Syringic acid suppresses Cutibacterium acnes-induced inflammation in human keratinocytes via regulating the NLRP3/caspase-1/IL-1ß signaling axis by activating PPARγ/Nrf2-antioxidant pathway.

BACKGROUND: Our previous studies have demonstrated a strong relationship betweenCutibacterium acnes(C. acnes), oxidative stress, and acne inflammation. Syringic acid (SA) is a plant widely used for its antimicrobial, anti-inflammatory, and antioxidant activities, but lacking data on acne. This study aims to investigate the effect and mechanism of SA on acne inflammation induced by C. acnes in vitro and in vivo. METHODS: After using the SA to expose HaCaT keratinocytes, we reevaluated the effect of the SA on cell viability, cell apoptosis, ROS, CAT, SOD, and other inflammatory variables in the heat-killed C. acnes-treated HaCaT cells. Next, to induce mice with acne inflammation, ICR mice were given an intradermal injection of live C. acnes into their right ears. The effect of SA on this inflammation was then examined. Moreover, we explored the mechanism of SA on PPARγ/Nrf2 and NLRP3/caspase-1/IL-1ß pathways by ELISA, immunofluorescence microscopy, and western blot assay. RESULTS: Heat-killed C. acnes triggered remarkable cell apoptosis, ROS production, interleukin (IL)-1ß, IL-18, IL-6, and TNF-α release, reduced SOD and CAT activity, and upregulated the expression of proteins in HaCaT cells, including up-regulating IL-1ß, PPARγ, Nrf2, HO-1, NQO1, NLRP3, and caspase-1, whereas SA inhibited these effects by partially impairing PPARγ activation. In addition, PPARγ silencing decreased C. acnes-induced IL-1ß secretion and the production of intracellular ROS, down-regulating the expression of Nrf2. Nrf2 activator (SFN) enhanced anti-inflammatory activity through antioxidant mechanisms, boosting intracellular ROS production, reducing SOD and CAT activity, and promoting the increase in ROS, HO-1, NQO1, and IL-1ß levels, while PPARγ inhibitor (GW662) effectively inhibited this effect in heat-killed C. acnes-treated cells. Finally, SA also exhibited notable improvements in ear redness, swelling, and the expression of PPARγ, NLRP3, and IL-1ß in vivo. CONCLUSIONS: SA inhibited C. acnes-induced inflammation via regulating the NLRP3/caspase-1/IL-1ß signaling axis by activating the PPARγ/Nrf2-antioxidant pathway, suggesting a new treatment possibility for acne vulgaris.

Characteristics of the tmexC2-tmexD2-toprJ2 carrying of multi-drug resistant Hypervirulent Klebsiella Pneumoniae belonging ST29 and K212.

BACKGROUND: This study aimed to characterize a tigecycline-resistant Hypervirulent Klebsiella pneumoniae (HvKP) strain, identified as KLZT, carrying a tigecycline resistance gene cluster tmexC2-tmexD2-toprJ2 belonging to ST29 and serotype K212. MATERIALS AND METHODS: Antimicrobial susceptibility and virulence phenotypes were assessed, followed by whole genome sequencing (WGS) using PacBio II and MiSeq sequencers. Genome annotation was conducted using the RAST server, and bioinformatics analysis revealed the genetic characteristics for this strain. RESULTS: Antimicrobial and virulence phenotype testing indicated that K.pneumoniae strain KLZT can be considered as the Multi-Drug Resistant HvKP. WGS analysis showed that it has one 5,536,506 bp chromosome containing three plasmids in the size of 290963bp (pKLZT-1), 199,302bp (pKLZT-2) and 4820bp (pKLZT-3). KLZT was classified as ST29 and K212 serotype. Five (blaSHV-187, oqxA, oqxB, fosA6) and 6 resistance genes (tmexC2-tmxeD2-toprJ2, blaOXA-1, aac(6')-Ib-cr, catB3, arr-3 and blaLEN27) were identified from the chromosome and plasmid pKLZT-1, respectively. Resistance genes genetic context of plasmid pKLZT-1 analysis showed that tigecycline resistance gene cluster carrying region was flanked by umuC and umuD (umuD-hps-IS5-tmexC2-tmexD2-toprJ2-umuC) and other resistance genes and virulence factors (ureB, ureC and ureG) were carried by a (IS5075-Tn3-intI1 -aac(6')-Ib-cr-blaOXA-1-catB3-arr-3-blaLEN27-Tn3-ISkpn26-ureBCG- IS5075). CONCLUSIONS: Here, we present the WGS of a Multi-Drug Resistant HvKP KLZT belonging to ST29 with capsular serotype K212 contains a muti-drug resistance plasmid.

