Importin-7-dependent nuclear translocation of the Flavivirus core protein is required for infectious virus production.

Flaviviridae is a family of positive-stranded RNA viruses, including human pathogens, such as Japanese encephalitis virus (JEV), dengue virus (DENV), Zika virus (ZIKV), and West Nile virus (WNV). Nuclear localization of the viral core protein is conserved among Flaviviridae, and this feature may be targeted for developing broad-ranging anti-flavivirus drugs. However, the mechanism of core protein translocation to the nucleus and the importance of nuclear translocation in the viral life cycle remain unknown. We aimed to identify the molecular mechanism underlying core protein nuclear translocation. We identified importin-7 (IPO7), an importin-ß family protein, as a nuclear carrier for Flaviviridae core proteins. Nuclear import assays revealed that core protein was transported into the nucleus via IPO7, whereas IPO7 deletion by CRISPR/Cas9 impaired their nuclear translocation. To understand the importance of core protein nuclear translocation, we evaluated the production of infectious virus or single-round-infectious-particles in wild-type or IPO7-deficient cells; both processes were significantly impaired in IPO7-deficient cells, whereas intracellular infectious virus levels were equivalent in wild-type and IPO7-deficient cells. These results suggest that IPO7-mediated nuclear translocation of core proteins is involved in the release of infectious virus particles of flaviviruses.

Molecular epidemiology of multidrug-resistant Acinetobacter baumannii isolates from a hospital in Nepal.

OBJECTIVES: The emergence of multidrug-resistant (MDR) Acinetobacter baumannii has become a serious worldwide medical problem. This study was designed to clarify the genetic and epidemiological properties of MDR A. baumannii clinical isolates. METHODS: A total of 66 MDR A. baumannii isolates were obtained from 66 inpatients between May 2019 and February 2020 in a university hospital in Nepal. Whole genomes of these isolates were sequenced using next-generation sequencing. Phylogenetic trees were constructed from single nucleotide polymorphism concatemers. Multilocus sequence typing (MLST) and clonal complex (CC) analysis were conducted, and drug-resistance genes were identified. RESULTS: Of the 66 isolates, 26 harboured a gene encoding NDM-type metallo-ß-lactamase, and 55 harboured a gene encoding the 16S rRNA methyltransferase, ArmA. All isolates had point mutations in the quinolone-resistance-determining regions of gyrA and parC. Phylogenetic analysis showed that 55 isolates harboured armA, 26 harboured blaNDM-1, and14 harboured blaPER-7. Multilocus sequence typing and CC analysis revealed that 34 isolates belonged to CC2 (ST2), 10 to CC1 (nine ST1 and one ST623), and eight to CC149 (ST149). Compared to our previous study on MDR A. baumannii in Nepal in 2012, the isolation rate of CC2 increased, whereas that of CC149 decreased between 2012 and 2020. CONCLUSIONS: This study indicates that MDR A. baumannii producing carbapenemase and 16S rRNA methyltransferase, with high resistance to carbapenems and/or aminoglycosides, are spreading in medical settings in Nepal. The genetic backgrounds of MDR A. baumannii isolates have shifted to international clone 2 over several years.

Multidrug-resistant Klebsiella pneumoniae clinical isolates producing NDM- and OXA-type carbapenemase in Nepal.

OBJECTIVES: The emergence of multidrug-resistant Klebsiella pneumoniae has become a serious problem in medical settings worldwide. METHODS: A total of 46 isolates of multidrug-resistant K. pneumoniae were obtained from 2 hospitals in Nepal from October 2018 to April 2019. RESULTS: Most of these isolates were highly resistant to carbapenems, aminoglycosides, and fluoroquinolones with the minimum inhibitory concentrations (MICs) of more than 64 µg/mL. These isolates harboured carbapenemase-encoding genes, including blaNDM-1, blaNDM-5, blaOXA-181 and blaOXA-232, and 16S rRNA methyltransferase-encoding genes, including armA, rmtB, rmtC, and rmtF. Multilocus sequence typing revealed that 44 of 46 isolates were high-risk clones such as ST11 (2%), ST14 (4%), ST15 (11%), ST37 (2%), ST101 (2%), ST147 (28%), ST231 (13%), ST340 (4%), and ST395 (28%). In particular, ST395 isolates, which spread across medical settings in Nepal, co-harboured blaNDM-5 and rmtB on IncFII plasmids and co-harboured blaOXA-181/-232 and rmtF on ColKP3 plasmids. Several isolates harboured blaOXA-181 or blaNDM-5 on their chromosomes and multi-copies of blaNDM-1 or genes encoding 16S rRNA methyltransferases on their plasmids. CONCLUSIONS: The presented study demonstrates that the high-risk clones of multidrug-resistant K. pneumoniae spread in a clonal manner across hospitals in Nepal.

