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Abstract The spread of vancomycin-resistant enterococci (VRE) is a major threat in nosocomial settings. A large-scale multiclonal VRE outbreak has rarely been reported in Japan due to low VRE prevalence. We evaluated the transmission of vancomycin resistance in a multiclonal VRE outbreak, conducted biological and genomic analyses of VRE isolates, and assessed the implemented infection control measures. In total, 149 patients harboring VanA-type VRE were identified from April 2017 to October 2019, with 153 vancomycin-resistant Enterococcus faecium isolated being grouped into 31 pulsotypes using pulsed-field gel electrophoresis, wherein six sequence types belonged to clonal complex 17. Epidemic clones varied throughout the outbreak; however, they all carried vanA-plasmids (pIHVA). pIHVA is a linear plasmid, carrying a unique structural Tn1546 containing vanA; it moves between different Enterococcus spp. by genetic rearrangements. VRE infection incidence among patients in the “hot spot” ward correlated with the local VRE colonization prevalence. Local prevalence also correlated with vancomycin usage in the ward. Transmission of a novel transferrable vanA-plasmid among Enterococcus spp. resulted in genomic diversity in VRE in a non-endemic setting. The prevalence of VRE colonization and vancomycin usage at the ward level may serve as VRE cross-transmission indicators in non-intensive care units for outbreak control.

Worldwide spread of Enterobacteriaceae resistant to colistin, a polypeptide antibacterial drug for last-resort treatment of carbapenemase-producing Enterobacteriaceae (CPE) infections, is concerning. This study aimed to elucidate colistin MICs and molecular characteristics of mcr-1 to mcr-9 of ESBL-producing Escherichia coli (ESBL-Ec) and CPE in Japan and clarify the genomic structure of strains harboring mcr genes (especially mcr-9). This study included 168 ESBL-Ec and 126 CPE strains isolated at Japanese medical facilities. Colistin susceptibility testing and multiplex PCR targeting mcr-1 to mcr-9 were performed for all strains with S1-nuclease pulsed-field gel electrophoresis, Southern blot hybridization, and whole-genome sequencing (WGS) with hybrid assembly performed for mcr gene-carrying strains. Two CPE strains showed a MIC ≥ 4 μg/ml in colistin susceptibility testing, with no known resistance mechanism detected. However, PCR conducted on all target strains detected three mcr-9-carrying strains showing colistin susceptibility. The blaCTX–M–62-positive E. coli THUN648 strain simultaneously carried blaCTX–M–62 and mcr-9 on a 275-kbp plasmid. Besides, blaIMP–6 + blaCTX–M–2-positive Klebsiella pneumoniae THUN262 and blaGES–24-positive Enterobacter kobei THUN627 had mcr-9 encoded on the chromosome. Only THUN627 encoded qseB/C, which is suggested to be a regulatory gene for mcr-9, downstream of mcr-9. However, this strain showed no increased expression of these genes in mRNA quantitative analysis under colistin exposure. Colistin MICs of ESBL-Ec and CPE in Japan were all below 2 μg/ml, which is below the epidemiological cutoff (ECOFF) value (https://eucast.org/) or clinical breakpoint (CB) (CLSI M100-S30) reported for colistin, indicating neither “microbiological” nor “clinical” resistance. Several colistin-susceptible Enterobacteriaceae carrying silent mcr-9 encoded on plasmids and chromosomes have already spread worldwide along with other antimicrobial resistance genes. However, the mechanism of colistin resistance by mcr-9 remains unclear.

Colistin is used as an alternative therapeutic for carbapenemase-producing Enterobacteriaceae (CPE) infections which are spreading at a very high rate due to the transfer of carbapenemase genes through mobile genetic elements. Due to the emergence of mcr-1, the plasmid-mediated colistin resistance gene, mcr-1-positive Enterobacteriaceae (MCRPEn) pose a high risk for the transfer of mcr-1-carrying plasmid to CPE, leading to a situation with no treatment alternatives for infections caused by Enterobacteriaceae possessing both mcr-1 and carbapenemase genes. Here, we report the application of PCR-dipstick-oriented surveillance strategy to control MCRPEn and CPE by conducting the PCR-dipstick technique for the detection of MCRPEn and CPE in a tertiary care hospital in Thailand and comparing its efficacy with conventional surveillance method. Our surveillance results showed a high MCRPEn (5.9%) and CPE (8.7%) carriage rate among the 219 rectal swab specimens examined. Three different CPE clones were determined by pulsed-field gel electrophoresis (PFGE) whereas only two MCRPEn isolates were found to be closely related as shown by single nucleotide polymorphism-based phylogenetic analysis. Whole genome sequencing (WGS) and plasmid analysis showed that MCRPEn carried mcr-1 in two plasmids types—IncX4 and IncI2 with ~99% identity to the previously reported mcr-1-carrying plasmids. The identification of both MCRPEn and CPE in the same specimen indicates the plausibility of plasmid-mediated transfer of mcr-1 genes leading to the emergence of colistin- and carbapenem-resistant Enterobacteriaceae. The rapidity (

