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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 harbored a gene encoding NDM-type metallo-ß-lactamase, and 55 harbored 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 harbored armA, 26 harbored blaNDM-1, and14 harbored blaPER-7. MLST 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.
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Mycobacteroides (Mycobacterium) abscessus, which causes a variety of infectious diseases in humans, is becoming detected more frequently in clinical specimens as cases are spreading worldwide. Taxonomically, M. abscessus is composed of three subspecies of M. abscessus subsp. abscessus, M. abscessus subsp. bolletii, and M. abscessus subsp. massiliense, with different susceptibilities to macrolides. In order to identify rapidly these three subspecies, we determined useful biomarker proteins, including ribosomal protein L29, L30, and hemophore-related protein, for distinguishing the subspecies of M. abscessus using the matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) profiles. Thirty-three clinical strains of M. abscessus were correctly identified at the subspecies-level by the three biomarker protein peaks. This study ultimately demonstrates the potential of routine MALDI-MS-based laboratory methods for early identification and treatment for M. abscessus infections.
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Proteínas Bacterianas , Mycobacterium abscessus , Proteínas Ribosómicas , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Proteínas Ribosómicas/metabolismo , Proteínas Ribosómicas/análisis , Mycobacterium abscessus/metabolismo , Proteínas Bacterianas/metabolismo , Humanos , Infecciones por Mycobacterium no Tuberculosas/microbiología , Infecciones por Mycobacterium no Tuberculosas/diagnóstico , Biomarcadores/análisis , Biomarcadores/metabolismoRESUMEN
Streptococcus suis is a gram-positive bacterium that causes meningitis, septicemia, endocarditis, and other disorders in pigs and humans. We obtained 42 and 50 S. suis isolates from lesions of porcine endocarditis and palatine tonsils, respectively, of clinically healthy pigs in Japan; we then determined their sequence types (STs) by multilocus sequence typing (MLST), cps genotypes, serotypes, and presence of classical major virulence-associated marker genes (mrp, epf, and sly). The 42 isolates from endocarditis lesions were assigned to a limited number of STs and clonal complexes (CCs). On the other hand, the 50 isolates from tonsils were diverse in these traits and seemingly in the degree of virulence, suggesting that tonsils can accommodate a variety of S. suis isolates. The goeBURST full algorithm using tonsil isolates obtained in this study and those retrieved from the database showed that major CCs as well as many other clusters were composed of isolates originating from different countries, and some of the STs were very similar to each other despite the difference in country of origin. These findings indicate that S. suis with not only different but also similar mutations in the genome have survived in tonsils independently across different geographical locations. Therefore, unlike the lesions of endocarditis, the tonsils of pigs seemingly accommodate various S. suis lineages. The present study suggests that S. suis acquired its diversity by natural mutations during colonization and persistence in the tonsils of pigs.
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Endocarditis , Infecciones Estreptocócicas , Streptococcus suis , Enfermedades de los Porcinos , Humanos , Porcinos , Animales , Tipificación de Secuencias Multilocus/veterinaria , Tonsila Palatina/microbiología , Streptococcus suis/genética , Infecciones Estreptocócicas/microbiología , Infecciones Estreptocócicas/veterinaria , Enfermedades de los Porcinos/microbiología , Endocarditis/veterinariaRESUMEN
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.
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Acinetobacter , Antiinfecciosos , Meropenem/farmacología , Pseudomonas , Tienamicinas/farmacología , Carbapenémicos/farmacología , Pruebas de Sensibilidad Microbiana , Antibacterianos/farmacología , beta-LactamasasRESUMEN
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.
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Colistina , Klebsiella pneumoniae , Humanos , Klebsiella pneumoniae/genética , Mianmar , Colistina/farmacología , ARN Ribosómico 16S , Antibacterianos/farmacología , Aminoglicósidos , CarbapenémicosRESUMEN
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.
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Colistina , Escherichia coli , Animales , Gatos , Colistina/farmacología , Escherichia coli/genética , Lípido A/farmacología , Etanolaminofosfotransferasa/genética , Proteínas Bacterianas/genética , Farmacorresistencia Bacteriana/genética , Antibacterianos/farmacología , Klebsiella pneumoniaeRESUMEN
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 82â% of pMRCP2 was 92â% similar to the sequence of a plasmid of P. aeruginosa PA83, whereas the sequence of 79â% of pMRCP1333 was >95â% 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.