Evaluation of antimicrobial susceptibility tests for Acinetobacter and Pseudomonas species using disks containing a high dose of meropenem.

The emergence and dissemination of carbapenem-resistant species of Acinetobacter and Pseudomonas have become a serious health concern. Routine antimicrobial disk susceptibility tests in clinical laboratories cannot distinguish between isolates that are highly carbapenem-resistant and those that are moderately carbapenem-resistant. The present study describes antimicrobial susceptibility tests using disks containing high doses (1000 µg) of meropenem. The diameters of inhibition zones were significantly negatively correlated with the MICs of Pseudomonas and Acinetobacter species for meropenem (R2: 0.93 and 0.91, respectively) and imipenem (R2: 0.75 and 0.84, respectively). Double disk synergy tests using clavulanic acid or sodium mercaptoacetate can detect ESBL or MBL producers. Susceptibility tests using disks containing high doses of meropenem can easily detect highly carbapenem-resistant isolates in a quantitative manner. These disks may be useful in bacteriological laboratories because of their technical ease, stability, and relatively low cost.

Klebsiella pneumoniae co-harbouring bla NDM-1 , armA and mcr-10 isolated from blood samples in Myanmar.

Background. The spread of Enterobacteriaceae coproducing carbapenemases, 16S rRNA methylase and mobile colistin resistance proteins (MCRs) has become a serious public health problem worldwide. This study describes two clinical isolates of Klebsiella pneumoniae coharbouring bla IMP-1, armA and mcr-10.Methods. Two clinical isolates of K. pneumoniae resistant to carbapenems and aminoglycosides were obtained from two patients at a hospital in Myanmar. Their minimum inhibitory concentrations (MICs) were determined by broth microdilution methods. The whole-genome sequences were determined by MiSeq and MinION methods. Drug-resistant factors and their genomic environments were determined.Results. The two K. pneumoniae isolates showed MICs of ≥4 and ≥1024 µg ml-1 for carbapenems and aminoglycosides, respectively. Two K. pneumonaie harbouring mcr-10 were susceptible to colistin, with MICs of ≤0.015 µg ml-1 using cation-adjusted Mueller-Hinton broth, but those for colistin were significantly higher (0.5 and 4 µg ml-1) using brain heart infusion medium. Whole-genome analysis revealed that these isolates coharboured bla NDM-1, armA and mcr-10. These two isolates showed low MICs of 0.25 µg ml-1 for colistin. Genome analysis revealed that both bla NDM-1 and armA were located on IncFIIs plasmids of similar size (81 kb). The mcr-10 was located on IncM2 plasmids of sizes 220 or 313 kb in each isolate. These two isolates did not possess a qseBC gene encoding a two-component system, which is thought to regulate the expression of mcr genes.Conclusion. This is the first report of isolates of K. pneumoniae coharbouring bla NDM-1, armA and mcr-10 obtained in Myanmar.

Emergence of colistin-resistant Acinetobacter modestus harbouring the intrinsic phosphoethanolamine transferase EptA.

OBJECTIVES: Colistin-resistant Gram-negative pathogens have become a serious worldwide medical problem. This study was designed to reveal the effects of an intrinsic phosphoethanolamine transferase from Acinetobacter modestus on Enterobacterales. METHODS: A strain of colistin-resistant A. modestus was isolated from a sample of nasal secretions taken in 2019 from a hospitalised pet cat in Japan. The whole genome was sequenced by next generation sequencing, and transformants of Escherichia coli, Klebsiella pneumoniae, and Enterobacter cloacae harbouring the phosphoethanolamine transferase-encoding gene from A. modestus were constructed. Lipid A modification in E. coli transformants was analysed using electrospray ionization mass spectrometry. RESULTS: Sequencing of the entire genome revealed that the isolate harboured a phosphoethanolamine transferase-encoding gene, eptA_AM, on its chromosome. Transformants of E. coli, K. pneumoniae, and E. cloacae harbouring both the promoter and eptA_AM gene from A. modestus had 32-fold, 8-fold, and 4-fold higher minimum inhibitory concentrations (MICs) for colistin, respectively, than transformants harbouring a control vector. The genetic environment surrounding eptA_AM in A. modestus was similar to that surrounding eptA_AM in Acinetobacter junii and Acinetobacter venetianus. Electrospray ionization mass spectrometry analysis revealed that EptA_AM modified lipid A in Enterobacterales. CONCLUSION: This is the first report to describe the isolation of an A. modestus strain in Japan and show that its intrinsic phosphoethanolamine transferase, EptA_AM, contributes to colistin resistance in Enterobacterales and A. modestus.

