Pseudomonas paralcaligenes sp. nov., isolated from a hospitalized patient.

A Gram-stain-negative, aerobic, rod-shaped, non-endospore-forming bacterium, designated as strain MRCP1333T, was isolated from a faecal sample from a hospital patient in Japan. MRCP1333T grew at temperatures of 15-40 °C (optimum 25-35 °C), with 1.0-3.0 % (w/v, 171-513 mM) NaCl [optimum 1-2 % (w/v), 171-342 mM], and at pH 6.0-9.5 (optimum pH 7.0-8.0). The results of phylogenetic analysis based on the sequences of the 16S rRNA gene and the 53 genes encoding the bacterial ribosome protein subunits indicated that MRCP1333T represented a member of the Pseudomonas aeruginosa group, most closely related to Pseudomonas alcaligenes. Whole-genome comparisons, using average nucleotide identity, digital DNA-DNA hybridization and average amino acid identity, confirmed that MRCP1333T represented a distinct species in the P. aeruginosa group. Phenotypic characterization tests demonstrated utilization by this strain of citrate, glycerol, and d-malic acid, the ability to reduce nitrite to nitrogen and the ability of this strain to grow in the presence of minocycline and tetrazolium blue, distinguishing this strain from P. alcaligenes and other closely related species of the P. aeruginosa group. The major fatty acids of MRCP1333T were summed feature 8 (C18 : 1ω7c/C18 : 1ω6c; 38.4 %), summed feature 3 (C16 : 1ω7c/C16 : 1ω6c; 21.1 %) and C16 : 0 (20.6 %). The DNA G+C content of MRCP1333T was 66.5 mol%. Genetic and phenotypic evidence indicated that MRCP1333T should be classified as representing a novel species, for which the name Pseudomonas paralcaligenes sp. nov. is proposed. The type strain is MRCP1333T (=LMG 32254T,=JCM 34250T).

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.

Antibodies against spike protein of SARS-CoV-2 variants in bovine whey IgG enriched fraction.

Bovine whey IgG enriched fraction contains IgG antibodies against bacterial and viral pathogens, including antibodies against the spike protein [amino acids (aa) 1-1274] of SARS-CoV-2 Wuhan strain (2019-nCoV WHU01). To date, 13 SARS-CoV-2 variants have been identified, including gamma, delta, kappa, and omicron, which contain 10, eight, seven, and over 30 mutations in the spike protein, respectively. We investigated whether bovine whey IgG enriched fraction contains antibodies against spike proteins of these variants, specifically recombinant partial length spike proteins (aa 177-512, aa 509-685, aa 177-324, aa 250-410 and aa 387-516) of these variants. Direct enzyme-linked immunosorbent assays revealed bovine whey IgG enriched fraction contained antibodies against all recombinant spike proteins of these variants with highest reactivity against aa 177-512 region of omicron spike protein. These results indicate bovine whey IgG enriched fraction contains antibodies against spike proteins of several SARS-CoV-2 variants, including omicron.

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).

A transferrable IncL/M plasmid harboring a gene encoding IMP-1 metallo-ß-lactamase in clinical isolates of Enterobacteriaceae.

BACKGROUND: The worldwide spread of carbapenemase-producing Enterobacteriaceae (CPE) has reduced the clinical utility of carbapenems. Plasmids often play an important role in the spread of genes encoding drug-resistance factors, especially in the horizontal transfer of these genes among species of Enterobacteriaceae. This study describes a patient infected with three species of CPE carrying an identical transferrable IncL/M plasmid. METHODS: Clinical isolates of CPE were collected at St. Luke's International Hospital, Tokyo, Japan, from 2015 to 2019. Three species of CPE isolates, Enterobacter cloacae, Klebsiella aerogenes and Serratia marcescens, were isolated from a patient who developed severe gallstone pancreatitis associated with bloodstream infection, with all three isolates producing IMP-1 metallo-ß-lactamase. The complete sequences of the plasmids of the three isolates were determined by both MiSeq and MinION. The medical chart of this patient was retrospectively reviewed conducted to obtain relevant clinical information. RESULTS: The three CPE species carried an IncL/M plasmid, pSL264, which was 81,133 bp in size and harbored blaIMP-1. The genetic environment surrounding blaIMP-1 consisted of int1-blaIMP-1-aac(6')-IIc-qacL-qacEdelta1-sul1-istB-IS21. Conjugation experiments showed that S. marcescens could transmit the plasmid to E. cloacae and K. aerogenes. In contrast, pSL264 could not transfer from E. cloacae or K. aerogenes to S. marcescens. CONCLUSION: The IncL/M plasmid pSL264 harboring blaIMP-1 was able to transfer among different species of Enterobacteriaceae in a patient receiving long-term antimicrobial treatment. The worldwide emergence and spread of IncL/M plasmids harboring carbapenemase-encoding genes among species of Enterobacteriaceae is becoming a serious public health hazard.