Co-occurrence of ST412 Klebsiella pneumoniae isolates with hypermucoviscous and non-mucoviscous phenotypes in a short-term hospitalized patient.

Hypermucoviscosity (HMV) is a phenotype that is commonly associated with hypervirulence in Klebsiella pneumoniae. The factors that contribute to the emergence of HMV subpopulations remain unclear. In this study, eight K. pneumoniae strains were recovered from an inpatient who had been hospitalized for 20 days. Three of the isolates exhibited a non-HMV phenotype, which was concomitant with higher biofilm formation than the other five HMV isolates. All eight isolates were highly susceptible to serum killing, albeit HMV strains were remarkably more infective than non-HMV counterparts in a mouse model of infection. Whole genome sequencing (WGS) showed that the eight isolates belonged to the K57-ST412 lineage. Average nucleotide identity (FastANIb) analysis indicated that eight isolates share 99.96% to 99.99% similarity and were confirmed to be the same clone. Through comparative genomics analysis, 12 non-synonymous mutations were found among these isolates, eight of which in the non-HMV variants, including rmpA (c.285delG) and wbaP (c.1305T > A), which are assumed to be associated with the non-HMV phenotype. Mutations in manB (c.1318G > A), dmsB (c.577C > T) and tkt (c.1928C > A) occurred in HMV isolates only. RNA-Seq revealed transcripts of genes involved in energy metabolism, carbohydrate metabolism and membrane transport, including cysP, cydA, narK, tktA, pduQ, aceB, metN, and lsrA, to be significantly dysregulated in the non-HMV strains, suggesting a contribution to HMV phenotype development. This study suggests that co-occurrence of HMV and non-HMV phenotypes in the same clonal population may be mediated by mutational mechanisms as well as by certain genes involved in membrane transport and central metabolism. IMPORTANCE: K. pneumoniae with a hypermucoviscosity (HMV) phenotype is a community-acquired pathogen that is associated with increased invasiveness and pathogenicity, and underlying diseases are the most common comorbid risk factors inducing metastatic complications. HMV was earlier attributed to the overproduction of capsular polysaccharide, and more data point to the possibility of several causes contributing to this bacterial phenotype. Here, we describe a unique event in which the same clonal population showed both HMV and non-HMV characteristics. Studies have demonstrated that this process is influenced by mutational processes and genes related to transport and central metabolism. These findings provide fresh insight into the mechanisms behind co-occurrence of HMV and non-HMV phenotypes in monoclonal populations as well as potentially being critical in developing strategies to control the further spread of HMV K. pneumoniae.

DNAzyme-RCA-based colorimetric and lateral flow dipstick assays for the point-of-care testing of exosomal m5C-miRNA-21.

Methylation of microRNAs (miRNAs) is a post-transcriptional modification that affects miRNA activity by altering the specificity of miRNAs to target mRNAs. Abnormal methylation of miRNAs in cancer suggests their potential as a tumor marker. However, the traditional methylated miRNA detection mainly includes mass spectrometry, sequencing and others; complex procedures and reliance on large instruments greatly limit their application in point-of-care testing (POCT). Based on this, we developed DNAzyme-RCA-based gold nanoparticle (AuNP) colorimetric and lateral flow dipstick (LFD) assays to achieve convenient detection of exosomal 5-methylcytosine miRNA-21 (m5C-miRNA-21) for the first time. The two assays achieved specific recognition and linear amplification of m5C-miRNA-21 through the DNAzyme triggered RCA reaction and color output with low background interference through AuNP aggregation induced by base complementary pairing. The lowest concentration of m5C-miRNA-21 visible to the naked eye of the two assays can reach 1 pM and 0.1 pM, respectively. Detection of exosomal m5C-miRNA-21 in clinical blood samples showed that the expression level of m5C-miRNA-21 in colorectal cancer patients was significantly higher than that in healthy individuals. This approach not only demonstrates a new strategy for the detection of colorectal cancer but also provides a reference for the development of novel diagnostic tools for other miRNA methylation-related diseases.