Emergence of carbapenem-resistant Pseudomonas alcaligenes and Pseudomonas paralcaligenes clinical isolates with plasmids harbouring bla IMP-1 in Japan.

Introduction. The emergence of carbapenem-resistant Pseudomonas species producing metallo-ß-lactamase (MBL) has become a serious medical problem worldwide. IMP-type MBL was firstly detected in 1991 in Japan. Since then, it has become one of the most prevalent types of MBLs.Hypothesis/Gap statement. Avirulent species of Pseudomonas, such as Pseudomonas alcaligenes, function as reservoirs of drug resistance-associated genes encoding carbapenemases in clinical settings.Methodology. Active surveillance for carbapenem-resistant Gram-negative pathogens was conducted in 2019 at a hospital in Tokyo, Japan. Of the 543 samples screened for carbapenem-resistant isolates, 2 were species of Pseudomonas. One was from a stool sample from a medical staff member, and the other was from a stool sample from a hospitalized patient.Results. Whole-genome sequencing showed that the former isolate was a strain of P. alcaligenes, and the latter was a strain of Pseudomonas paralcaligenes, a species close to P. alcaligenes. Both isolates were resistant to all carbapenems and harboured bla IMP-1 genes encoding IMP-1 MBL, which conferred resistance to carbapenems. The bla IMP-1 genes of P. alcaligenes and P. paralcaligenes were located on the plasmids, pMRCP2, 323125 bp in size, and pMRCP1333, 16592 bp in size, respectively. The sequence of 82 % of pMRCP2 was 92 % similar to the sequence of a plasmid of P. aeruginosa PA83, whereas the sequence of 79 % of pMRCP1333 was >95 % similar to the sequence of a plasmid of Achromobacter xylosoxidans FDAARGOS 162. The genomic environments surrounding the bla IMP-1 of pMRCP2 and pMRCP1333 differed completely from each other.Conclusions. These results indicate that the two isolates acquired bla IMP-1 from different sources and that P. alcaligenes and P. paralcaligenes function as vectors and reservoirs of carbapenem-resistant genes in hospitals.

CID12261165, a flavonoid compound as antibacterial agents against quinolone-resistant Staphylococcus aureus.

Flavonoids are plant-produced secondary metabolites that are found ubiquitously. We have previously reported that apigenin, a class of flavonoid, has unique antimicrobial activity against Staphylococcus aureus (S. aureus), one of the major human pathogens. Apigenin inhibited fluoroquinolone-resistant S. aureus with DNA gyrase harboring the quinolone-resistant S84L mutation but did not inhibit wild-type DNA gyrase. In this study, we describe five flavonoids, quercetin, luteolin, kaempferol, baicalein, and commercially available CID12261165, that show similar antimicrobial activity against fluoroquinolone-resistant S. aureus. Among them, CID12261165 was the most effective with MIC values of ≤ 4 mg/L against quinolone-resistant S. aureus strains. In vitro DNA cleavage and supercoiling assays demonstrated inhibitory activity of CID12261165 against mutated DNA gyrase, whereas activity against wild-type DNA gyrase was not observed. CID12261165 also inhibited quinolone-resistant Enterococci with an MIC value of 8 mg/L. While fluoroquinolone-resistant amino acid replacements can improve the fitness of bacterial cells, it is unknown why quinolone-susceptible S. aureus strains were predominant before the introduction of fluoroquinolone. The present study discusses the current discrepancies in the interpretation of antimicrobial activities of flavonoids, as well as the possible reasons for the preservation of wild-type DNA gyrase wherein the environmental flavonoids cannot be ignored.

