A longitudinal epidemiology study of fluoroquinolone-nonsusceptible Klebsiella pneumoniae reveals an increasing prevalence of qnrB and qnrS in Taiwan.

BACKGROUND: Our objective was to investigate the prevalence of plasmid-mediated quinolone resistance (PMQR) genes in fluoroquinolone-nonsusceptible Klebsiella pneumoniae (FQNSKP) in Taiwan, 1999-2022. METHODS: A total of 938 FQNSKP isolates were identified from 1966 isolates. The presence of PMQR and virulence genes, antimicrobial susceptibility, capsular types, and PMQR-plasmid transferability were determined. RESULTS: An increasing number of PMQR-containing FQNSKP isolates were observed over the study period. Our results showed that 69.0% (647 isolates) of FQNSKP isolates contained at least one PMQR gene, and 40.6%, 37.0%, and 33.9% of FQNSKP carried aac(6')-Ib-cr, qnrB, and qnrS, respectively. None of FQNSKP carried qepA and qnrC. The most common combination of PMQR genes was aac(6')-Ib-cr and qnrB (12.3%). The presence of PMQR genes is strongly related to resistance to aminoglycoside, cephalosporin, tetracycline, and sulfamethoxazole/trimethoprim in FQNSKP. The capsular serotype K64 is the most common serotype we tested in both the non-PMQR and PMQR FQNSKP isolates, while K20 showed a higher prevalence in PMQR isolates. The magA and peg-344 genes showed a significantly higher prevalence rate in non-PMQR isolates than in PMQR isolates. Eleven isolates that carried the PMQR and carbapenemase genes were identified; however, three successful transconjugants showed that the PMQR and carbapenemase genes were not located on the same plasmid. CONCLUSIONS: Our results indicated an increasing prevalence of PMQR genes, especially qnrB and qnrS, in FQNSKP in Taiwan. Moreover, the distribution of PMQR genes was associated with capsular serotypes and antimicrobial resistance gene and virulence gene distribution in FQNSKP.

A 24-year longitudinal study of Klebsiella pneumoniae isolated from patients with bacteraemia and urinary tract infections reveals the association between capsular serotypes, antibiotic resistance, and virulence gene distribution.

Longitudinal studies on the variations of phenotypic and genotypic characteristics of K. pneumoniae across two decades are rare. We aimed to determine the antimicrobial susceptibility and virulence factors for K. pneumoniae isolated from patients with bacteraemia or urinary tract infection (UTI) from 1999 to 2022. A total of 699 and 1,267 K. pneumoniae isolates were isolated from bacteraemia and UTI patients, respectively, and their susceptibility to twenty antibiotics was determined; PCR was used to identify capsular serotypes and virulence-associated genes. K64 and K1 serotypes were most frequently observed in UTI and bacteraemia, respectively, with an increasing frequency of K20, K47, and K64 observed in recent years. entB and wabG predominated across all isolates and serotypes; the least frequent virulence gene was htrA. Most isolates were susceptible to carbapenems, amikacin, tigecycline, and colistin, with the exception of K20, K47, and K64 where resistance was widespread. The highest average number of virulence genes was observed in K1, followed by K2, K20, and K5 isolates, which suggest their contribution to the high virulence of K1. In conclusion, we found that the distribution of antimicrobial susceptibility, virulence gene profiles, and capsular types of K. pneumoniae over two decades were associated with their clinical source.

Sequential isolation of metabolites and lipids from a single sample to achieve multiomics by using TRIzol reagent.

Simultaneous extraction of various types of biomolecule from a single sample can be beneficial for multiomics studies of unique specimens. An efficient and convenient sample preparation approach must be developed that can comprehensively isolate and extract biomolecules from one sample. TRIzol reagent is widely used in biological studies for DNA, RNA, and protein isolation. This study evaluated the feasibility of using TRIzol reagent for the simultaneous isolation of not only DNA, RNA, and proteins but also metabolites and lipids from a single sample. Through the comparison of known metabolites and lipids obtained using the conventional methanol (MeOH) and methyl-tert-butyl ether (MTBE) extraction methods, we determined the presence of metabolites and lipids in the supernatant during TRIzol sequential isolation. Finally, we performed untargeted metabolomics and lipidomics to examine metabolite and lipid alterations associated with the jhp0417 mutation in Helicobacter pylori by using the TRIzol sequential isolation protocol and MeOH and MTBE extraction methods. Metabolites and lipids with significant differences isolated using the TRIzol sequential isolation protocol were consistent with those obtained using the conventional MeOH and MTBE extraction methods. These results indicated that TRIzol reagent can be used to simultaneously isolate metabolites and lipids from a single sample. Thus, TRIzol reagent can be used in biological and clinical research, especially in multiomics studies.

