The Association between Transformation Ability and Antimicrobial Resistant Potential in Haemophilus influenzae.

The prevalence of quinolone low-susceptible Haemophilus influenzae has increased in Japan. Low quinolone susceptibility is caused by point mutations in target genes; however, it can also be caused by horizontal gene transfer via natural transformation. In this study, we examined whether this horizontal gene transfer could be associated with resistance to not only quinolones but also other antimicrobial agents. Horizontal transfer ability was quantified using the experimental transfer assay method for low quinolone susceptibility. Further, the association between horizontal transfer ability and resistance to ß-lactams, the first-choice drugs for H. influenzae infection, was investigated. The transformation efficiency of 50 clinical isolates varied widely, ranging from 102 to 106 colony forming unit (CFU) of the colonies obtained by horizontal transfer assay. Efficiency was associated with ß-lactam resistance caused by ftsI mutations, indicating that strains with high horizontal transfer ability acquired quinolone low-susceptibility as well as ß-lactam resistance more easily. Strains with high transformation efficiency increased the transcript level of comA, suggesting that enhanced com operon was associated with a high DNA uptake ability. Overall, this study revealed that the transformation ability of H. influenzae was associated with multiple antimicrobial resistance. Increase in the number of strains with high horizontal transformation ability has raised concerns regarding the rapid spread of antimicrobial-resistant H. influenzae.

Brain-wide network analysis of resting-state neuromagnetic data.

The present study performed a brain-wide network analysis of resting-state magnetoencephalograms recorded from 53 healthy participants to visualize elaborate brain maps of phase- and amplitude-derived graph-theory metrics at different frequencies. To achieve this, we conducted a vertex-wise computation of threshold-independent graph metrics by combining proportional thresholding and a conjunction analysis and applied them to a correlation analysis of age and brain networks. Source power showed a frequency-dependent cortical distribution. Threshold-independent graph metrics derived from phase- and amplitude-based connectivity showed similar or different distributions depending on frequency. Vertex-wise age-brain correlation maps revealed that source power at the beta band and the amplitude-based degree at the alpha band changed with age in local regions. The present results indicate that a brain-wide analysis of neuromagnetic data has the potential to reveal neurophysiological network features in the human brain in a resting state.

Contribution of amino acid substitutions in ParE to quinolone resistance in Haemophilus haemolyticus revealed through a horizontal transfer assay using Haemophilus influenzae.

BACKGROUND: In 2019, a high-level quinolone-resistant Haemophilus haemolyticus strain (levofloxacin MIC = 16 mg/L) was isolated from a paediatric patient. In this study, we aimed to determine whether the quinolone resistance of H. haemolyticus could be transferred to Haemophilus influenzae and to identify the mechanism underlying the high-level quinolone resistance of H. haemolyticus. METHODS: A horizontal gene transfer assay to H. influenzae was performed using genomic DNA or PCR-amplified quinolone-targeting genes from the high-level quinolone-resistant H. haemolyticus 2019-19 strain. The amino acids responsible for conferring quinolone resistance were identified through site-directed mutagenesis. RESULTS: By adding the genomic DNA of H. haemolyticus 2019-19, resistant colonies were obtained on agar plates containing quinolones. Notably, H. influenzae grown on levofloxacin agar showed the same level of resistance as H. haemolyticus. Sequencing analysis showed that gyrA, parC and parE of H. influenzae were replaced by those of H. haemolyticus, suggesting that horizontal transfer occurred between the two strains. When the quinolone-targeting gene fragments were added sequentially, the addition of parE, as well as gyrA and parC, contributed to high-level resistance. In particular, amino acid substitutions at both the 439th and 502nd residues of ParE were associated with high-level resistance. CONCLUSIONS: These findings indicate that quinolone resistance can be transferred between species and that amino acid substitutions at the 439th and 502nd residues of ParE, in addition to amino acid substitutions in both GyrA and ParC, contribute to high-level quinolone resistance.

Japanese Spotted Fever Associated with Mild Encephalitis/Encephalopathy with a Reversible Splenial Lesion.

