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.

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.

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).

RNA Sequencing Identifies a Common Physiology in Vancomycin- and Ciprofloxacin-Tolerant Staphylococcus aureus Induced by ileS Mutations.

Little is known about the mechanisms by which ileS mutations induce vancomycin tolerance in Staphylococcus aureus This study showed that transcriptome profiles were similar in vancomycin-tolerant mutants and the IleRS-inhibitor-treated parent. Notably, ileS and relA, which induce a stringent response, were upregulated. The same mechanism was responsible for cross-tolerance to vancomycin and ciprofloxacin. These findings suggest that the accumulation of uncharged isoleucyl-tRNA following ileS mutations in S. aureus was responsible for drug tolerance.

Emergence of Carbapenem-Resistant Providencia rettgeri and Providencia stuartii Producing IMP-Type Metallo-ß-Lactamase in Japan.

Four Providencia rettgeri isolates and one Providencia stuartii isolate were obtained from urine samples of five patients in 2018 in Japan. All of the isolates were resistant to imipenem and meropenem, and three were highly resistant to both carbapenems, with MICs of 512 µg/ml. The three highly carbapenem-resistant isolates harbored blaIMP-70, encoding a variant of IMP-1 metallo-ß-lactamase with two amino acid substitutions (Val67Phe and Phe87Val), and the other two harbored blaIMP-1 and blaIMP-11, respectively. Whole-genome sequencing revealed that an isolate harbored two copies of blaIMP-1 on the chromosome and that the other four harbored a copy of blaIMP-11 or blaIMP-70 in a plasmid. Expression of blaIMP-70 conferred carbapenem resistance in Escherichia coli Recombinant IMP-70 and an IMP-1 variant with Val67Phe but without Phe87Val had significant higher hydrolytic activities against meropenem than recombinant IMP-1, indicating that an amino acid substitution of Val67Phe affects increased activities against meropenem in IMP-70. These results suggest that Providencia spp. become more highly resistant to carbapenems by acquisition of two copies of blaIMP-1 or by mutation of blaIMP genes with amino acid substitutions, such as blaIMP-70.

Pseudomonas izuensis sp. nov., a novel species isolated from Izu Oshima, Japan.

An aerobic, Gram-stain-negative, rod-shaped bacterial strain, IzPS43_3003T, was isolated from Izu Oshima, an active volcanic island located 22 km east of the Izu Peninsula, Japan. The sequence of its 16S rRNA gene indicated that IzPS43_3003T belongs to the Pseudomonas fluorescens lineage, with its sequence being most similar to that of Pseudomonas vancouverensis DhA-51T (99.79 %). Phylogenetic analysis based on whole genome sequences showed that IzPS43_3003T was a member of the Pseudomonas jessenii subgroup. The average nucleotide identity values and genome-to genome distances between the whole genome sequences of IzPS43_3003T and other type strains showed that the highest correlations were with Pseudomonas moorei DSM 12647T (87.3 and 33.5% respectively). These genotypic and phenotypic analyses indicated that IzPS43_3003T belongs to a novel species, Pseudomonas izuensis sp. nov. Its type strain is IzPS43_3003T (=LMG 31527T,=CECT 9963T).

A Novel VIM-Type Metallo-ß-Lactamase Variant, VIM-60, with Increased Hydrolyzing Activity against Fourth-Generation Cephalosporins in Pseudomonas aeruginosa Clinical Isolates in Japan.

A novel VIM-type metallo-ß-lactamase variant, VIM-60, was identified in multidrug-resistant Pseudomonas aeruginosa clinical isolates in Japan. Compared with VIM-2, VIM-60 had two amino acid substitutions (Arg228Leu and His252Arg) and higher catalytic activities against fourth-generation cephalosporins. The genetic context for blaVIM-60 was intI1-blaVIM-60-aadA1-aacA31-qacEdeltaI-sulI on the chromosome.

Identification of a Novel Gene Associated with High-Level ß-Lactam Resistance in Heterogeneous Vancomycin-Intermediate Staphylococcus aureus Strain Mu3 and Methicillin-Resistant S. aureus Strain N315.