Bacteriophage-resistant carbapenem-resistant Klebsiella pneumoniae shows reduced antibiotic resistance and virulence.

Phage therapy has shown great promise in the treatment of bacterial infections. However, the effectiveness of phage therapy is compromised by the inevitable emergence of phage-resistant strains. In this study, a phage-resistant carbapenem-resistant Klebsiella pneumoniae strain SWKP1711R, derived from parental carbapenem-resistant K. pneumoniae strain SWKP1711 was identified. The mechanism of bacteriophage resistance in SWKP1711R was investigated and the molecular determinants causing altered growth characteristics, antibiotic resistance, and virulence of SWKP1711R were tested. Compared to SWKP1711, SWKP1711R showed slower growth, smaller colonies, filamentous cells visible under the microscope, reduced production of capsular polysaccharide (CPS) and lipopolysaccharide, and reduced resistance to various antibiotics accompanied by reduced virulence. Adsorption experiments showed that phage vB_kpnM_17-11 lost the ability to adsorb onto SWKP1711R, and the adsorption receptor was identified to be bacterial surface polysaccharides. Genetic variation analysis revealed that, compared to the parental strain, SWKP1711R had only one thymine deletion at position 78 of the open reading frame of the lpcA gene, resulting in a frameshift mutation that caused alteration of the bacterial surface polysaccharide and inhibition of phage adsorption, ultimately leading to phage resistance. Transcriptome analysis and quantitative reverse transcriptase PCR revealed that genes encoding lipopolysaccharide synthesis, ompK35, blaTEM-1, and type II and Hha-TomB toxin-antitoxin systems, were all downregulated in SWKP1711R. Taken together, the evidence presented here indicates that the phenotypic alterations and phage resistance displayed by the mutant may be related to the frameshift mutation of lpcA and altered gene expression. While evolution of phage resistance remains an issue, our study suggests that the reduced antibiotic resistance and virulence of phage-resistant strain derivatives might be beneficial in alleviating the burden caused by multidrug-resistant bacteria.

Vitamin K1 ameliorates lipopolysaccharide-triggered skeletal muscle damage revealed by faecal bacteria transplantation.

BACKGROUND: Sepsis-associated muscle weakness is common in patients of intensive care units (ICUs), and it is closely associated with poor outcomes. The mechanism of sepsis-induced muscle weakness is unclear. Recent studies have found that gut microbiota and metabolites are involved in the regulation of skeletal muscle mass and metabolism. This study aimed to investigate the effects of gut microbiota and metabolites on sepsis-associated muscle weakness. METHODS: In a lipopolysaccharide (LPS)-induced inflammation mouse model, mice with different sensitivities to LPS-induced inflammation were considered as donor mice for the faecal microbiota transplantation (FMT) assay, and recipient mice were divided into sensitive (Sen) and resistant (Res) groups. Skeletal muscle mass and function, as well as colonic barrier integrity were tested and gut microbiota and metabolite composition were analysed in both groups of mice. The effect of intestinal differential metabolite vitamin K1 on LPS-triggered muscle damage was investigated, and the underlying mechanism was explored. RESULTS: Recipients exhibited varying LPS-triggered muscle damage and intestinal barrier disruption. Tibialis anterior (TA) muscle of Sen exhibited upregulated expression levels of MuRF-1 (0.825 ± 0.063 vs. 0.304 ± 0.293, P = 0.0141) and MAFbx (1.055 ± 0.079 vs. 0.456 ± 0.3, P = 0.0092). Colonic tight junction proteins ZO-1 (0.550 ± 0.087 vs. 0.842 ± 0.094, P = 0.0492) and occludin (0.284 ± 0.057 vs. 0.664 ± 0.191, P = 0.0487) were significantly downregulated in the Sen group. Metabolomic analysis showed significantly higher vitamin K1 in the faeces (P = 0.0195) and serum of the Res group (P = 0.0079) than those of the Sen group. After vitamin K1 intervention, muscle atrophy-related protein expression downregulated (P < 0.05). Meanwhile SIRT1 protein expression were upregulated (0.320 ± 0.035 vs. 0.685 ± 0.081, P = 0.0281) and pNF-κB protein expression were downregulated (0.815 ± 0.295 vs. 0.258 ± 0.130, P = 0.0308). PI3K (0.365 ± 0.142 vs. 0.763 ± 0.013, P = 0.0475), pAKT (0.493 ± 0.159 vs. 1.183 ± 0.344, P = 0.0254) and pmTOR (0.509 ± 0.088 vs. 1.110 ± 0.190, P = 0.0368) protein expression levels were upregulated in TA muscle. Meanwhile, vitamin K1 attenuated serum inflammatory factor levels. CONCLUSIONS: Vitamin K1 might ameliorate LPS-triggered skeletal muscle damage by antagonizing NF-κB-mediated inflammation through upregulation of SIRT1 and regulating the balance between protein synthesis and catabolism.