Genome-based characterization of conjugative IncHI1B plasmid carrying carbapenemase genes blaVIM-1, blaIMP-23, and truncated blaOXA-256in Klebsiella pneumoniae NTU107224.

The wide dissemination of plasmids carrying antibiotic resistance determinants among bacteria is a severe threat to global public health. Here, we characterized an extensively drug-resistant (XDR) Klebsiella pneumoniae NTU107224 by whole genome sequencing (WGS) in combination with phenotypic tests. Broth dilution method was used to determine the minimal inhibitory concentrations (MICs) of NTU107224 to 24 antibiotics. The whole genome sequence of NTU107224 was determined by Nanopore/Illumina hybrid genome sequencing. Conjugation assay was performed to determine the transferability of plasmids in NTU107224 to recipient K. pneumoniae 1706. Larvae infection model was used to determine the effect(s) of conjugative plasmid pNTU107224-1 on bacterial virulence. Among the 24 antibiotics tested, XDR K. pneumoniae NTU107224 had low MICs only for amikacin (≤1 µg/mL), polymyxin B (0.25 µg/mL), colistin (0.25 µg/mL), eravacycline (0.25 µg/mL), cefepime/zidebactam (1 µg/mL), omadacycline (4 µg/mL), and tigecycline (0.5 µg/mL). Whole genome sequencing showed that the closed NTU107224 genome comprises a 5,076,795-bp chromosome, a 301,404-bp plasmid named pNTU107224-1, and a 78,479-bp plasmid named pNTU107224-2. IncHI1B plasmid pNTU107224-1 contained three class 1 integrons accumulated various antimicrobial resistance genes (including carbapenemase genes blaVIM-1, blaIMP-23, and truncated blaOXA-256) and the blast results suggested the dissemination of IncHI1B plasmids in China. By day 7 after infection, larvae infected with K. pneumoniae 1706 and transconjugant had 70% and 15% survival rates, respectively. We found that the conjugative plasmid pNTU107224-1 is closely related to IncHI1B plasmids disseminated in China and contributes to the virulence and antibiotic resistance of pathogens.

Anti-Helicobacter pylori activity of potential probiotic Lactiplantibacillus pentosus SLC13.

BACKGROUND: Here, we aimed to evaluate and compare the anti-Helicobacter pylori activity of potential probiotic Lactiplantibacillus pentosus SLC13 to Lactobacillus gasseri BCRC 14619 T and Lacticaseibacillus rhamnosus LGG. Phenotypic assays including growth curve, cell adhesion, and cellular cytotoxicity were performed to characterize SLC13. Anti-H. pylori activity of lactobacilli was determined by the disk diffusion method and co-culture assay. Exopolysaccharide (EPS) was extracted from lactobacilli to test its immune modulation activity, and IL-8 expression in AGS and GES-1 was determined by RT-qPCR. RESULTS: All three lactobacilli strains were tolerant to the simulated gastrointestinal conditions. SLC13 showed the highest adhesion ability to AGS and GES-1 cells, compared to LGG and BCRC 14619 T. The coculture assays of SLC13, LGG, and BCRC 14619 T with cells for 4 h showed no significant cytotoxic effects on cells. All tested strains exhibited an inhibitory effect against H. pylori J99. The cell-free supernatant (CFS) of three strains showed activity to inhibit H. pylori urease activity in a dose-dependent manner and the CFS of SLC13 had the highest urease inhibitory activity, compared to LGG and BCRC 14619 T. Only the treatment of AGS cells with SLC13 EPS significantly decreased the IL-8 expression induced by H. pylori infection as compared to cells treated with LGG and BCRC 14619 T EPS. CONCLUSIONS: SLC13 possesses potent antimicrobial activity against H. pylori growth, infection, and H. pylori-induced inflammation. These results suggest that SLC13 and its derivatives have the potential as alternative agents against H. pylori infection and alleviate inflammatory response.

Whole-genome-sequence-based characterization of an NDM-5-producing uropathogenic Escherichia coli EC1390.

