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Introduction. The emergence of carbapenem-resistant Pseudomonas species producing metallo-β-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.

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.

The aim of this study was to clarify the biological and clinical significance of a tandem duplicate of

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.

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.

Objective The aim of this study was to clarify the genetic and epidemiological properties of multidrug-resistant Acinetobacter baumannii in medical settings in Myanmar. Methods A total of 45 A. baumannii clinical isolates were obtained in medical settings in Myanmar. The whole genomes were sequenced by a next generation sequencer, and the phylogenetic tree was constructed from single nucleotide polymorphism concatemers. Multilocus sequence types were deduced and drug resistance genes were identified. Results Thirty-eight MDR Acinetobacter baumannii isolates were obtained from seven hospitals in Myanmar. The majority of MDR A. baumannii isolates belonged to ST2. Of the 38 isolates, 5 harbored blaNDM-1, and 28 did armA or armA2 Conclusions A. baumannii ST2 producing 16S rRNA methylase ArmA has been spreading in medical settings in Myanmar.

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).

Morganella morganiican harbour extended-spectrum β-lactamases and carbapenemases, resulting in increased resistance to multiple antibiotics and a high mortality rate. This study describes the emergence of highly multidrug-resistant clinical isolates ofM. morganiifrom Nepal co-producing NDM-type metallo-β-lactamases, including NDM-1 and NDM-5, and the 16S rRNA methylase ArmA. This is the first report ofM. morganiiclinical isolates from Nepal co-producing NDM-1/-5 and ArmA. It is important to establish infection control systems and effective treatments against multidrug-resistantM. morganii.

Objectives: The aim of this study was to describe the emergence in Nepal of clinical isolates of Klebsiella pneumoniae harboring both blaNDM-5 and blaOXA-181/-232. Methods: Six clinical isolates of K. pneumoniae highly resistant to carbapenems and aminoglycosides were obtained from inpatients in Nepal. Their whole genomes were sequenced by a next generation sequencer. Results: The minimum inhibitory concentrations of meropenem, amikacin and ciprofloxacin were ≥128 μg/ml, >1024 μg/ml and ≥256 μg/ml, respectively. All six isolates co-harbored blaNDM-5, blaOXA-181 or -232 and rmtB. Of them, 1 also harbored rmtF. The blaNDM-5, blaOXA-232 and rmtB in all six isolates were located on plasmids. Of the six isolates tested, one isolate harbored two copies of blaOXA-181 and rmtF on the chromosome. Conclusions: This is the first report of clinical isolates of K. pneumoniae co-harboring blaNDM-5, blaOXA-181 or -232 and rmtB in Nepal. These strains were highly carbapenem- and aminoglycoside-resistant, and belonged to ST147 or ST395. Of them, ST147 isolate harbored two copies of blaOXA-181 on the chromosome. Keywords: Klebsiella pneumoniae, Multidrug resistance, Carbapenemase, 16S rRNA methylase

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

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).

This study aimed to describe a clinical isolate of Aeromonas jandaei (A. jandaei) in Nepal that harboured four types of genes encoding phosphoethanolamine transferases.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.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 were80% identical to MCR-3.1, and that of EptAv7.2 was79% 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.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.

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 usinga€€ 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 1i½ž3, 4i½ž9, and ≥ 10 days after symptom onset from COVID-19 patients diagnosed by RT-PCR showed positivity rates of 100 %, >80 %, and

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 bla(IMP-70), encoding a variant of IMP-1 metallo-β-lactamase with two amino acid substitutions (Val67Phe and Phe87Val), and the other two harbored bla(IMP-1) and bla(IMP-11), respectively. Whole-genome sequencing revealed that an isolate harbored two copies of bla(IMP-1) on the chromosome and that the other four harbored a copy of bla(IMP-11) or bla(IMP-70) in a plasmid. Expression of bla(IMP-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 bla(IMP-1) or by mutation of bla(IMP) genes with amino acid substitutions, such as bla(IMP-70).

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.