ß-Lactam resistance levels vary among methicillin-resistant Staphylococcus aureus (MRSA) clinical isolates, mediated by chromosomal mutations and exogenous resistance gene mecA However, MRSA resistance mechanisms are incompletely understood. A P440L mutation in the RNA polymerase ß' subunit (RpoC) in slow-vancomycin-intermediate S. aureus (sVISA) strain V6-5 is associated with conversion of heterogeneous VISA (hVISA) to sVISA. In this study, we found a V6-5-derivative strain (L4) with significantly decreased MICs to oxacillin (OX) and vancomycin. Whole-genome sequencing revealed that L4 has nonsense mutations in two genes, relQ, encoding (p)ppGpp synthetase, an alarmone of the stringent response, and a gene of unknown function. relQ deletion in the hVISA strain Mu3 did not affect OX MIC. However, introducing nonsense mutation of the unknown gene into Mu3 decreased OX MIC, whereas wild-type gene recovered high-level resistance. Thus, mutation of this unknown gene (ehoM) decreased ß-lactam resistance in Mu3 and L4. Presence of relQ in a multicopy plasmid restored high-level resistance in strain L4 but not in the ehoM mutant Mu3 strain, indicating a genetic interaction between ehoM and relQ depending on the L4 genetic background. While mupirocin (a stringent response inducer) can increase the ß-lactam resistance of MRSA, mupirocin supplementation in an ehoM deletion mutant of N315 did not elevate resistance. ehoM expression in N315 was induced by mupirocin, and the relative amount of ehoM transcript in Mu3 was higher than in N315 induced by the stringent response. Our findings indicate that ehoM plays an essential role in high-level ß-lactam resistance in MRSA via the stringent response.

Genetic and Transcriptomic Analyses of Ciprofloxacin-Tolerant Staphylococcus aureus Isolated by the Replica Plating Tolerance Isolation System (REPTIS).

We developed a simple, efficient, and cost-effective method, named the replica plating tolerance isolation system (REPTIS), to detect the antibiotic tolerance potential of a bacterial strain. This method can also be used to quantify the antibiotic-tolerant subpopulation in a susceptible population. Using REPTIS, we isolated ciprofloxacin (CPFX)-tolerant mutants (mutants R2, R3, R5, and R6) carrying a total of 12 mutations in 12 different genes from methicillin-sensitive Staphylococcus aureus (MSSA) strain FDA209P. Each mutant carried multiple mutations, while few strains shared the same mutation. The R2 strain carried a nonsense mutation in the stress-mediating gene, relA Additionally, two strains carried the same point mutation in the leuS gene, encoding leucyl-tRNA synthetase. Furthermore, RNA sequencing of the R strains showed a common upregulation of relA Overall, transcriptome analysis showed downregulation of genes related to translation; carbohydrate, fat, and energy metabolism; nucleotide synthesis; and upregulation of amino acid biosynthesis and transportation genes in R2, R3, and R6, similar to the findings observed for the FDA209P strain treated with mupirocin (MUP0.03). However, R5 showed a unique transcription pattern that differed from that of MUP0.03. REPTIS is a unique and convenient method for quantifying the level of tolerance of a clinical isolate. Genomic and transcriptomic analyses of R strains demonstrated that CPFX tolerance in these S. aureus mutants occurs via at least two distinct mechanisms, one of which is similar to that which occurs with mupirocin treatment.

Molecular Characterization of Multidrug-Resistant Pseudomonas aeruginosa Isolates in Hospitals in Myanmar.

The emergence of multidrug-resistant (MDR) Pseudomonas aeruginosa has become a serious worldwide medical problem. This study was designed to clarify the genetic and epidemiological properties of MDR P. aeruginosa strains isolated from hospitals in Myanmar. Forty-five MDR P. aeruginosa isolates obtained from different patients in seven hospitals in Myanmar were screened using the broth microdilution method. The whole genomes of the MDR isolates were sequenced using a MiSeq platform (Illumina). Phylogenetic trees were constructed from single nucleotide polymorphism concatemers. Multilocus sequence types were deduced, and drug resistance genes were identified. Of the 45 isolates, 38 harbored genes encoding carbapenemases, including DIM-1, IMP-1, NDM-1, VIM-2, and VIM-5, and 9 isolates had genes encoding 16S rRNA methylases, including RmtB, RmtD3, RmtE, and RmtF2. Most MDR P. aeruginosa strains isolated in Myanmar belonged to sequence type 1047 (ST1047). This is the first molecular epidemiological analysis of MDR P. aeruginosa clinical isolates in Myanmar. These findings strongly suggest that P. aeruginosa ST1047 strains harboring carbapenemases, including DIM-, IMP-, NDM-, and VIM-type metallo-ß-lactamases, have been spreading throughout medical settings in Myanmar.