BACKGROUND: Urinary tract infection (UTI) is one of the most common outpatient bacterial infections. In this study, we isolated and characterized an extensively-drug resistant (XDR) NDM-5-producing Escherichia coli EC1390 from a UTI patient by using whole-genome sequencing (WGS) in combination with phenotypic assays. METHODS: Antimicrobial susceptibility to 23 drugs was determined by disk diffusion method. The genome sequence of EC1390 was determined by Nanopore MinION MK1C platform. Conjugation assays were performed to test the transferability of EC1390 plasmids to E. coli recipient C600. Phenotypic assays, including growth curve, biofilm formation, iron acquisition ability, and cell adhesion, were performed to characterize the function of EC1390 plasmids. RESULTS: Our results showed that EC1390 was only susceptible to tigecycline and colistin, and thus was classified as XDR E. coli. A de novo genome assembly was generated using Nanopore 73,050 reads with an N50 value of 20,936 bp and an N90 value of 7,624 bp. WGS analysis showed that EC1390 belonged to the O101-H10 serotype and phylogenetic group A E. coli. Moreover, EC1390 contained 2 conjugative plasmids with a replicon IncFIA (pEC1390-1 with 156,286 bp) and IncFII (pEC1390-2 with 71,840 bp), respectively. No significant difference was observed in the bacterial growth rate in LB broth and iron acquisition ability between C600, C600 containing pEC1390-1, C600 containing pEC1390-2, and C600 containing pEC1390-1 and pEC1390-2. However, the bacterial growth rate in nutrition-limited M9 broth was increased in C600 containing pEC1390-2, and the cell adhesion ability was increased in C600 containing both pEC1390-1 and pEC1390-2. Moreover, these plasmids modulated the biofilm formation under different conditions. CONCLUSIONS: In summary, we characterized the genome of XDR-E. coli EC1390 and identified two plasmids contributing to the antimicrobial resistance, growth of bacteria in a nutrition-limited medium, biofilm formation, and cell adhesion.

Plasmid-mediated quinolone resistance determinants in fluoroquinolone-nonsusceptible Escherichia coli isolated from patients with urinary tract infections in a university hospital, 2009-2010 and 2020.

OBJECTIVES: This study aimed to characterize the plasmid-mediated quinolone resistance (PMQR) in fluoroquinolone nonsusceptible E. coli (FQNSEC) isolated from patients with urinary tract infections (UTIs) in 2019-2010 and 2020. METHODS: A total of 844 E. coli isolates were collected from UTI patients at National Cheng Kung University Hospital. The antimicrobial susceptibility of E. coli isolates to 21 antibiotics was determined by disk diffusion tests. The distribution of phylogenetic groups, virulence factor, and PMQR genes was determined by PCR. Conjugation assays were performed to investigate the transferability of qnr genes from FQNSEC isolates to E. coli C600. RESULTS: We found 211 (41.9%) and 152 (44.7%) E. coli isolates were FQNSEC in 2009-2010 and 2020, respectively. Phylogenetic group B2 was dominant in FQNSEC isolates (52.34%), followed by group F (10.47%), group B1 (9.64%), and group D (9.64%). FQNSEC isolates were more resistant to 17 of 19 tested antimicrobial agents, compared to the fluoroquinolone susceptible E. coli. PMQR screening results showed 34, 22, and 10 FQNSEC isolates containing aac(6')-Ib-cr, qnr genes, and efflux pump genes (qepA or oqxAB), respectively. PMQR E. coli isolates were more nonsusceptible to gentamicin, amoxicillin, ampicillin/sulbactam, imipenem, cefazolin, cefuroxime, cefmetazole, ceftriaxone, ceftazidime, and cefepime compared to non-PMQR FQNSEC. Moreover, 16 of 22 qnr-carrying plasmids were transferrable to the recipient C600. CONCLUSION: Here, we reported the high prevalence of MDR- and XDR-E. coli in FQNSEC isolates. Moreover, qnr-carrying plasmids were highly transferable and led to the resistance to other classes of antibiotics in the transconjugants.

A Longitudinal Nine-Year Study of the Molecular Epidemiology of Carbapenemase-Producing Enterobacterales Isolated From a Regional Hospital in Taiwan: Predominance of Carbapenemase KPC-2 and OXA-48.

Enterobacterales clinical isolates are now being resistant to clinically achievable concentrations of most commonly used antibiotics that makes treatment of hospitalized patients very challenging. We hereby determine the molecular characteristics of carbapenemase genes in carbapenem-resistant Enterobacterales (CRE) isolates in Taiwan. A total of 455 CRE isolates were identified between August 2011 to July 2020. Minimum inhibitory concentrations for selected carbapenems were tested using Vitek 2, and carbapenemase genes were determined using polymerase chain reaction in combination with sequencing. Phenotypic detection of carbapenemase was determined by modified carbapenem inactivation method (mCIM) and EDTA-modified carbapenem inactivation method (eCIM) to validate our PCR screening results. Pulsed-field gel electrophoresis (PFGE) was used to determine the clonality of carbapenemase-producing Enterobacterales (CPE) isolates, and the transferability of carbapenemase-carrying plasmids was determined by conjugation assays. A slight increase in carbapenem-resistant E. coli (CREC) was observed, however, the prevalence of carbapenem-resistant K. pneumoniae (CRKP) was steady, during 2011-2020. The dominant species among our CRE was K. pneumoniae (270/455, 59.3%), followed by E. coli (81/455, 17.8%), Morganella morganii (32/455, 7.0%), and Enterobacter cloacae (25/455, 5.5%). From 2011 to 2020, the total percentage of CPE increased steadily, accounting for 61.0% of CRE in 2020. Moreover, 122 of 455 CRE isolates (26.8%) were CPE. Among the CPE isolates, the dominant carbapenemase gene was bla OXA-48-like (54/122, 44.3%), and the second most common carbapenemase gene was bla KPC-2 (47/122, 38.5%). The sensitivity and specificity for mCIM to detect carbapenemase in the 455 isolates were both 100% in this study. The PFGE results showed that 39 carbapenemase-producing E. coli and 69 carbapenemase-producing K. pneumoniae isolates carrying bla KPC-2 and/or bla NDM-5 could be classified into 5 and 12 clusters, respectively. In conclusion, our results showed an increase in CPE isolates in Taiwan. Moreover, the distribution of carbapenemase and antimicrobial susceptibility in CPE were associated with PFGE typing.