Presence of antibodies against SARS-CoV-2 spike protein in bovine whey IgG enriched fraction.

Bovine whey IgG enriched fraction contains antibodies against various human bacterial pathogens. It contains antibodies against some viral antigens, including human respiratory syncytial virus and influenza virus. We investigated whether the IgG enriched fraction has cross-reactivity with IgG antibodies against SARS-CoV-2 spike (S) and nucleocapsid (N) proteins. The full-length and partial-length SARS-CoV-2 S, N, a recombinant protein of the receptor binding domain (RBD) and nine peptides covering the receptor binding motif (RBM) of S were prepared. Direct enzyme-linked immunosorbent assays were conducted using these recombinant proteins and peptides as coating antigens and revealed the IgG enriched fraction contained antibodies against partial-length S [amino acids (aa) 177-512, 288-512, 348-578, 387-516 and 408-664], full-length N (aa 1-419) and partial-length N (aa 1-120, 111-220, 1-220 and 210-419), two RBD peptides, covering aa 427-446 and 502-520 of S, and recombinant RBD of S. These results indicate IgG enriched fraction contains antibodies against SARS-CoV-2.

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.

Genome analysis-based reclassification of Pseudomonas fuscovaginae and Pseudomonas shirazica as later heterotypic synonyms of Pseudomonas asplenii and Pseudomonas asiatica, respectively.

This study was conducted to clarify the taxonomic status of the species Pseudomonas fuscovaginae and Pseudomonas shirazica. Whole genome sequences for the type strains of P. fuscovaginae and P. shirazica were compared against the closely related type strains of the Pseudomonas putida group and the Pseudomonas fluorescens group species. Average nucleotide identity and digital DNA-DNA hybridization values between P. fuscovaginae LMG 2158T and Pseudomonas asplenii ATCC 23835T were 98.4 and 85.5 %, and between P. shirazica VM14T and Pseudomonas asiatica RYU5T were 99.3 and 95.3 %. These values were greater than recognized thresholds for bacterial species delineation, indicating that they belong to the same genomospecies, respectively. Therefore, P. fuscovaginae and P. shirazica should be reclassified as later heterotypic synonyms of P. asplenii and P. asiatica, respectively.

Pseudomonas yangonensis sp. nov., isolated from wound samples of patients in a hospital in Myanmar.

Strains of a Gram-negative, aerobic, rod-shaped, non-spore-forming bacterium, designated MY50T, MY63 and MY101, were isolated from wound samples of three hospitalized patients in Yangon, Myanmar. Strains MY50T, MY63 and MY101 grew at temperatures of 4-44 °C, in media containing 1.0-7.0 % (w/v) NaCl and at pH 6.0-9.5. Phylogenetic analysis based on 16S rRNA gene and whole genome sequences showed that these strains belonged to the genus Pseudomonas and were part of the Pseudomonas oleovorans group and located close to Pseudomonas guguanensis and Pseudomonas mendocina. Whole-genome comparisons, using average nucleotide identity and digital DNA-DNA hybridization analyses, confirmed that strains MY50T, MY63 and MY101 were the same strain and they were a distinct species in the P. oleovorans group. Results of phenotypic characterization tests demonstrated that utilization of p-hydroxy-phenylacetic acid, glycerol, l-pyroglutamic acid and quinic acid could distinguish these strains from other species of the P. oleovorans group. These genetic and phenotypic characteristics suggest that they should be classified as representing a novel species, under the proposed name Pseudomonas yangonensis sp. nov. The type strain is MY50T (=LMG 31602T,=JCM 33396T), with a DNA G+C content of 62.82 mol%.