The global dissemination of carbapenemase-producing Enterobacteriaceae (CPE) is a major concern in public health. Due to the existence of the diversity of carbapenemases, development of an easily available, cost-effective multiplex detection assay for CPE is required worldwide. Using clinically available and reliable equipment, COBAS® z480 (Roche Diagnostics K.K., Tokyo, Japan), we developed a multiplex real-time PCR assay for the detection of two combinations of carbapenemases; first, blaNDM, blaKPC, and blaIMP (Set 1), and second, blaGES, blaOXA-48, and blaVIM (Set 2). We constructed standard curves for each carbapenemase gene using serial dilutions of DNA standards, then applied reference or clinical isolates with each carbapenemase gene to this assay. The multiplex assay showed satisfactory accuracy to detect CPE genes, with the correlation coefficients of greater than 0.99 for all genotypes. The assay appropriately differentiated the reference or clinical strains harboring each carbapenemase gene without cross reactivity. Lastly, the assay successfully detected multiple genes without false-positive reactions by applying six clinical isolates carrying both NDM and OXA-48-like carbapenemase genes. Major advantages of our assay include multiplicity, simple operation, robustness, and speed (1 h). We believe that the multiplex assay potentially contributes to early diagnosis of CPE with a diverse genetic background.

Roseomonas species is rarely found to be pathogenic to humans and there are few clinical cases that have been described in the literature. We report a case of Roseomonas mucosa bacteremia that involved a 9-year-old Japanese boy who was in a condition of febrile neutropenia caused by chemotherapy for cerebellar medulloblastoma. Conventional phenotyping failed to identify the organism; however, genetic analysis using 16S rDNA sequencing confirmed the pathogen to be R. mucosa. The patient recovered following treatment by meropenem without any complications. A literature review of pediatric cases of Roseomonas bacteremia identified 12 other documented cases, and these revealed that a common clinical situation for the infection is an immunocompromised state with malignancy and/or an indwelling intravenous catheter. Because of the low number of cases, the overall picture of Roseomonas bacteremia in children remains to be elucidated; however, the prognosis of the infection appears to be satisfactory. Keywords: Bloodstream infection, Febrile neutropenia, Roseomonas mucosa

The bacterial enzyme New Delhi metallo-β-lactamase hydrolyzes almost all β-lactam antibiotics, including carbapenems, which are drugs of last resort for severe bacterial infections. The spread of carbapenem-resistant Enterobacteriaceae that carry the New Delhi metallo-β-lactamase gene, blaNDM, poses a serious threat to public health. In this study, we genetically characterized eight carbapenem-resistant Escherichia coli isolates from a tertiary care hospital in Yangon, Myanmar. The eight isolates belonged to five multilocus-sequence types and harbored multiple antimicrobial-resistance genes, resulting in resistance against nearly all of the antimicrobial agents tested, except colistin and fosfomycin. Nine plasmids harboring blaNDM genes were identified from these isolates. Multiple blaNDM genes were found in the distinct Inc-replicon types of the following plasmids: an IncA/C2 plasmid harboring blaNDM-1 (n = 1), IncX3 plasmids harboring blaNDM-4 (n = 2) or blaNDM-7 (n = 1), IncFII plasmids harboring blaNDM-4 (n = 1) or blaNDM-5 (n = 3), and a multireplicon F plasmid harboring blaNDM-5 (n = 1). Comparative analysis highlighted the diversity of the blaNDM-harboring plasmids and their distinct characteristics, which depended on plasmid replicon types. The results indicate circulation of phylogenetically distinct strains of carbapenem-resistant E. coli with various plasmids harboring blaNDM genes in the hospital.