Untargeted Microbial Exometabolomics and Metabolomics Analysis of Helicobacter pylori J99 and jhp0106 Mutant.

Untargeted metabolomic profiling provides the opportunity to comprehensively explore metabolites of interest. Herein, we investigated the metabolic pathways associated with Jhp0106, a glycosyltransferase enzyme in Helicobacter pylori. Through untargeted exometabolomic and metabolomic profiling, we identified 9 and 10 features with significant differences in the culture media and pellets of the wild-type (WT) J99 and jhp0106 mutant (Δjhp0106). After tentative identification, several phosphatidylethanolamines (PEs) were identified in the culture medium, the levels of which were significantly higher in WT J99 than in Δjhp0106. Moreover, the reduced lysophosphatidic acid absorption from the culture medium and the reduced intrinsic diacylglycerol levels observed in Δjhp0106 indicate the possibility of reduced PE synthesis in Δjhp0106. The results suggest an association of the PE synthesis pathway with flagellar formation in H. pylori. Further investigations should be conducted to confirm this finding and the roles of the PE synthesis pathway in flagellar formation. This study successfully demonstrates the feasibility of the proposed extraction procedure and untargeted exometabolomic and metabolomic profiling strategies for microbial metabolomics. They may also extend our understanding of metabolic pathways associated with flagellar formation in H. pylori.

Distinct Characteristics of Escherichia coli Isolated from Patients with Urinary Tract Infections in a Medical Center at a Ten-Year Interval.

Escherichia coli causing urinary tract infections (UTIs) are one of the most common outpatient bacterial infections. This study aimed to compare the characteristics of E. coli isolated from UTI patients in a single medical center in 2009-2010 (n = 504) and 2020 (n = 340). The antimicrobial susceptibility of E. coli was determined by the disk diffusion method. PCRs were conducted to detect phylogenetic groups, ST131, K1 capsule antigen, and 15 virulence factors. Phylogenetic group B2 dominated in our 2009-2010 and 2020 isolates. Moreover, no phylogenetic group E strains were isolated in 2020. E. coli isolates in 2020 were more susceptible to amoxicillin, ampicillin/sulbactam, cefuroxime, cefmetazole, ceftazidime, cefoxitin, tetracycline, and sulfamethoxazole/trimethoprim, compared to the isolates in 2009-2010. Extensively drug-resistant (XDR)-E. coli in 2009-2010 were detected in groups B1 (5 isolates), B2 (12 isolates), F (8 isolates), and unknown (1 isolate). In 2020, XDR-E. coli were only detected in groups A (2 isolates), B2 (5 isolates), D (1 isolate), and F (4 isolates). The prevalence of virulence factor genes aer and fimH were higher in E. coli in 2009-2010 compared to those in 2020. In contrast, afa and sat showed higher frequencies in E. coli isolates in 2020 compared to E. coli in 2009-2010.

Preparation of porous scaffolds by using freeze-extraction and freeze-gelation methods.

Freeze-fixation and freeze-gelation methods are presented in this paper which can be used to prepare highly porous scaffolds without using the time and energy consuming freeze-drying process. The porous structure was generated during the freeze of a polymer solution, following which either the solvent was extracted by a non-solvent or the polymer was gelled under the freezing condition; thus, the porous structure would not be destructed during the subsequent drying stage. Compared with the freeze-drying method, the presented methods are time and energy-saving, with less residual solvent, and easier to be scaled up. Besides, the problem of formation of surface skin can be resolved and the limitation of using solvent with low boiling point can be lifted by the presented methods. With the freeze-extraction and freeze-gelation methods, porous PLLA, PLGA, chitosan and alginate scaffolds were successfully fabricated. In addition to the presentation of the morphologies of the fabricated scaffolds, preliminary data of cell culture on them are as well included in the present work.