplantation. The prevalence of IFI was low in our population given that over 80% of liver recipients received antifungal prophylaxis. The prognosis of late-onset IA remains poor, and predictors associated with late-onset IA, such as steroid use before liver transplantation, bloodstream infection and reoperation after liver transplantation, may help clinicians to optimize prevention measures for these devastating infections.

Oral administration and younger age decrease plasma concentrations of voriconazole in pediatric patients.

Voriconazole is used for treating or preventing invasive aspergillosis and other invasive fungal infections. To minimize adverse reactions and to maximize treatment effects, therapeutic drug monitoring should be performed. However, it is challenging to optimize daily voriconazole dosing because limited data have been published so far on pediatric patients. We retrospectively analyzed voriconazole concentrations in patients aged 0-18 years. In addition, a literature review was conducted. In our study cohort, younger age and oral administration were significantly associated with lower plasma voriconazole concentrations (P < 0.01). An unfavorable outcome was associated with low concentrations of voriconazole (P = 0.01). Reports of voriconazole administration in pediatric patients show that higher doses are required in younger children and in patients receiving oral administration. Hence, the current data suggest that we should escalate both initial and maintenance doses of voriconazole in pediatric patients, particularly in patients of younger age receiving an oral administration of voriconazole.