A 58-year-old woman had disseminated intravascular coagulation (DIC) and septic shock caused by Japanese spotted fever (JSF). Following treatment with minocycline, her general condition gradually improved; however, her disorientation persisted. Mild encephalitis/encephalopathy with a reversible splenial lesion (MERS) type II was diagnosed based on brain magnetic resonance imaging (MRI) showing a hyperintense area in the splenium of the corpus callosum and bilateral cerebral white matter on diffusion-weighted imaging. Thereafter, her consciousness gradually improved, but she continued to experience difficulty concentrating and attention deficits. MERS type II may take longer to improve than type I, and long-term follow-up is required.

Alternative quinolone-resistance pathway caused by simultaneous horizontal gene transfer in Haemophilus influenzae.

BACKGROUND: Quinolone-resistant bacteria are known to emerge via the accumulation of mutations in a stepwise manner. Recent studies reported the emergence of quinolone low-susceptible Haemophilus influenzae ST422 isolates harbouring two relevant mutations, although ST422 isolates harbouring one mutation were never identified. OBJECTIVES: To investigate if GyrA and ParC from quinolone low-susceptible isolates can be transferred horizontally and simultaneously to susceptible isolates. METHODS: Genomic DNA was extracted from an H. influenzae isolate harbouring amino acid substitutions in both gyrA and parC and mixed with clinical isolates. The emergence of resistant isolates was compared, and WGS analysis was performed. RESULTS: By adding the genomic DNA harbouring both mutated gyrA and parC, resistant bacteria exhibiting recombination at gyrA only or both gyrA and parC loci were obtained on nalidixic acid and pipemidic acid plates, and the frequency was found to increase with the amount of DNA. Recombination events in gyrA only and in both gyrA and parC occurred with at least 1 and 1-100 ng of DNA, respectively. The genome sequence of a representative strain showed recombination events throughout the genome. The MIC of quinolone for the resulting strains was found to be similar to that of the donor. Although the recombination efficacy was different among the various strains, all strains used in this study obtained multiple genes simultaneously. CONCLUSIONS: These findings indicate that H. influenzae can simultaneously obtain more than two mutated genes. This mechanism of horizontal transfer could be an alternative pathway for attaining quinolone resistance.

A case of Shewanella algae-induced bacteremia in Japan: Case report and literature review.

Shewanella algae (S. algae) is a rare bacterium that causes infectious diseases in humans. Herein, we present a case of an 84-year-old man with S. algae-induced bacteremia and performed a review of 12 cases identified via a literature search and this case. Literature review of previous reports in Japan have revealed that 69.2% of patients with S. algae-induced bacteremia had a history of contact with fresh fish. Appropriate interviews of patients, especially in the hot season, and the accurate identification of the causative bacterium, by using techniques such as MALDI-TOF-MS and genetic testing, are necessary if S. algae or other bacteria from the genus Shewanella are detected in blood-culture tests.

Identification and characterisation of a novel multidrug-resistant streptococcus, Streptococcus toyakuensis sp. nov., from a blood sample.

OBJECTIVES: In 2018, we isolated multidrug-resistant α-haemolytic streptococci TP1632 from the blood of a 34-year-old patient with bacteraemia. This study aimed to characterise α-haemolytic streptococci TP1632 and elucidate its multidrug resistance mechanisms. METHODS: TP1632 was characterised by whole genome sequencing and biochemical testing. Based on the genome sequence, digital DNA-DNA hybridization (dDDH) and average nucleotide identity based on BLAST (ANIb) values were calculated. In addition, antimicrobial-resistance mechanisms were evaluated by ResFinder and transformation assay using Streptococcus mitis. RESULTS: TP1632 showed resistance to ß-lactams, macrolides, and quinolones. Genomic analysis revealed that dDDH and ANIb values between TP1632 and S. mitis were 56.3% and 93.63%, respectively, indicating TP1632 as the novel species. TP1632 exhibited macrolide resistance genes such as mef(A), msr(D), and erm(B) and tetracycline resistance gene tet(M). In addition, amino acids at position 81 in GyrA and 79 in ParC were tyrosine and isoleucine, respectively. When penicillin-binding proteins of TP1632 were introduced into S. mitis, recombinants showed ß-lactam resistance. Thus, acquired resistance genes and low affinities towards antimicrobial agents contribute to multidrug resistance. CONCLUSION: Our findings indicated that multidrug-resistant TP1632 is a novel species in the genus Streptococcus. Furthermore, antimicrobial resistance of this strain could transfer horizontally among α-haemolytic streptococci. These data indicated the risk of emergence of multidrug-resistant streptococci not only as a pathogen but also a reservoir of antimicrobial resistance. This isolate was proposed as a novel species, Streptococcus toyakuensis sp. nov. The type strain is TP1632T (= JCM 34623T = CCUG 75492T).