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Infecciones por Pseudomonas , Pseudomonas alcaligenes , Humanos , Carbapenémicos/farmacología , Pseudomonas/genética , Antibacterianos/farmacología , Pseudomonas alcaligenes/genética , Japón , Pruebas de Sensibilidad Microbiana , beta-Lactamasas/genética , Pseudomonas aeruginosa/genética , Plásmidos/genéticaRESUMEN
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 1.0-3.0â% (w/v, 171-513 mM) NaCl [optimum 1-2â% (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 (C18â:â1ω7c/C18â:â1ω6c; 38.4â%), summed feature 3 (C16â:â1ω7c/C16â:â1ω6c; 21.1 %) and C16â:â0 (20.6â%). 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).
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Ácidos Grasos , Fosfolípidos , Humanos , Ácidos Grasos/química , Fosfolípidos/química , Pseudomonas , Filogenia , ARN Ribosómico 16S/genética , Análisis de Secuencia de ADN , ADN Bacteriano/genética , Composición de Base , Técnicas de Tipificación BacterianaRESUMEN
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.
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Infecciones por Pseudomonas , Pseudomonas aeruginosa , Humanos , Pseudomonas aeruginosa/genética , Antibacterianos/farmacología , Cefepima/farmacología , Japón , Infecciones por Pseudomonas/tratamiento farmacológico , Pruebas de Sensibilidad Microbiana , Plásmidos/genética , beta-Lactamasas/genética , Carbapenémicos/farmacologíaRESUMEN
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.
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A strain of Clostridium perfringens was isolated from the bile sample of a patient with emphysematous cholecystitis who underwent a laparoscopic cholecystectomy, followed by treatment with meropenem and recovery. Metagenomic analysis of the bile sample showed that 99.73% of the bile microbiota consisted of C. perfringens, indicating that C. perfringens JUM001 was the causative pathogen of acute emphysematous cholecystitis in this patient. Complete genome sequencing showed that C. perfringens JUM001 contained a circular chromosome of 3,231,023 bp and two circular plasmids, pJUM001-1 of 49,289 bp and pJUM001-2 of 47,855 bp. JUM001 was found to possess a typing toxin gene, plc, but no other typing toxin genes, indicating that its toxinotype is type A. The plasmids pJUM001-1 and pJUM001-2 belonged to the pCP13-like and pCW3-like families of plasmids, respectively, which are characteristic conjugative and archetypical plasmids of C. perfringens. Phylogenetic analysis showed that JUM001 was closely related to C. perfringens strain JXNC-DD isolated from a dog in China. To our knowledge, this is the first report of whole-genome sequences of a clinical isolate of C. perfringens causing acute emphysematous cholecystitis.
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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.
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Colistina , Proteínas de Escherichia coli , Humanos , Colistina/farmacología , Enterobacteriaceae , Antibacterianos/farmacología , Agar , Caseínas/genética , Caseínas/farmacología , Escherichia coli/genética , Peptonas/farmacología , Farmacorresistencia Bacteriana/genética , Pruebas de Sensibilidad Microbiana , Plásmidos , Proteínas de Escherichia coli/genéticaRESUMEN
Background Bile inhibits bacterial growth because it is rich in bacteriostatic compounds such as bile acids. Analytical techniques using a high-intensity sequencer recently revealed bacterial flora in the bile of normal gallbladders in brain-dead patients. Therefore, we performed a microbial flora analysis of bile collected from pathologically normal gallbladders surgically removed from patients with hepatobiliary pancreatic diseases and normal liver function. Methods Bacterial DNA was extracted from bile samples and analyzed using 16S rRNA sequencing. Results The culture results of all 12 bile samples were negative. However, the results of the 16S ribosome gene analysis suggested the presence of bacterial flora in all samples. The phyla Firmicutes, Proteobacteria, Actinobacteria, and, more specifically, the genera Anaerobacillus, Delftia, Bacillus, Ralstonia, Ochrobactrum, Acidovorax, and Curvibacter were detected in all 12 samples. The results of the 16S rRNA gene profile analysis revealed that Anaerobacillus and Delftia accounted for 58.62%-87.63% of the bacteria identified in each sample. Conclusion In a bacterial flora analysis targeting the 16S ribosomal gene, a specific bacterial flora was detected in bile collected from the pathologically normal gallbladders of patients with hepatobiliary pancreatic diseases. Although a diverse bacterial flora was previously reported in the bile of brain-dead patients, the present results revealed a simple bacterial flora with no diversity in the bile samples.