Plasmids Harboring a Tandem Duplicate of blaVIM-24 in Carbapenem-Resistant ST1816 Pseudomonas aeruginosa in Japan.

The aim of this study was to clarify the biological and clinical significance of a tandem duplicate of blaVIM-24 in Pseudomonas aeruginosa ST1816 isolates. Thirteen ST1816 isolates carrying a plasmid harboring blaVIMs were obtained from two medical settings in Japan between 2016 and 2019. Complete sequencing revealed that, of the 13 plasmids, four had a tandem duplicate of blaVIM-24. These four plasmids harbored a replicon, a relaxase gene, and T4SS genes belonging to IncP-9, MOBF, and MPFT, respectively. All four plasmids transferred to PAO1 by filter mating. Cefepime marginally affected the growth of PAO1, carrying a pUCP19 harboring the tandem duplicate. Western blotting analysis showed that the relative intensity of VIM-24 metallo-ß-lactamase produced by a PAO1 transformant containing a tandem duplicate was 2.6-fold higher than that produced by a PAO1 transformant containing a single copy. These results suggest that the tandem duplicate of blaVIM-24 in plasmids may confer resistance against cefepime, enabling P. aeruginosa ST1816 strains to proliferate in hospitals in Japan.

Highly Colistin-resistant Aeromonas jandaei from a Human Blood Sample.

Aeromonas species are Gram-negative rods known to cause infections such as gastroenteritis, bacteremia and wound infections. Colistin is one of few treatments for multidrug-resistant Gram-negative bacteria. However, colistin-resistant bacteria carrying the mobilized colistin resistance (mcr) gene are a threat in healthcare settings worldwide. In recent years, colistin-resistant Aeromonas species have been detected in environmental and clinical samples. We analyzed the genomic characteristics of one highly colistin-resistant A. jandaei isolated from a blood sample in Nepal, which harbored four novel mcr-like genes on its chromosome. Our study strongly suggests that A. jandaei is a reservoir of colistin-resistant genes. Inappropriate use of drugs in medicine and food production should be reduced and continued global surveillance for colistin-resistant bacteria is necessary.

Modified Drug-Susceptibility Testing and Screening Culture Agar for Colistin-Susceptible Enterobacteriaceae Isolates Harboring a Mobilized Colistin Resistance Gene mcr-9.

Three isolates of the Enterobacter cloacae complex harboring mcr-9, a member of the colistin resistance mcr gene family encoded on plasmids, were susceptible to colistin, with MICs of 0.125 to 0.5 µg/mL in standard broth microdilution (BMD) tests using cation-adjusted Mueller-Hinton broth (CA-MHB) in accordance with European Committee on Antimicrobial Susceptibility Testing guidelines. In contrast, their MICs for colistin were significantly higher (4 to 128 µg/mL) when BMD tests were performed using brain-heart infusion (BHI) medium, Luria-Bertani (LB) broth, tryptic soy broth (TSB), or CA-MHB supplemented with casein, tryptonen or peptone. Colistin significantly induced mcr-9 expression in a dose-dependent manner when these mcr-9-positive isolates were cultured in BHI or CA-MHB supplemented with peptone/casein. Pretreatment of mcr-9-positive isolates and Escherichia coli DH5α harboring mcr-9 with colistin significantly increased their survival rates against LL-37, a human antimicrobial peptide. Electrospray ionization time-of-flight mass spectrometry analysis showed that a lipid A moiety of lipopolysaccharide was partially modified by phosphoethanolamine in E. coli DH5α harboring mcr-9 when treated with colistin. Of 93 clinical isolates of Enterobacteriaceae, only the mcr-9-positive isolates showed MICs to colistin that were at least 32 times higher in BHI than in CA-MHB. These mcr-9-positive isolates grew on a modified BHI agar, MCR9-JU, containing 3 µg/mL colistin. These results suggest that the BMD method using BHI is useful when performed together with the BMD method using CA-MHB to detect mcr-9-positive isolates and that MCR9-JU agar is useful in screening for Enterobacteriaceae isolates harboring mcr-9 and other colistin-resistant isolates.

Stenotrophomonas maltophilia from Nepal Producing Two Novel Antibiotic Inactivating Enzymes, a Class A ß-Lactamase KBL-1 and an Aminoglycoside 6'-N-Acetyltransferase AAC(6')-Iap.