Molecular characterisation of carbapenem- and tigecycline-resistant Klebsiella pneumoniae strains isolated from blood and bile samples.

INTRODUCTION: The number of carbapenem-resistant Klebsiella pneumoniae (CRKP) strains are increasing, further raising healthcare concerns worldwide. In this study, we isolated three CRKP strains from bile and blood samples of an elderly patient (90s) with acute cholangitis and characterised the features and antimicrobial resistance mechanism of CRKP isolates. METHODS: Three CRKP isolates were characterised by Pulsed-field gel electrophoresis (PFGE), whole genome sequencing using the NovaSeq 6000, and antimicrobial susceptibility testing. Transcriptional levels of resistance-associated genes were measured by real-time RT-qPCR. RESULTS: PFGE analysis revealed highly similar patterns for these isolates. Furthermore, they showed resistance to not only carbapenem but also tigecycline. Genomic analysis of the blood isolate identified the exogenous resistance genes blaCTX-M14, tet(A), tet(D), opxAB, and qnrS1 but not any carbapenemase-encoding genes. In addition, nonsense mutations were found in both the outer membrane protein K36 (ompK36) and transcriptional regulator ramR, suggesting that this isolate developed multidrug resistance by acquiring both exogenous resistance genes and nonsense mutations. The extended-spectrum ß-lactamase-producing carbapenem-susceptible K. pneumoniae isolate exhibited the same susceptibility pattern, except to ß-lactams, as prior CRKP isolates. CONCLUSIONS: Antimicrobial susceptibility to carbapenem and tigecycline should be continuously monitored, because it might change from susceptible to resistant during another antimicrobial treatment, even if an isolate initially shows susceptibility, and the patient has not been exposed to these agents.

Theobromine enhances the conversion of white adipocytes into beige adipocytes in a PPARγ activation-dependent manner.

The adipocytes play an important role in driving the obese-state-white adipose tissue (WAT) stores the excess energy as fat, wherein brown adipose tissue (BAT) is responsible for energy expenditure via the thermoregulatory function of uncoupling protein 1 (UCP1)-the imbalance between these two onsets obesity. Moreover, the anti-obesity effects of brown-like-adipocytes (beige) in WAT are well documented. Browning, the process of transformation of energy-storing into energy-dissipating adipocytes, is a potential preventive strategy against obesity and its related diseases. In the present study, to explore an alternative source of natural products in the regulation of adipocyte transformation, we assessed the potential of theobromine (TB), a bitter alkaloid of the cacao plant, inducing browning in mice (in vivo) and primary adipocytes (in vitro). Dietary supplementation of TB significantly increased skin temperature of the inguinal region in mice and induced the expression of UCP1 protein. It also increased the expression levels of mitochondrial marker proteins in subcutaneous adipose tissues but not in visceral adipose tissues. The microarray analysis showed that TB supplementation upregulated multiple thermogenic and beige adipocyte marker genes in subcutaneous adipose tissue. Furthermore, in mouse-derived primary adipocytes, TB upregulated the expression of the UCP1 protein and mitochondrial mass in a PPARγ ligand-dependent manner. It also increased the phosphorylation levels of PPARγ coactivator 1α without affecting its protein expression. These results indicate that dietary supplementation of TB induces browning in subcutaneous WAT and enhances PPARγ-induced UCP1 expression in vitro, suggesting its potential to treat obesity.

Mediastinal Lymph Node Metastasis of Esophageal Cancer with Esophageal Stenosis Diagnosed via Transesophageal Endoscopic Ultrasound with Bronchoscope-guided Fine-needle Aspiration.