Comparative genomics analysis of Stenotrophomonas maltophilia strains from a community.

Background: Stenotrophomonas maltophilia is a multidrug-resistant (MDR) opportunistic pathogen with high resistance to most clinically used antimicrobials. The dissemination of MDR S. maltophilia and difficult treatment of its infection in clinical settings are global issues. Methods: To provide more genetic information on S. maltophilia and find a better treatment strategy, we isolated five S. maltophilia, SMYN41-SMYN45, from a Chinese community that were subjected to antibiotic susceptibility testing, biofilm formation assay, and whole-genome sequencing. Whole-genome sequences were compared with other thirty-seven S. maltophilia sequences. Results: The five S. maltophilia strains had similar antibiotic resistance profiles and were resistant to ß-lactams, aminoglycosides, and macrolides. They showed similar antimicrobial resistance (AMR) genes, including various efflux pumps, ß-lactamase resistance genes (blaL1/2), aminoglycoside resistance genes [aac(6'), aph(3'/6)], and macrolide-resistant gene (MacB). Genome sequencing analysis revealed that SMYN41-SMYN45 belonged to sequence type 925 (ST925), ST926, ST926, ST31, and ST928, respectively, and three new STs were identified (ST925, ST926, and ST928). Conclusion: This study provides genetic information by comparing genome sequences of several S. maltophilia isolates from a community of various origins, with the aim of optimizing empirical antibiotic medication and contributing to worldwide efforts to tackle antibiotic resistance.

Circulation of enterotoxigenic Escherichia coli (ETEC) isolates expressing CS23 from the environment to clinical settings.

IMPORTANCE: The importance of clean water cannot be overstated. It is a vital resource for maintaining health and well-being. Unfortunately, water sources contaminated with fecal discharges from animal and human origin due to a lack of wastewater management pose a significant risk to communities, as they can become a means of transmission of pathogenic bacteria like enterotoxigenic E. coli (ETEC). ETEC is frequently found in polluted water in countries with a high prevalence of diarrheal diseases, such as Bolivia. This study provides novel insights into the circulation of ETEC between diarrheal cases and polluted water sources in areas with high rates of diarrheal disease. These findings highlight the Choqueyapu River as a potential reservoir for emerging pathogens carrying antibiotic-resistance genes, making it a crucial area for monitoring and intervention. Furthermore, the results demonstrate the feasibility of a low-cost, high-throughput method for tracking bacterial pathogens in low- and middle-income countries, making it a valuable tool for One Health monitoring efforts.

Characteristics of a ceftadine/avibatam resistance KPC-33-producing Klebsiella Pneumoniae strain with capsular serotype K19 belonging to ST15.

OBJECTIVES: The aim of this study was to characterize the blaKPC-33 in a ST15-K19 ceftazidime-avibactam (CAZ-AVI)-resistant Klebsiella pneumoniae strain after the antibiotic CAZ-AVI was approved for use in Wuxi No. 2 People's Hospital, China. METHODS: Antimicrobial susceptibility testing was performed by the microdilution broth method. Whole genome sequencing (WGS) was performed using PacBio II and MiSeq sequencers. High-quality reads were assembled using the SOAPdenovo and GapCloser v1.12, and genome annotation was performed using the NCBI Prokaryotic Genome Annotation Pipeline (PGAP). Genomic characteristics were analysed by using bioinformatics methods. RESULTS: K. pneumoniae strain KPHRJ showed resistance to CAZ-AVI. WGS analysis showed that strain KPHRJ had one 5 536 506 bp chromosome (57.25% G+C content) and one plasmid (133 451 bp, G+C 54.29%). KPHRJ was classified as ST15 and K19 serotype. Resistome analysis showed that KPHRJ carries seven antimicrobial resistance genes (ARGs). WGS analysis and conjugation experiments demonstrated that the blaKPC-33 gene was carried by plasmid pKPHRJ, flanked by two copies of IS26 mobile elements (IS26-ISKpn27-blaKPC-33-ISKpn6-korC-TnAs1-tetR-tetA-Tn3-IS26). Besides these acquired resistance genes, mutations in porin protein-coding genes, such as OmpK36 and OmpK37, which may reduce susceptibility to the CAZ-AVI, were also identified from the genome. CONCLUSION: Here, we present the WGS of a CAZ-AVI resistant K. pneumoniae isolate, strain KPHRJ, with capsular serotype K19 and belonging to ST15. CAZ-AVI resistance is likely conferred by a KPC-2 variant, blaKPC-33 and mutations in porin-coding genes. We speculate that the approval of the CAZ-AVI in hospital could contribute to the emergence of these genomic features by providing a selective pressure leading to the emergence of CAZ-AVI resistant bacteria.