Seven drug-resistant strains of Stenotrophomonas maltophilia were isolated from patients at two university hospitals in Nepal. S. maltophilia JUNP497 was found to encode a novel class A ß-lactamase, KBL-1 (Kathmandu ß-lactamase), consisting of 286 amino acids with 52.98% identity to PSV-1. Escherichia coli transformants expressing blaKBL-1 were less susceptible to penicillins. The recombinant KBL-1 protein efficiently hydrolyzed penicillins. The genomic environment surrounding blaKBL-1 was a unique structure, with the upstream region derived from strains in China and the downstream region from strains in India. S. maltophilia JUNP350 was found to encode a novel 6'-N-aminoglycoside acetyltransferase, AAC(6')-Iap, consisting of 155 amino acids with 85.0% identity to AAC(6')-Iz. E. coli transformants expressing aac(6')-Iap were less susceptible to arbekacin, amikacin, dibekacin, isepamicin, neomycin, netilmicin, sisomicin and tobramycin. The recombinant AAC(6')-Iap protein acetylated all aminoglycosides tested, except for apramycin and paromomycin. The genomic environment surrounding aac(6')-Iap was 90.99% identical to that of S. maltophilia JV3 obtained from a rhizosphere in Brazil. Phylogenetic analysis based on whole genome sequences showed that most S. maltophilia isolates in Nepal were similar to those isolates in European countries, including Germany and Spain. IMPORTANCE The emergence of drug-resistant S. maltophilia has become a serious problem in medical settings worldwide. The present study demonstrated that drug-resistant S. maltophilia strains in Nepal harbored novel genes encoding a class A ß-lactamase, KBL-1, or a 6'-N-aminoglycoside acetyltransferase, AAC(6')-Iap. Genetic backgrounds of most S. maltophilia strains in Nepal were similar to those in European countries. Surveillance of drug-resistant S. maltophilia in medical settings in Nepal is necessary.

Whole-Genome Sequencing-Based Re-Identification of Pseudomonas putida/fluorescens Clinical Isolates Identified by Biochemical Bacterial Identification Systems.

The genus Pseudomonas, a complex Gram-negative genus, includes species isolated from various environments, plants, animals, and humans. We compared whole-genome sequencing (WGS) with clinical bacteriological methods and evaluated matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) to identify Pseudomonas species. Clinical isolates (N = 42) identified as P. putida or P. fluorescens by a bacterial identification system based on biochemical properties were reexamined by another identification system based on biochemical properties, two systems based on MALDI-TOF MS, and WGS. WGS revealed that 30 of the 42 isolates belonged to one of 14 known Pseudomonas species, respectively. The remaining 12 belonged to one of 9 proposed novel Pseudomonas species, respectively. MALDI-TOF MS analysis showed that the 9 novel species had unique major peaks. These results suggest that WGS is the optimal method to identify Pseudomonas species and that MALDI-TOF MS may complement WGS in identification. Based on their morphologic, physiologic, and biochemical properties, we propose nine novel Pseudomonas species. IMPORTANCE Most of the clinical isolates, identified as P. putida or P. fluorescens, were misidentified in clinical laboratories. Whole-genome sequencing (WGS) revealed that these isolates belonged to different Pseudomonas species, including novel species. WGS is a gold-standard method to identify Pseudomonas species, and MALDI-TOF MS analysis has the potential to complement WGS to reliably identify them.

Emergence of a highly colistin-resistant Aeromonas jandaei clinical isolate harbouring four genes encoding phosphoethanolamine transferases in Nepal.