Assessment of a newly developed immunochromatographic assay for NDM-type metallo-ß-lactamase producing Gram-negative pathogens in Myanmar.

BACKGROUND: To detect carbapenemase-producing Gram-negative bacteria in bacterial laboratories at medical settings, a new immunochromatographic assay for New Delhi metallo-ß-lactamases (NDMs) was developed. METHODS: The immunochromatographic assay for New Delhi metallo-ß-lactamases producers was developed using rat monoclonal antibodies against NDMs. The assessment was performed using 350 isolates of Gram-negative bacteria, including Acinetobacter baumannii (51 isolates), Enterobacteriaceae (163 isolates), and Pseudomonas aeruginosa (136 isolates) obtained from 2015 to 2017 in medical settings in Myanmar. Of them, 302 isolates were resistant to carbapenems, including imipenem and/or meropenem. The blaNDM genes were identified by PCR and sequencing. RESULTS: Of the 350 clinical isolates tested, 164 (46.9%) (60 isolates of Escherichia coli, 51 isolates of Klebsiella pneumoniae, 25 isolates of Enterobacter cloacae, 23 isolates of P. aeruginosa, and 5 isolates of A. baumannii) were positive on this assay, and all the positive isolates harbored genes encoding NDM-1, - 4, - 5 and - 7. The remaining 186 (53.1%) isolates negative on the assay did not harbor genes encoding NDMs. The assay had a specificity of 100% and a sensitivity of 100%. The assessment revealed that more than 90% of carbapenem-resistant Enterobacteriaceae produced NDMs. CONCLUSIONS: The immunochromatographic assay is an easy-to-use and reliable kit for detection of NDMs-producing Gram-negative bacteria. The assay revealed that NDM-producing Enterobacteriaceae isolates are wide-spread in medical settings in Myanmar.

In Vitro Tolerance of Drug-Naive Staphylococcus aureus Strain FDA209P to Vancomycin.

The mechanisms underlying bacterial tolerance to antibiotics are unclear. A possible adaptation strategy was explored by exposure of drug-naive methicillin-susceptible Staphylococcus aureus strain FDA209P to vancomycin in vitro Strains surviving vancomycin treatment (vancomycin survivor strains), which appeared after 96 h of exposure, were slow-growing derivatives of the parent strain. Although the vancomycin MICs for the survivor strains were within the susceptible range, the cytokilling effects of vancomycin at 20-fold the MIC were significantly lower for the survivor strains than for the parent strain. Whole-genome sequencing demonstrated that ileS, encoding isoleucyl-tRNA synthetase (IleRS), was mutated in two of the three vancomycin survivor strains. The IleRS Y723H mutation is located close to the isoleucyl-tRNA contact site and potentially affects the affinity of IleRS binding to isoleucyl-tRNA, thereby inhibiting protein synthesis and leading to vancomycin tolerance. Introduction of the mutation encoding IleRS Y723H into FDA209P by allelic replacement successfully transferred the vancomycin tolerance phenotype. We have identified mutation of ileS to be one of the bona fide genetic events leading to the acquisition of vancomycin tolerance in S. aureus, potentially acting via inhibition of the function of IleRS.

Complete Reconstitution of the Vancomycin-Intermediate Staphylococcus aureus Phenotype of Strain Mu50 in Vancomycin-Susceptible S. aureus.