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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.
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Stenotrophomonas maltophilia , Acetiltransferasas , Aminoácidos/metabolismo , Antibacterianos/farmacología , Escherichia coli/metabolismo , Humanos , Pruebas de Sensibilidad Microbiana , Nepal , Penicilinas/metabolismo , Filogenia , Stenotrophomonas maltophilia/genética , beta-Lactamasas/genéticaRESUMEN
A total of 38 isolates of carbapenem-resistant Klebsiella pneumoniae harboring blaNDM were obtained during surveillance of 10 hospitals in Myanmar. Of these 38 isolates, 19 (50%) harbored genes encoding 16S rRNA methylases, such as armA or rmtB. The K. pneumoniae strains tested belonged to 17 sequence types (STs), including the high-risk clonal lineages ST101 and ST147. The ST101 and ST147 isolates carried IncFII plasmids harboring blaNDM-5 and IncFIB(pQil) plasmids harboring blaNDM-1, respectively. These results indicate that IncFII plasmids harboring blaNDM-5 and IncFIB(pQil) plasmids harboring blaNDM-1 have been spreading in K. pneumoniae ST101 and ST147 isolates, respectively, in Myanmar. IMPORTANCE The emergence of carbapenem-resistant K. pneumoniae has become a serious problem in medical settings worldwide. The present study demonstrated that carbapenem-resistant K. pneumoniae strains have been spreading in medical settings in Myanmar. In particular, plasmid genes encoding NDMs and 16S rRNA methylases have been spreading in K. pneumoniae high-risk clones.
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Infecciones por Klebsiella , Klebsiella pneumoniae , Antibacterianos/farmacología , Proteínas Bacterianas/genética , Carbapenémicos/farmacología , Humanos , Infecciones por Klebsiella/epidemiología , Klebsiella pneumoniae/genética , Pruebas de Sensibilidad Microbiana , Mianmar/epidemiología , Plásmidos , ARN Ribosómico 16S , beta-Lactamasas/genéticaRESUMEN
Streptococcus ruminantium is a close relative of Streptococcus suis, an important zoonotic pathogen that causes various diseases in pigs and humans. Here, we report the complete genome sequences of three S. ruminantium strains isolated from bovine endocarditis in Japan.
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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.
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Pseudomonas fluorescens , Pseudomonas putida , Animales , Técnicas Bacteriológicas/métodos , Pseudomonas fluorescens/genética , Pseudomonas putida/genética , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Secuenciación Completa del GenomaRESUMEN
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.
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Aeromonas , Proteínas de Escherichia coli , Aeromonas/genética , Antibacterianos/farmacología , Colistina/farmacología , Farmacorresistencia Bacteriana/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Etanolaminofosfotransferasa/genética , Etanolaminofosfotransferasa/metabolismo , Etanolaminas , Pruebas de Sensibilidad Microbiana , Nepal , PlásmidosRESUMEN
Background: Escherichia coli is thought to cause xanthogranulomatous cholecystitis (XGC). However, it is unclear whether other pathogens are associated with the cause and progression of XGC. Case presentation: Patient 1 was a 55-year-old man with a previous surgical history of right lung cancer. He presented with abdominal pain and was diagnosed with acute cholecystitis. He underwent endoscopic nasogallbladder drainage (ENGBD), antimicrobial therapy, and endoscopic sphincterotomy (EST). He underwent cholecystectomy on day 59. The patient was pathologically diagnosed with chronic phase XGC. Acinetobacter baumannii was isolated from the bile sample during the operation. Patient 2 was a 58-year-old man with no previous medical history. He presented with abdominal pain and was diagnosed with acute cholecystitis. He underwent endoscopic retrograde biliary drainage (ERGBD) and antimicrobial therapy. His symptoms improved, but acute cholecystitis became exacerbated on day 53. The patient was treated with antimicrobial therapy. He underwent cholecystectomy on day 88. The patient was pathologically diagnosed with focal acute inflammatory phase XGC. Staphylococcus capitis was isolated from the bile during the operation. This study describes two patients with XGC, one infected with A. baumannii and the other with S. capitis, in their gallbladders, which was identified by bacterial culture. Metagenomic analysis revealed that the genera Acinetobacter and Staphylococcus predominated and that other genera, including Delftia and Anaerobacillus, were also present, suggesting that these bacteria play a significant role in the pathological changes associated with XGC. Conclusions: This is the first report of A. baumannii and S. capitis infections in patients with XGC.