OBJECTIVES: This study aimed to describe a clinical isolate of Aeromonas jandaei (A. jandaei) in Nepal that harboured four types of genes encoding phosphoethanolamine transferases. METHODS: An isolate of colistin-resistant A. jandaei was obtained from a blood sample of an inpatient in a hospital in Nepal, and its complete genome sequence was determined. Escherichia coli (E. coli) and Aeromonas hydrophila (A. hydrophila) transformants expressing genes encoding novel phosphoethanolamine transferase variants were constructed and colistin-susceptibility profiles were determined. RESULTS: The isolate harboured four genes encoding phosphoethanolamine transferases on the chromosome, which were designated eptAv3.2, eptAv3.3, eptAv3.4 and eptAv7.2. The amino acid sequences of EptAv3.2, 3.3 and 3.4 were > 80% identical to MCR-3.1, and that of EptAv7.2 was > 79% identical to MCR-7.1. E. coli expressing eptAv3.2, 3.3 and 3.4 showed reduced susceptibility to colistin, whereas E. coli expressing eptAv7.2 did not. In contrast, A. hydrophila expressing eptAv7.2 showed reduced susceptibility to colistin, whereas A. hydrophila expressing eptAv3.2, 3.3 and 3.4 did not; eptAv3.3 and 3.4 formed a tandem structure. The genomic environments surrounding eptAv3.2, 3.3 and 3.4 were similar to Aeromonas veronii obtained from the effluent of a treatment plant in Japan in 2018. The genomic environment surrounding eptAv7.2 was similar to that of A. jandaei obtained from a chicken in the USA in 2019. CONCLUSIONS: The highly colistin-resistant A. jandaei clinical isolate harboured four chromosomal genes encoding phosphoethanolamine transferases, suggesting that Aeromonas spp. harbouring eptAv genes with strong similarities to mcr-3 and mcr-7 are emerging in medical settings as well as environments.

Emergence of Carbapenem-resistant Clinical Isolates of Providencia Species.

Providencia is a genus of Gram-negative and non-spore forming bacteria belonging to the family Morganellaceae, which causes opportunistic infections in humans. Of the 10 Providencia species identified to date, three, P. alcalifaciens, P. rettgeri and P. stuartii, are clinically important. P. alcalifaciens causes diarrhea, including outbreaks arising from food-borne infections, and P. stuartii and P. rettgeri have been found to cause hospital acquired urinary tract infections. Four isolates of P. rettgeri and one isolate of P. stuartii were obtained from urine samples of five patients in Japan in 2018. All five isolates were highly resistant to carbapenems. Three isolates harbored bla IMP-70, encoding a variant of IMP-1 metallo-ß-lactamase, with two amino acid substitutions (Val67Phe and Phe87Val), one isolate harbored two copies of bla IMP-1 and one isolate harbored bla IMP-11. Expression of bla IMP-70 conferred carbapenem resistance in Escherichia coli. Recombinant IMP-10, an IMP-1 variant with Val67Phe but without Phe87Val, had significant higher hydrolytic activities against meropenem than recombinant IMP-1, indicating that the Val67Phe amino acid substitution alters activities against meropenem in IMP-70. These results suggest that Providencia species. become more highly resistant to carbapenems by acquisition of two copies of bla IMP-1 or by mutations in bla IMP that result in amino acid substitutions, such as bla IMP-70.

Three novel species of the Bacillus cereus group isolated from clinical samples in Japan.

Three Gram-positive bacterial strains, BML-BC004, BML-BC017 and BML-BC059, isolated from blood samples from three inpatients in Japan, were identified as members of Bacillus cereus using matrix-assisted laser desorption ionization time-of-flight MS. The 16S rRNA gene sequences of these three strains were more than 97.1 % similar to 18 type strains belonging to the B. cereus group. Whole-genome comparisons, using average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH), confirmed that the three strains represented three individual distinct species belonging to the B. cereus group. A phylogenetic tree showed that BML-BC004, BML-BC017 and BML-BC059 were located close to B. luti, B. mobilis and B. paramycoides, respectively. Based on these phylogenetic and phenotypic data, including values below the threshold for ANI and dDDH, the three strains should be classified as representing three different novel species of the B. cereus group: Bacillus sanguinis sp. nov., with type strain BML-BC004T (=DSM 111102T=JCM 34122T), Bacillus paramobilis sp. nov., with type strain BML-BC017T (=DSM 111100T=JCM 34124T) and Bacillus hominis sp. nov., with type strain BML-BC059T (=DSM 111101T=JCM 34125T).

Molecular characterisation of carbapenem-resistant Pseudomonas aeruginosa clinical isolates in Nepal.