Complete reconstitution of the vancomycin-intermediate Staphylococcus aureus (VISA) phenotype of strain Mu50 was achieved by sequentially introducing mutations into six genes of vancomycin-susceptible S. aureus (VSSA) strain N315ΔIP. The six mutated genes were detected in VISA strain Mu50 but not in N315ΔIP. Introduction of the mutation Ser329Leu into vraS, encoding the sensor histidine kinase of the vraSR two-component regulatory (TCR) system, and another mutation, Glu146Lys, into msrR, belonging to the LytR-CpsA-Psr (LCP) family, increased the level of vancomycin resistance to that detected in heterogeneous vancomycin-intermediate S. aureus (hVISA) strain Mu3. Introduction of two more mutations, Asn197Ser into graR of the graSR TCR system and His481Tyr into rpoB, encoding the ß subunit of RNA polymerase, converted the hVISA strain into a VISA strain with the same level of vancomycin resistance as Mu50. Surprisingly, however, the constructed quadruple mutant strain ΔIP4 did not have a thickened cell wall, a cardinal feature of the VISA phenotype. Subsequent study showed that cell wall thickening was an inducible phenotype in the mutant strain, whereas it was a constitutive one in Mu50. Finally, introduction of the Ala297Val mutation into fdh2, which encodes a putative formate dehydrogenase, or a 67-amino-acid sequence deletion into sle1 [sle1(Δ67aa)], encoding the hydrolase of N-acetylmuramyl-l-alanine amidase in the peptidoglycan, converted inducible cell wall thickening into constitutive cell wall thickening. sle1(Δ67aa) was found to cause a drastic decrease in autolysis activity. Thus, all six mutated genes required for acquisition of the VISA phenotype were directly or indirectly involved in the regulation of cell physiology. The VISA phenotype seemed to be achieved through multiple genetic events accompanying drastic changes in cell physiology.

A mutation of RNA polymerase ß' subunit (RpoC) converts heterogeneously vancomycin-intermediate Staphylococcus aureus (hVISA) into "slow VISA".

Various mutations in the rpoB gene, which encodes the RNA polymerase ß subunit, are associated with increased vancomycin (VAN) resistance in vancomycin-intermediate Staphylococcus aureus (VISA) and heterogeneously VISA (hVISA) strains. We reported that rpoB mutations are also linked to the expression of the recently found "slow VISA" (sVISA) phenotype (M. Saito, Y. Katayama, T. Hishinuma, A. Iwamoto, Y. Aiba, K Kuwahara-Arai, L. Cui, M. Matsuo, N. Aritaka, and K. Hiramatsu, Antimicrob Agents Chemother 58:5024-5035, 2014, http://dx.doi.org/10.1128/AAC.02470-13). Because RpoC and RpoB are components of RNA polymerase, we examined the effect of the rpoC(P440L) mutation on the expression of the sVISA phenotype in the Mu3fdh2*V6-5 strain (V6-5), which was derived from a previously reported hVISA strain with the VISA phenotype. V6-5 had an extremely prolonged doubling time (DT) (72 min) and high vancomycin MIC (16 mg/liter). However, the phenotype of V6-5 was unstable, and the strain frequently reverted to hVISA with concomitant loss of low growth rate, cell wall thickness, and reduced autolysis. Whole-genome sequencing of phenotypic revertant strain V6-5-L1 and comparison with V6-5 revealed a second mutation, F562L, in rpoC. Introduction of the wild-type (WT) rpoC gene using a multicopy plasmid resolved the sVISA phenotype of V6-5, indicating that the rpoC(P440L) mutant expressed the sVISA phenotype in hVISA. To investigate the mechanisms of resistance in the sVISA strain, we independently isolated an additional 10 revertants to hVISA and VISA. In subsequent whole-genome analysis, we identified compensatory mutations in the genes of three distinct functional categories: the rpoC gene itself as regulatory mutations, peptidoglycan biosynthesis genes, and relQ, which is involved in the stringent response. It appears that the rpoC(P440L) mutation causes the sVISA phenotype by augmenting cell wall peptidoglycan synthesis and through the control of the stringent response.

"Slow VISA," a novel phenotype of vancomycin resistance, found in vitro in heterogeneous vancomycin-intermediate Staphylococcus aureus strain Mu3.