ABSTRACT Avian haemosporidian parasites and avian pox virus (APV) are well‐known pathogens for their impact on avian populations, especially in oceanic islands where introduced pathogens show strong virulence for endemic and naïve birds. The Bonin Islands are a group of oceanic islands 1000 km south of Tokyo. Like the Hawaiian Islands, there are many endemic and endangered species as well as introduced species, which have greatly affected the native avian fauna. However, pathogens in wild birds of this archipelago had not been investigated. In this study, we investigated the prevalence of avian haemosporidian parasites and APV among birds and mosquitoes in this unique ecosystem of the Bonin Islands. From 2014 to 2020, 524 birds of 39 species either rescued, deceased, or caught by mist‐netting were sampled. APV‐like lesions were sampled from nine birds. 262 mosquitoes were collected by sweeping nets or CDC traps. All samples were tested via PCR for haemosporidian infection, and lesions were tested for APV.209 birds (39.9%) of 11 species were positive for haemosporidian parasite DNA, and all three parasite genera were detected. Prevalence was particularly high for Plasmodium elongatum (pGRW06) and Prelictum relictum (pGRW04). The former was detected from both resident birds and mosquitoes, suggesting local transmission. An introduced species, the warbling white‐eye (Zosterops japonicus), had a particularly high prevalence of pGRW06 (68.3%) and may be a reservoir of this lineage. Both APV and Plasmodium spp. were detected from all APV‐tested birds, suggesting that these two pathogens may be transmitted simultaneously via mosquitoes. The presence of avian haemosporidian parasites and APV was confirmed in the Bonin Islands for the first time. However, the virulence and origin of these pathogens remain unknown, and many bird species are still understudied. Further investigations are required to contribute to the conservation of this unique avifauna.

In Japan, the native population of the Oriental White Stork Ciconia boyciana Swinhoe, 1873 had been extinct in 1986, when the last captured stork died in a breeding facility. Later on, some wild or bred storks from Russia and mainland China (clear origin unknown) were transported to Japan, and their descendants were repeatedly released into natural fields to establish wild populations in this country. In this study, the feather mite Pelargolichus orientalis Waki, Mironov, and Shimano, 2023 has been collected from a taxidermy specimen of the Oriental White Stork belonging to a Japanese native population and preserved in the Himeji City Science Museum, Japan. Pelargolichus orientalis is the only feather mite species detected and described from the introduced storks bred in a Japanese zoo in our previous study. The present study shows that P. orientalis did occur in the Japanese native host population.

Background: Identification of carbapenemase-producing Enterobacteriaceae (CPE) in faecal specimens is challenging. This fact is particularly critical because low-level carbapenem-resistant organisms such as IMP-producing CPE are most prevalent in Japan. We developed a modified selective medium more suitable for IMP-type CPE.Methods: Fifteen reference CPE strains producing different types of β-lactamases were used to evaluate the commercially available CHROMagar KPC and chromID CARBA as well as the newly prepared MC-ECC medium (CHROMagar ECC supplemented with meropenem, cloxacillin, and ZnSO4) and M-ECC medium (CHROMagar ECC supplemented with meropenem and ZnSO4). A total of 1035 clinical samples were then examined to detect CPE using chromID CARBA and M-ECC medium.Results: All tested strains producing NDM-, KPC-, and OXA-48-carbapenemases were successfully cultured in the media employed. Although most of the IMP-positive strains did not grow in CHROMagar KPC, chromID CARBA, or MC-ECC, all tested strains grew on M-ECC. When faecal samples were applied to the media, M-ECC medium allowed the best growth of IMP-type CPE with a significantly higher sensitivity (99.3%) than that of chromID CARBA (13.9%).Conclusions: M-ECC medium was determined as the most favourable selective medium for the detection of IMP-type CPE as well as other types of CPE. [ABSTRACT FROM AUTHOR]

Surveillance is very important to prevent the nosocomial spread of methicillin-resistant Staphylococcus aureus (MRSA), and infection sources and routes have historically been identified using molecular and epidemiological genotyping with pulsed-field gel electrophoresis. However, phage-open reading frame typing (POT) has recently been developed. Here, we investigated whether POT would be useful to survey MRSA outbreaks and transmission. We therefore applied POT to 91 MRSA isolates detected in cultures from inpatients at our hospital between May and October 2014. Among the 91 isolates, 12 POT types comprising 38 isolated MRSA strains were considered as overlapping. Five of them were detected in different wards, whereas the remaining seven were found in the same ward, including the emergency department. Three of seven POT number 93-155-111 strains were detected in the surgical ward, and all of four POT number 93-157-61 strains were detected in the cardiosurgical ward. These data suggested that transmission of the MRSA strains with the same POT-types from the same wards was nosocomial, and that POT accurately and rapidly identified MRSA strains, which allowed effective control of infection and transmission. [ABSTRACT FROM AUTHOR]