An 80-year-old man underwent follow-up examinations after endoscopic submucosal dissection (ESD) for esophageal cancer. Computed tomography showed enlarged lymph nodes of the right recurrent nerve. The patient had esophageal stenosis due to repeated ESD for multiple esophageal tumors. The stenosis made the passage of an endoscopic ultrasound (EUS) scope through the esophagus difficult. Thus, an endobronchial ultrasound bronchoscope, which had a thinner diameter than that of the EUS scope, was used for transesophageal endoscopic ultrasound with bronchoscope-guided fine-needle aspiration. This technique led to the diagnosis of mediastinal lymph node metastasis of esophageal cancer.

Pancreatic Exocrine Insufficiency in Intraductal Papillary Mucinous Carcinoma Presenting with Leg Edema Treated with Pancreatic Exocrine Replacement Therapy.

An 89-year-old woman underwent examinations for leg edema. Blood tests indicated low nutrition and low pancreatic enzymes, and a stool examination indicated fatty stool. Computed tomography showed pleural effusion, ascites, and cystic lesions in the pancreatic head and mural nodules within the cysts. Pancreatic juice cytology revealed adenocarcinoma. The diagnosis was pancreatic exocrine insufficiency caused by intraductal papillary mucinous carcinoma. The patient did not wish to undergo surgery. Therefore, diuretics, component nutrients, and pancreatic exocrine replacement therapy using pancrelipase were initiated. After starting treatment, her leg edema, pleural effusion, and ascites disappeared, and her activities of daily living improved markedly.

High-Level Quinolone-Resistant Haemophilus haemolyticus in Pediatric Patient with No History of Quinolone Exposure.

The prevalence of antimicrobial resistance among Haemophilus spp. is a critical concern, but high-level quinolone-resistant strains had not been isolated from children. We isolated high-level quinolone-resistant H. haemolyticus from the suction sputum of a 9-year-old patient. The patient had received home medical care with mechanical ventilation for 2 years and had not been exposed to any quinolones for >3 years. The H. haemolyticus strain we isolated, 2019-19, shared biochemical features with H. influenzae. However, whole-genome analysis found this strain was closer to H. haemolyticus. Phylogenetic and mass spectrometry analyses indicated that strain 2019-19 was in the same cluster as H. haemolyticus. Comparison of quinolone resistance-determining regions showed strain 2019-19 possessed various amino acid substitutions, including those associated with quinolone resistance. This report highlights the existence of high-level quinolone-resistant Haemophilus species that have been isolated from both adults and children.

Quinolone Resistance Is Transferred Horizontally via Uptake Signal Sequence Recognition in Haemophilus influenzae.

The presence of Haemophilus influenzae strains with low susceptibility to quinolones has been reported worldwide. However, the emergence and dissemination mechanisms remain unclear. In this study, a total of 14 quinolone-low-susceptible H. influenzae isolates were investigated phylogenetically and in vitro resistance transfer assay in order to elucidate the emergence and dissemination mechanisms. The phylogenetic analysis based on gyrA sequences showed that strains with the same sequence type determined by multilocus sequence typing were classified into different clusters, suggesting that H. influenzae quinolone resistance emerges not only by point mutation, but also by the horizontal transfer of mutated gyrA. Moreover, the in vitro resistance transfer assay confirmed the horizontal transfer of quinolone resistance and indicated an active role of extracellular DNA in the resistance transfer. Interestingly, the horizontal transfer of parC only occurred in those cells that harbored a GyrA with amino acid substitutions, suggesting a possible mechanism of quinolone resistance in clinical settings. Moreover, the uptake signal and uptake-signal-like sequences located downstream of the quinolone resistant-determining regions of gyrA and parC, respectively, contributed to the horizontal transfer of resistance in H. influenzae. Our study demonstrates that the quinolone resistance of H. influenzae could emerge due to the horizontal transfer of gyrA and parC via recognition of an uptake signal sequence or uptake-signal-like sequence. Since the presence of quinolone-low-susceptible H. influenzae with amino acid substitutions in GyrA have been increasing in recent years, it is necessary to focus our attention to the acquisition of further drug resistance in these isolates.

Comparison of the bactericidal effects of quinolones against low-susceptible Haemophilus influenzae.