Heterogeneous vancomycin-intermediate Staphylococcus aureus (hVISA) clinical strain Mu3 spontaneously generates VISA strains at an extremely high frequency (≥1×10(-6)). The generated VISA strains usually grow more slowly than does the parent hVISA strain, but they form colonies on vancomycin-containing agar plates before 48 h of incubation. However, we noticed a curious group of VISA strains, designated "slow VISA" (sVISA), whose colonies appear only after 72 h of incubation. They have extremely prolonged doubling times but have vancomycin MICs of 8 to ∼24 mg/liter when determined after 72 to ∼144 h of incubation. We established strain Mu3-6R-P (6R-P), which has a vancomycin MIC of 16 mg/liter (at 72 h), as a representative sVISA strain. Its cell wall was thickened and autolytic activity was decreased compared to the respective qualities of the parent hVISA strain Mu3. Whole-genome sequencing of 6R-P revealed only one mutation, encoded by rpoB (R512P), which replaced the 512th arginine of the RNA polymerase ß-subunit with proline. Its VISA phenotype was unstable, and the strain frequently reverted to hVISA with concomitant losses of pinpoint colony morphology and cell wall thickness and reduced autolytic activity. Sequencing of the rpoB genes of the phenotypic revertant strains revealed mutations affecting the 512th codon, where the proline of 6R-P was replaced with leucine, serine, or histidine. Slow VISA generated in the tissues of an infected patient serves as a temporary shelter for hVISA to survive vancomycin therapy. The sVISA strain spontaneously returns to hVISA when the threat of vancomycin is lifted. The rpoB(R512P) mutation may be regarded as a regulatory mutation that switches the reversible phenotype of sVISA on and off.

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.

Mutation of RNA polymerase ß-subunit gene promotes heterogeneous-to-homogeneous conversion of ß-lactam resistance in methicillin-resistant Staphylococcus aureus.

Three types of phenotypic expression of ß-lactam resistance have been reported in methicillin-resistant Staphylococcus aureus (MRSA): heterogeneous, homogeneous, and Eagle-type resistance. Heterogeneous-to-homogeneous conversion of ß-lactam resistance is postulated to be caused by a chromosomal mutation (chr*) in addition to the expression of the mecA gene. Eagle-type resistance is a unique phenotype of chr* occurring in pre-MRSA strain N315 whose mecA gene expression is strongly repressed by an intact mecI gene. We here report that certain mutations of the rpoB gene, encoding the RNA polymerase ß subunit, belong to chr*. We studied homogeneous MRSA (homo-MRSA) strain N315ΔIP-H5 (abbreviated as ΔIP-H5), which was obtained from hetero-MRSA strain N315ΔIP by selection with 8 mg/liter imipenem. Whole-genome sequencing of ΔIP-H5 revealed the presence of a unique mutation in the rpoB gene, rpoB(N967I), causing the amino acid replacement of Asn by Ile at position 967 of RpoB. The effect of the rpoB(N967I) mutation was confirmed by constructing a revertant H5 rpoB(I967N) strain as well as an N315-derived mutant, N315 rpoB(N967I). H5 rpoB(I967N) regained the hetero-resistance phenotype, and the N315 rpoB(N967I) strain showed an Eagle-type phenotype similar to that of the typical Eagle-type MRSA strain N315h4. Furthermore, subsequent whole-genome sequencing revealed that N315h4 also had a missense mutation of rpoB(R644H). Introduction of the rpoB(N967I) mutation was accompanied by decreased autolysis, prolonged doubling time, and tolerance to bactericidal concentrations of methicillin. We consider that rpoB mutations are the major cause for heterogeneous-to-homogeneous phenotypic conversion of ß-lactam resistance in MRSA strain N315 and its derived strains.

Transcription and translation products of the cytolysin gene psm-mec on the mobile genetic element SCCmec regulate Staphylococcus aureus virulence.

The F region downstream of the mecI gene in the SCCmec element in hospital-associated methicillin-resistant Staphylococcus aureus (HA-MRSA) contains two bidirectionally overlapping open reading frames (ORFs), the fudoh ORF and the psm-mec ORF. The psm-mec ORF encodes a cytolysin, phenol-soluble modulin (PSM)-mec. Transformation of the F region into the Newman strain, which is a methicillin-sensitive S. aureus (MSSA) strain, or into the MW2 (USA400) and FRP3757 (USA300) strains, which are community-acquired MRSA (CA-MRSA) strains that lack the F region, attenuated their virulence in a mouse systemic infection model. Introducing the F region to these strains suppressed colony-spreading activity and PSMα production, and promoted biofilm formation. By producing mutations into the psm-mec ORF, we revealed that (i) both the transcription and translation products of the psm-mec ORF suppressed colony-spreading activity and promoted biofilm formation; and (ii) the transcription product of the psm-mec ORF, but not its translation product, decreased PSMα production. These findings suggest that both the psm-mec transcript, acting as a regulatory RNA, and the PSM-mec protein encoded by the gene on the mobile genetic element SCCmec regulate the virulence of Staphylococcus aureus.