The emergence of multidrug-resistant E. cloacae complex has become a public health threat worldwide. E. xiangfangensis is a recently classified species belonging to E. cloacae complex. Here, we report a clonal dissemination of multidrug-resistant E. xiangfangensis ST200 producing two types of New Delhi metallo-β-lactamase (NDM-type MBL), NDM-1 and -4, and three types of 16S rRNA methylases, ArmA, RmtC, and RmtE, in hospitals in Myanmar. The observation of these multidrug-resistant E. xiangfangensis ST200 isolates stresses the urgency to continue molecular epidemiological surveillance of these pathogens in Myanmar and in South Asian countries., Surveillance of 10 hospitals and a regional public health laboratory in Myanmar identified 31 isolates of carbapenem-resistant Enterobacter cloacae complex harboring blaNDM-type. Of these isolates, 19 were highly resistant to aminoglycosides and harbored one or more genes encoding 16S rRNA methylases, including armA, rmtB, rmtC, and/or rmtE. Of the 19 isolates, 16 were Enterobacter xiangfangensis ST200, with armA on the chromosome and a plasmid harboring blaNDM-1 and rmtC, indicating that these isolates were clonally disseminated nationwide in Myanmar. IMPORTANCE The emergence of multidrug-resistant E. cloacae complex has become a public health threat worldwide. E. xiangfangensis is a recently classified species belonging to E. cloacae complex. Here, we report a clonal dissemination of multidrug-resistant E. xiangfangensis ST200 producing two types of New Delhi metallo-β-lactamase (NDM-type MBL), NDM-1 and -4, and three types of 16S rRNA methylases, ArmA, RmtC, and RmtE, in hospitals in Myanmar. The observation of these multidrug-resistant E. xiangfangensis ST200 isolates stresses the urgency to continue molecular epidemiological surveillance of these pathogens in Myanmar and in South Asian countries.

The aim of this study was to describe the emergence in Nepal of clinical isolates of Klebsiella pneumoniae harboring both blaSix clinical isolates of K. pneumoniae highly resistant to carbapenems and aminoglycosides were obtained from inpatients in Nepal. Their whole genomes were sequenced by a next generation sequencer.The minimum inhibitory concentrations of meropenem, amikacin and ciprofloxacin were ≥128 μg/ml,1024 μg/ml and ≥256 μg/ml, respectively. All six isolates co-harbored blaThis is the first report of clinical isolates of K. pneumoniae co-harboring bla

Several species of Aeromonas produce the enzyme CphA metallo-β-lactamase. This study describes an isolate of Aeromonas hydrophila harbouring an acquired gene encoding the carbapenemase GES-24. This isolate was obtained from an inpatient in Okinawa, Japan, with no apparent record of travelling overseas. The minimum inhibitory concentrations of carbapenems against this isolate were 8 µg ml−1 for imipenem and 16 µg ml−1 for meropenem. Recombinant GES-24 hydrolyzed all of the tested β-lactams, including imipenem and meropenem. The genomic environment surrounding bla GES-24 was intI1-bla GES-24 -aac(6′)-IIc-qacEdelta1-sulI-orfX-tetR-tetE. This is the first report of A. hydrophila producing a GES-type carbapenemase.

Background To detect carbapenemase-producing Gram-negative bacteria in bacterial laboratories at medical settings, a new immunochromatographic assay for New Delhi metallo-β-lactamases (NDMs) was developed. Methods The immunochromatographic assay for New Delhi metallo-β-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. Electronic supplementary material The online version of this article (10.1186/s12879-019-4147-4) contains supplementary material, which is available to authorized users.

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-β-lactamases, have been spreading throughout medical settings in Myanmar.

CRE-JU is a novel selective agar for carbapenem-resistant Enterobacteriaceae that contains ceftazidime, cloxacillin, meropenem, and vancomycin. This study evaluated the ability of 63 carbapenem-resistant isolates and 53 non-carbapenem-resistant Enterobacteriaceae strains clinically isolated in Japan, Myanmar, Nepal, and Vietnam to grow on CRE-JU. CRE-JU showed 92.1% sensitivity and 100% specificity for detecting carbapenem-resistant Enterobacteriaceae compared with dug susceptibility profiles.

β-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 β′ 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 β-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 β-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 β-lactam resistance in MRSA via the stringent response.

We developed a simple, efficient, and cost-effective method, named the re plica p lating t olerance i solation s ystem (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.

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-β-lactamases, have been spreading throughout medical settings in Myanmar.

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 β 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.

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.

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.

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 β-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.