Background: The epidemiology of Clostridium difficile is not well known in Japan because C. difficile cultures are not routinely performed.Methods: We analyzed the toxigenicity and the polymerase chain reaction (PCR) ribotype of C. difficile strains isolated from 2 geographically separate hospitals in Japan.Results: The results of PCR ribotyping of toxigenic C. difficile strains isolated from these 2 hospitals indicated that a single type (type smz) was predominant at hospitals located in diverse areas of Japan. Toxin A-negative, toxin B-positive (A-B+) variant C. difficile comprised 8 (18.6%) of 43 and 11 (16.4%) of 67 toxigenic C. difficile isolates from 2 hospitals, respectively. Of the 19 A-B+ strains, 18 (94.7%) showed the same PCR ribotype pattern (type z), which was identical to that of previously reported outbreak strains. Only 1 A+B+ strain possessed the binary toxin genes.Conclusion: PCR ribotype smz, which has been reported as a responsible ribotype for multiple outbreaks at Japanese hospitals in the late 1990s, is still the major epidemic ribotype in Japan. PCR ribotype z strains of A-B+ variant C. difficile also have disseminated within Japan and represent a substantial proportion of C. difficile isolates. [ABSTRACT FROM AUTHOR]

We report a case of severe Chlamydophila (Chlamydia) psittaci pneumonia rapidly diagnosed by detection of antigen in sputum with an immunochromatography assay. The patient was admitted to our hospital because of shock, disturbance of consciousness, accidental hypothermia, and multiple organ dysfunction syndrome, and he recovered after administration of intravenous erythromycin and high-dose methylpredonisolone therapy. Psittacosis was confirmed by detection of 16S rRNA gene of C.psittasi in sputum with multiplex-polymerase chain reaction analysis. Serological responses to C. psittasi, C. trachomatis, and C. pneumoniae were also evaluated, and serological cross-reactivity was observed between each species. We consider that the commercially available immunochromatography assay for Chlamydia species can be helpful for rapid diagnosis of Chlamydia infection of the respiratory tract. Hereafter, further examination will be necessary regarding pretreatment of specimens or detection sensitivity and specificity. [ABSTRACT FROM AUTHOR]

Owing to the continual depletion of resources, low-grade nickel laterite ores are being treated nowadays to extract Ni. High-pressure acid leaching (HPAL) is a suitable process for treating nickel laterite ores. HPAL has the advantage of having a high extraction rate and low chemical consumption associated with the neutralization of iron; the low chemical consumption is owing to the formation of hematite precipitate in the residue. By studying the behavior of iron under pressure acid leaching, it was observed that sulfur and carbon contained in the ore decreased the redox potential and lead to Fe (II) dissolution. Iron concentration in the leached solution was decreased by oxygen rich leaching.

We report the first case of pseudomembranous colitis (PMC) due to a toxin A-negative, toxin B-positive strain (toxin A variant strain) of Clostridium difficile in Japan. The toxin A variant strain of C. difficile is capable of causing PMC and is undetectable in clinical laboratories that use only toxin A immunoassays for C. difficile testing. If C. difficile-associated diarrhea is clinically suspected and toxin A is not detected, then the possibility of a toxin A variant strain should be considered, and further diagnostic testing, such as polymerase chain reaction (PCR) analysis for the detection of C. difficile toxin genes should be performed. [ABSTRACT FROM AUTHOR]

A key characteristic of MHC genes is the persistence of allelic lineages over macroevolutionary periods, often through multiple speciation events. This phenomenon, known as trans-species polymorphism (TSP), is well documented in several major taxonomic groups, but has less frequently been observed in birds. The order Passeriformes is arguably the most successful terrestrial vertebrate order in terms of diversity of species and ecological range, but the reasons for this success remain unclear. Passerines exhibit the most highly duplicated MHC genes of any major vertebrate taxonomic group, which may generate increased immune response relative to other avian orders with fewer MHC loci. Here, we describe phylogenetic patterns of the MHC IIB in the passerine family Corvidae. Our results indicate wide-spread TSP within this family, with at least four supported MHC IIB allelic lineages that predate speciation by many millions of years. Markov chain Monte Carlo simulations indicate that divergence of these lineages occurred near the time of the divergence of the Passeriformes and other avian orders. We suggest that the current MHC diversity observed in passerines is due in part to the multiple duplication of a single MHC locus, DAB1, early in passerine evolution and that subsequent duplications of these paralogues have contributed to the enormous success of this order by increasing their ability to recognize and mount immune responses to novel pathogens.