Emergence of carbapenem-resistant and colistin-susceptible Enterobacter cloacae complex co-harboring blaIMP-1 and mcr-9 in Japan.

BACKGROUND: The spread of Enterobacteriaceae producing both carbapenemases and Mcr, encoded by plasmid-mediated colistin resistance genes, has become a serious public health problem worldwide. This study describes three clinical isolates of Enterobacter cloacae complex co-harboring blaIMP-1 and mcr-9 that were resistant to carbapenem but susceptible to colistin. METHODS: Thirty-two clinical isolates of E. cloacae complex non-susceptible to carbapenems were obtained from patients at 14 hospitals in Japan. Their minimum inhibitory concentrations (MICs) were determined by broth microdilution methods and E-tests. Their entire genomes were sequenced by MiSeq and MinION methods. Multilocus sequence types were determined and a phylogenetic tree constructed by single nucleotide polymorphism (SNP) alignment of whole genome sequencing data. RESULTS: All 32 isolates showed MICs of ≥2 µg/ml for imipenem and/or meropenem. Whole-genome analysis revealed that all these isolates harbored blaIMP-1, with three also harboring mcr-9. These three isolates showed low MICs of 0.125 µg/ml for colistin. In two of these isolates, blaIMP-1 and mcr-9 were present on two separate plasmids, of sizes 62 kb and 280/290 kb, respectively. These two isolates did not possess a qseBC gene encoding a two-component system, which is thought to regulate the expression of mcr-9. In the third isolate, however, both blaIMP-1 and mcr-9 were present on the chromosome. CONCLUSION: The mcr-9 is silently distributed among carbapenem-resistant E. cloacae complex isolates, of which are emerging in hospitals in Japan. To our knowledge, this is the first report of isolates of E. cloacae complex harboring both blaIMP-1 and mcr-9 in Japan.

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.

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.

Emergence of Carbapenem-Resistant Pseudomonas asiatica Producing NDM-1 and VIM-2 Metallo-ß-Lactamases in Myanmar.

Pseudomonas asiatica is a recently proposed species of the genus Pseudomonas This study describes eight isolates of carbapenem-resistant P. asiatica harboring blaNDM-1 and blaVIM-2, genes encoding metallo-ß-lactamase (MBL). These isolates were obtained from urine samples of patients hospitalized in Myanmar. These isolates were resistant to carbapenems but susceptible to colistin. All eight isolates were positive for a carbapenemase inactivation method, CIMTrisII, and seven were positive on an immunochromatographic assay for NDM-type MBL. One isolate was highly resistant to aminoglycosides. Whole-genome sequencing showed that seven isolates harbored blaNDM-1 and one harbored blaVIM-2, with these genes located on the chromosome. One isolate harbored blaNDM-1 and rmtC, a gene encoding 16S rRNA methylase. Five types of genomic environments surrounding blaNDM-1 and blaVIM-2 were detected in these eight isolates, with four isolates having the same type. These data indicate that P. asiatica isolates harboring genes encoding carbapenemases, including blaNDM-1 and blaVIM-2, are spreading in medical settings in Myanmar.

Pseudomonas juntendi sp. nov., isolated from patients in Japan and Myanmar.

A Gram-negative, aerobic, rod-shaped, non-spore-forming bacterium, designated as strain BML3T, was isolated from a sputum sample of a hospital patient in Japan. Strain BML3T grew at temperatures from 4 to 40 °C, in 1.0-7.0 % (w/v) NaCl and at pH 6.0-9.0. Results of phylogenetic analysis based on the sequences of housekeeping genes, including the 16S rRNA gene and rpoB, rpoD and gyrB, showed that strain BML3T was part of the Pseudomonas putida group and located close to Pseudomonas asiatica, Pseudomonas monteiliiand P. putida . Whole-genome comparisons, using average nucleotide identity and digital DNA-DNA hybridization, confirmed strain BML3T to be a distinct species among the P. putida group. Phenotypic characterization tests demonstrated that the utilization of phenylmercuric acetate could distinguish this strain from other closed species of the P. putida group. Based on genetic and phenotypic evidence, strain BML3T should be classified as a novel species, for which the name Pseudomonas juntendi sp. nov. is proposed. The type strain is BML3T (=DSM 109244T,=JCM 33395T), with a DNA G+C content of 62.66 mol %.