The increasing incidence of Haemophilus influenzae with decreased susceptibility to quinolones (quinolone low-susceptible H. influenzae) in Japan has raised concerns about therapeutic failure. Thus, assessment of effective antimicrobial agents is necessary to establish an effective therapeutic strategy against resulting infections. In this study, in vitro bactericidal effects of quinolones on low-susceptible H. influenzae strains were evaluated using time-kill curve analysis. For tosufloxacin, log reduction values for low-susceptible strains were significantly lower than those for susceptible strains at both Cmax and 1/2 Cmax. Conversely, although the log reduction values were lower for susceptible strains, the Cmax of levofloxacin and ß-lactams (amoxicillin and cefditoren) indicated bactericidal effects. In addition, higher concentrations of tosufloxacin at 2×Cmax and 4×Cmax had bactericidal effects on not only susceptible but also low-susceptible strains. These data strongly suggest that we should consider the presence of low-susceptible strains and reconsider the appropriate dosage of tosufloxacin for treatment, especially for paediatric patients.

Dissemination of quinolone low-susceptible Haemophilus influenzae ST422 in Tokyo, Japan.

INTRODUCTION: Haemophilus influenzae with a reduced susceptibility to quinolones (quinolone low-susceptible H. influenzae) has recently emerged in Japan. In addition, the regional outbreak of the quinolone low-susceptible H. influenzae ST422 clone has been reported. In this study, we isolated this clone from an acute care hospital located in a geographically different area from the previous outbreak and characterised the nature of this clone. METHODS: Eighty-nine H. influenzae isolated between 2017 and 2019 were tested. The antimicrobial susceptibility was determined by the broth dilution method. The genetic background was analysed by pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing. Growth ability and ß-lactamase acquisition were evaluated by growth curve analysis and conjugative transfer experiments, respectively. RESULTS: Quinolone low-susceptible isolates accounted for 4.2% (1/24) in 2018 and 13.9% (5/36) in 2019. Most of the quinolone low-susceptible strains (83.3%) were classified as ST422 and had amino acid substitutions in quinolone resistance-determining regions in both GyrA and ParC. The patients' backgrounds were highly diverse. In addition, these isolates showed the same PFGE pattern as outbreak strains. The growth of ST422 clone was relatively faster than other clones. Furthermore, ST422 clone was able to acquire ß-lactamase from a ß-lactamase positive strain by horizontal transfer, becoming highly resistant to ß-lactams. CONCLUSION: Our study indicated that the quinolone low-susceptible H. influenzae ST422 clone has been spreading in the community undetected. In addition, this clone has the potential to grow faster and become more resistant through exogenous gene transfer. Therefore, ST422 clone should be monitored attention throughout Japan.

ASAS-NANP SYMPOSIUM: prospects for interactive and dynamic graphics in the era of data-rich animal science1.

Statistical graphics, and data visualization, play an essential but under-utilized, role for data analysis in animal science, and also to visually illustrate the concepts, ideas, or outputs of research and in curricula. The recent rise in web technologies and ubiquitous availability of web browsers enables easier sharing of interactive and dynamic graphics. Interactivity and dynamic feedback enhance human-computer interaction and data exploration. Web applications such as decision support systems coupled with multimedia tools synergize with interactive and dynamic graphics. However, the importance of graphics for effectively communicating data, understanding data uncertainty, and the state of the field of interactive and dynamic graphics is underappreciated in animal science. To address this gap, we describe the current state of graphical methodology and technology that might be more broadly adopted. This includes an explanation of a conceptual framework for effective graphics construction. The ideas and technology are illustrated using publicly available animal datasets. We foresee that many new types of big and complex data being generated in precision livestock farming create exciting opportunities for applying interactive and dynamic graphics to improve data analysis and make data-supported decisions.

Brain damage caused by neonatal hypoxia-ischemia and the effects of hypothermia in severe combined immunodeficient (SCID) mice.