A distinguishing characteristic of genes that code for the major histocompatibility complex (MHC) is that alleles often share more similarity between, rather than within species. There are two likely mechanisms that can explain this pattern: convergent evolution and trans-species polymorphism (TSP), in which ancient allelic lineages are maintained by balancing selection and retained by descendant species. Distinguishing between these two mechanisms has major implications in how we view adaptation of immune genes. In this study we analyzed exon 2 of the MHC class IIB in three passerine bird species in the genus Corvus: jungle crows (Corvus macrorhynchos japonensis) American crows (C. brachyrhynchos) and carrion crows (C. corone orientalis). Carrion crows and American crows are recently diverged, but allopatric, sister species, whereas carrion crows and jungle crows are more distantly related but sympatric species, and possibly share pathogens linked to MHC IIB polymorphisms. These patterns of evolutionary divergence and current geographic ranges enabled us to test for trans-species polymorphism and convergent evolution of the MHC IIB in crows. Phylogenetic reconstructions of MHC IIB sequences revealed several well supported interspecific clusters containing all three species, and there was no biased clustering of variants among the sympatric carrion crows and jungle crows. The topologies of phylogenetic trees constructed from putatively selected sites were remarkably different than those constructed from putatively neutral sites. In addition, trees constructed using non-synonymous substitutions from a continuous fragment of exon 2 had more, and generally more inclusive, supported interspecific MHC IIB variant clusters than those constructed from the same fragment using synonymous substitutions. These phylogenetic patterns suggest that recombination, especially gene conversion, has partially erased the signal of allelic ancestry in these species. While clustering of positively selected amino acids by supertyping revealed a single supertype shared by only jungle and carrion crows, a pattern consistent with convergence, the overall phylogenetic patterns we observed suggest that TSP, rather than convergence, explains the interspecific allelic similarity of MHC IIB genes in these species of crows.

The pulse wave transit time (PWTT) is easily measured as the time from the R wave of an electrocardiogram to the arrival of the pulse wave measured by an oxygen saturation monitor at the earlobe. We investigated whether the change of PWTT during exercise testing reflects cardiopulmonary function. Eighty-nine cardiac patients who underwent cardiopulmonary exercise testing (CPX) were enrolled. We analyzed the change of PWTT during exercise and the relationship between the shortening of the PWTT and CPX parameters. PWTT was significantly shortened from rest to peak exercise (204.6 ± 33.6 vs. 145.6 ± 26.4 msec, p < 0.001) in all of the subjects. The patients with heart failure had significantly higher PWTT at peak exercise than the patients without heart failure (152.7 ± 27.1 vs. 140.4 ± 24.8 msec, p = 0.031). The shortening of PWTT from rest to peak exercise showed significant positive correlations with the peak O2 uptake (VO2) (r = 0.56, p < 0.001), anaerobic threshold (r = 0.40, p = 0.016), and % increase of systolic blood pressure during exercise (r = 0.75, p < 0.001), and a negative correlation with the slope of the increase in ventilation versus the increase in CO2 output (VE-VCO2 slope) (r = - 0.42, p = 0.010) in the patients with heart failure. PWTT was shortened during exercise as the exercise intensity increased. In the patients with heart failure, the shortening of PWTT from rest to peak exercise was smaller in those with lower exercise capacity and those with higher VE-VCO2 slope, an established index known to reflect the severity of heart failure. [ABSTRACT FROM AUTHOR]

Ventilation (VE) increases linearly with the increase of carbon dioxide output (VCO2) during cardiopulmonary exercise testing. VE-VCO2 slope rises in parallel with exercise intensity, reaches a turning point (called the RC point), then steepens because of respiratory compensation for lactic acidosis. While this RC point can be identified universally, it is undetectable in some patients. In this study we evaluated whether the respiratory compensation during exercise testing has clinical significance in cardiac patients. In total, 152 cardiac patients with a respiratory exchange ratio at peak exercise (peak R) of between 1.10 and 1.20 were enrolled. Cardiopulmonary parameters were compared between patients who manifested the RC point (n = 118) and those who did not (n = 34). The peak R did not significantly differ between these two groups. Compared to the patients without the RC point, those with the RC point had a higher oxygen uptake at peak exercise (peak VO2) (20.2 ± 5.3 vs 13.6 ± 3.4 mL/min/kg, p < 0.001), higher anaerobic threshold (AT) (12.4 ± 3.2 vs 9.2 ± 2.3 mL/min/kg, p < 0.001), and lower VE-VCO2 slope (31.7 ± 5.8 vs 37.8 ± 9.6, p = 0.001). Brain natriuretic peptide (BNP) tended to be lower in the patients with the RC point (175.4 ± 364.7 vs 327.9 ± 381.1 pg/mL, p = 0.067). Peak VO2, the marker of cardiopulmonary function, was found to be the independent predictor of the presence of the RC point. The present findings suggest that the phenomenon of respiratory compensation during heavy exercise indicates better cardiopulmonary function in cardiac patients within a prescribed range of effort. [ABSTRACT FROM AUTHOR]