OBJECTIVES: The emergence of carbapenem-resistant Pseudomonas aeruginosa has become a serious worldwide medical problem. The aim of this study was to determine the genetic and epidemiological properties of carbapenem-resistant P. aeruginosa strains isolated from hospitals in Nepal. METHODS: A total of 43 carbapenem-resistant P. aeruginosa isolates obtained from patients in two hospitals in Nepal between 2018 and 2020 were analysed. Their whole genomes were sequenced by next-generation sequencing. A phylogenetic tree was constructed from single nucleotide polymorphism (SNP) concatemers. Multilocus sequence typing (MLST) was performed and antimicrobial resistance genes were identified. RESULTS: Of the 43 isolates, 17 harboured genes encoding carbapenemases, including IMP-1, IMP-26, KPC-2, NDM-1, VIM-2 and VIM-5, and 12 harboured genes encoding 16S rRNA methylases, including RmtB4 and RmtF2. The carbapenem-resistant P. aeruginosa isolated in Nepal belonged to various sequence types (STs), including ST235 (5 isolates), ST244 (7 isolates), ST274 (1 isolate), ST357 (10 isolates), ST654 (3 isolates), ST664 (1 isolate), ST773 (1 isolate), ST823 (3 isolates), ST1047 (8 isolates), ST1203 (2 isolates) and ST3453 (2 isolates). CONCLUSION: To the best of our knowledge, this is the first molecular epidemiological analysis of carbapenem-resistant P. aeruginosa clinical isolates from Nepal. The findings strongly suggest that P. aeruginosa isolates producing carbapenemases and 16S rRNA methylases have spread throughout medical settings in Nepal.

Development of an immunochromatographic kit to detect severe acute respiratory syndrome coronavirus 2.

BACKGROUND: The novel severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is responsible for the worldwide coronavirus disease-19 (COVID-19) pandemic, starting in late 2019. The standard diagnostic methods to detect SARS-CoV-2 are PCR-based genetic assays. Antigen-antibody-based immunochromatographic assays are alternative methods of detecting this virus. Rapid diagnosis kits to detect SARS-CoV-2 are urgently needed. STUDY DESIGN: Three monoclonal antibodies against SARS-CoV-2 nucleocapsid (N) protein were used to develop an antigen-antibody-based immunochromatographic kit to detect SARS-CoV-2. These assays were evaluated using　 nasopharyngeal swab specimens collected from patients suspected of having COVID-19. RESULTS: These assays detected recombinant SARS-CoV-2 N protein at concentrations >0.2 ng/mL within 10 min after protein loading, but did not detect the N proteins of Middle East respiratory syndrome coronavirus (MERS-CoV), human coronaviruses OC43 (HCoV-OC43) and 299E (HCoV-229E) and other pathogens causing respiratory infections. Nasopharyngeal swab specimens obtained 1～3, 4～9, and ≥ 10 days after symptom onset from COVID-19 patients diagnosed by RT-PCR showed positivity rates of 100 %, >80 %, and <30 %, respectively. CONCLUSIONS: Kits using this immunochromatographic assay may be a rapid and useful tool for point-of-care diagnosis of COVID-19 when samples are obtained from patients 1～9 days after symptom onset.

Emergence of clinical isolates of Pseudomonas asiatica and Pseudomonas monteilii from Japan harbouring an acquired gene encoding a carbapenemase VIM-2.

Pseudomonas asiatica and Pseudomonas monteilii, belonging to the Pseudomonas putida phylogenetic group, are occasionally isolated from clinical samples, partly because they are often misidentified as P. putida in clinical laboratories. There are five reports describing carbapenem-resistant clinical isolates of these species. Carbapenem-resistant strains of P. asiatica and P. monteilii were isolated from stool samples. These isolates were sequenced using Illumina MiSeq and reidentified using average nucleotide identity (ANI) based on comparisons of their whole-genome sequences using the OrthoANI algorithm. The clonal relatedness of the isolates was assessed by pulse-field gel electrophoresis (PFGE). The size of plasmids conveying bla VIM-2 was examined by Southern blotting. A total of six carbapenem-resistant clinical isolates of P. asiatica (two isolates) and P. monteilii (four isolates) were obtained from stool samples from five patients in a Japanese hospital. All isolates harboured blaVIM-2. The two isolates of P. asiatica had a different pattern in the PFGE analysis, with both having a 23 kb plasmid. Of the four isolates of P. monteilii with similar patterns in the PFGE analysis, three had 320 kb plasmids and one had a 240 kb plasmid. The genetic environments of the 320/240 kb and 23 kb plasmids differed. The results strongly indicated that carbapenem-resistant P. asiatica and P. monteilii producing metallo-ß-lactamase are emerging in Japan. This is the first report of carbapenem-resistant P. asiatica and P. monteilii in Japan.