Pseudomonas asiatica sp. nov., isolated from hospitalized patients in Japan and Myanmar.

A novel Gram-negative, aerobic, rod-shaped, non-spore-forming bacterial strain, RYU5T, was isolated from a stool sample of an inpatient at a hospital in Okinawa, Japan. The optimal growth temperature of RYU5T was 30 °C. Phylogenetic analysis based on the sequences of housekeeping genes, including the 16S rRNA, rpoB, rpoD and gyrB genes, showed that RYU5T was a member of the Pseudomonas putida group and was located close to Pseudomonas monteilii and P. putida. Whole-genome comparisons, using average nucleotide identity and digital DNA-DNA hybridization, confirmed that strain RYU5T should be classified as a novel species of Pseudomonas. Phenotypic characterization tests showed that utilization of d-mannose, d-serine, l-arabinose and d-fructose could distinguish this strain from other related species of the genus Pseudomonas. Based on genetic and phenotypic evidence, strain RYU5T should be classified as a novel species, for which the name Pseudomonas asiatica sp. nov. is proposed. The type strain is RYU5T (=DSM 107182T, =JCM 32716T), with a DNA G+C content of 62.25 mol%.

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.

Emergence of a carbapenem-resistant and colistin-heteroresistant Enterobacter cloacae clinical isolate in Japan.

A carbapenem-resistant and colistin-heteroresistant clinical isolate of Enterobacter cloacae was obtained from an inpatient in Okinawa, Japan. The minimum inhibitory concentrations of both imipenem and meropenem were 32 µg/mL. The isolate showed heteroresistance to colistin using the Etest method and resistance to colistin using the broth microdilution method. It had a disrupted ompC and a mutation in the promoter region of blaACT-2, but did not harbor any genes encoding carbapenemase. The disruption of ompC and the mutation in blaACT-2 was associated with the carbapenem resistance of this isolate. This isolate also had mutations in pmrAB and phoPQ encoding two-component regulatory systems, which may be associated with colistin heteroresistance.

Emergence of ArmA, a 16S rRNA methylase in highly aminoglycoside-resistant clinical isolates of Klebsiella pneumoniae and Klebsiella oxytoca in Okinawa, Japan.

This study describes highly aminoglycoside-resistant Klebsiella pneumoniae and Klebsiella oxytoca clinical isolates obtained from an inpatient in Okinawa, Japan, with no known record of traveling overseas. The minimum inhibitory concentrations of amikacin and arbekacin against these strains were >1024 µg/ml. Whole-genome sequencing analysis revealed that these isolates harbored armA, which encodes a 16S rRNA methylase, ArmA, that confers pan-aminoglycoside resistance. This is the second report of K. pneumoniae harboring armA and the first report of K. oxytoca harboring a 16S rRNA methylase encoding gene in Japan.

PER-8, a Novel Extended-Spectrum ß-Lactamase PER Variant, from an Acinetobacter baumannii Clinical Isolate in Nepal.

A novel PER-type extended-spectrum ß-lactamase, PER-8, was identified in an Acinetobacter baumannii clinical isolate obtained in Nepal. The amino acid sequence of PER-8 has a substitution at position 39 (Gly to Glu) compared with that of PER-7. The kcat/Km ratio of PER-8 for aztreonam was lower than that of PER-7, while the kcat/Km ratio of PER-8 for imipenem was higher than that of PER-7. The genomic environment surrounding blaPER-8 was intI1 blaPSE-1qacEDI sulI ISCR1-blaPER-8gts sulI orfX on a 100-kb plasmid.

Pseudomonas aeruginosa Clinical Isolates in Nepal Coproducing Metallo-ß-Lactamases and 16S rRNA Methyltransferases.

A total of 11 multidrug-resistant Pseudomonas aeruginosa clinical isolates were obtained in Nepal. Four of these isolates harbored genes encoding one or more carbapenemases (DIM-1, NDM-1, and/or VIM-2), and five harbored genes encoding a 16S rRNA methyltransferase (RmtB4 or RmtF2). A novel RmtF variant, RmtF2, had a substitution (K65E) compared with the same gene in RmtF. To our knowledge, this is the first report describing carbapenemase- and 16S rRNA methyltransferase-coproducing P. aeruginosa clinical isolates in Nepal.