Neonatal hypoxic-ischemic encephalopathy (HIE) is a major cause of brain damage in newborns. Although therapeutic hypothermia has been shown to be neuroprotective against neonatal HIE in clinical trials, its effect is not satisfactory. Cell-based therapies have attracted much attention as novel treatments for HIE. Preclinical studies on a variety of human cell transplantation methods have been performed in immunodeficient/immunosuppressed animals, such as severe combined immunodeficient (SCID) mice, which lack functional T and B lymphocytes. The detailed characteristics of neonatal HIE in SCID mice, however, have not been delineated. In preclinical studies, novel therapies for neonatal HIE should be evaluated in combination with hypothermia, which has become a standard treatment for neonatal HIE. However, the effects of hypothermia in SCID mice have not been delineated. In the present study, we compared neonatal hypoxic-ischemic (HI) brain damage in SCID mice and wild-type mice treated with or without hypothermia. Male and female mouse pups were subjected to HI insult induced by unilateral common carotid artery ligation combined with systemic hypoxia on postnatal day 12. In the first 4 h after HI insult, body temperature was maintained at 36 °C for the normothermia groups or 32 °C for the hypothermia groups. The severity of brain damage in SCID mice did not differ from that in wild-type mice based on most evaluations, i.e., cerebral blood flow, hemiparesis, muscle strength, spontaneous activity, cerebral hemispheric volume, neuropathological injury, and serum cytokine levels, although spleen weight, brain weight, leukocyte counts and the levels of some cytokines in the peripheral blood were different between genotypes. The effects of hypothermia in SCID mice were comparable to those in wild-type mice based on most evaluations. Taken together, these findings indicate that SCID mice can be used as an appropriate preclinical model for cell therapies for neonatal HIE.

Metabolomic analysis and mass spectrometry imaging after neonatal stroke and cell therapies in mouse brains.

Ischemic brain injury provokes complex, time-dependent downstream pathways that ultimately lead to cell death. We aimed to demonstrate the levels of a wide range of metabolites in brain lysates and their on-tissue distribution following neonatal stroke and cell therapies. Postnatal day 12 mice underwent middle cerebral artery occlusion (MCAO) and were administered 1 × 105 cells after 48 h. Metabolomic analysis of the injured hemisphere demonstrated that a variety of amino acids were significantly increased and that tricarboxylic acid cycle intermediates and some related amino acids, such as glutamate, were decreased. With the exception of the changes in citric acid, neither mesenchymal stem/stromal cells nor CD34+ cells ameliorated these changes. On-tissue visualization with matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS) imaging revealed that the signal intensity of glutamate was significantly decreased in the infarct area, consistent with the metabolomic analysis, while its intensity was significantly increased in the peri-infarct area after MCAO. Although cell therapies did not ameliorate the changes in metabolites in the infarct area, mesenchymal stem cells ameliorated the increased levels of glutamate and carnitine in the peri-infarct area. MALDI-MS imaging showed the location-specific effect of cell therapies even in this subacute setting after MCAO. These methodologies may be useful for further investigation of possible treatments for ischemic brain injury.

Whole-genome sequence of Haemophilus influenzae ST422 outbreak clone strain 2018-Y40 with low quinolone susceptibility isolated from a paediatric patient.

OBJECTIVES: In recent years, Haemophilus influenzae strains with reduced susceptibility to quinolones have emerged and spread in Japan. In addition, an outbreak of isolates with low quinolone susceptibility among paediatric patients has also been reported. The aim of this study was to determine the molecular characteristics of an H. influenzae ST422 outbreak clone with low quinolone susceptibility isolated from a paediatric patient using whole-genome sequencing. METHODS: The PacBio RS II platform was used for sequencing, and de novo assembly was performed using RS HGAP assembly version 3.0. The assembled sequences were annotated using DFAST version 1.1.15. Prophages were estimated using the PHASTER program. RESULTS: Whole-genome sequencing of H. influenzae ST422 isolate 2018-Y40 revealed that the genome size was 1 957 393bp, comprising 1 926 protein-coding sequences, 19 rRNAs, and 57 tRNAs, with a guanine-cytosine (GC) content of 38.2%. This isolate had no relevant exogenous antimicrobial-resistant genes. However, amino acid substitutions were found in both GyrA and ParC, as well as at the 385th and 526th amino acid residues in penicillin-binding protein 3. In addition, four intact prophage regions and one incomplete prophage region were found. CONCLUSIONS: The whole-genome sequence of H. influenzae 2018-Y40 indicated that this clone emerged as a result of extensive genomic rearrangement by integration of multiple phages. As genomic rearrangement occasionally leads to a new phenotype, this clone could have acquired antimicrobial resistance and diversification via rearrangement events. These findings can form a basis to help clarify the mechanisms of low quinolone susceptibility and the spread of this outbreak clone.