Escherichia coli ST2797 Is Abundant in Wastewater and Might Be a Novel Emerging Extended-Spectrum Beta-Lactamase E. coli.

The increasing prevalence of antibiotic-resistant bacteria is an emerging threat to global health. The analysis of antibiotic-resistant enterobacteria in wastewater can indicate the prevalence and spread of certain clonal groups of multiresistant bacteria. In a previous study of Escherichia coli that were isolated from a pump station in Norway over 15 months, we found a recurring E. coli clone that was resistant to trimethoprim, ampicillin, and tetracycline in 201 of 3,123 analyzed isolates (6.1%). 11 representative isolates were subjected to whole-genome sequencing and were found to belong to the MLST ST2797 E. coli clone with plasmids carrying resistance genes, including blaTEM-1B, sul2, dfrA7, and tetB. A phenotypic comparison of the ST2797 isolates with the uropathogenic ST131 and ST648 that were repeatedly identified in the same wastewater samples revealed that the ST2797 isolates exhibited a comparable capacity for temporal survival in wastewater, greater biofilm formation, and similar potential for the colonization of mammalian epithelial cells. ST2797 has been isolated from humans and has been found to carry extended spectrum ß-lactamase (ESBL) genes in other studies, suggesting that this clonal type is an emerging ESBL E. coli. Collectively, these findings show that ST2797 was more ubiquitous in the studied wastewater than were the infamous ST131 and ST648 and that ST2797 may have similar abilities to survive in the environment and cause infections in humans. IMPORTANCE The incidence of drug-resistant bacteria found in the environment is increasing together with the levels of antibiotic-resistant bacteria that cause infections. The COVID-19 pandemic has shed new light on the importance of monitoring emerging threats and finding early warning systems. Therefore, to mitigate the antimicrobial resistance burden, the monitoring and early identification of antibiotic-resistant bacteria in hot spots, such as wastewater treatment plants, are required to combat the occurrence and spread of antibiotic-resistant bacteria. Here, we applied a PhenePlate system as a phenotypic screening method for genomic surveillance and discovered a dominant and persistent E. coli clone ST2797 with a multidrug resistance pattern and equivalent phenotypic characteristics to those of the major pandemic lineages, namely, ST131 and ST648, which frequently carry ESBL genes. This study highlights the continuous surveillance and report of multidrug resistant bacteria with the potential to spread in One Health settings.

Coexistence of tmexCD3-toprJ1b tigecycline resistance genes with two novel bla VIM-2-carrying and bla OXA-10-carrying transposons in a Pseudomononas asiatica plasmid.

Introduction: Tigecycline and carbapenems are considered the last line of defense against microbial infections. The co-occurrence of resistance genes conferring resistance to both tigecycline and carbapenems in Pseudomononas asiatica was not investigated. Methods: P. asiatica A28 was isolated from hospital sewage. Antibiotic susceptibility testing showed resistance to carbapenem and tigecycline. WGS was performed to analyze the antimicrobial resistance genes and genetic characteristics. Plasmid transfer by conjugation was investigated. Plasmid fitness costs were evaluated in Pseudomonas aeruginosa transconjugants including a Galleria mellonella infection model. Results: Meropenem and tigecycline resistant P. asiatica A28 carries a 199, 972 bp long plasmid PLA28.4 which harbors seven resistance genes. Sequence analysis showed that the 7113 bp transposon Tn7389 is made up of a class I integron without a 5'CS terminal and a complete tni module flanked by a pair of 25bp insertion repeats. Additionally, the Tn7493 transposon, 20.24 kp long, with a complete 38-bp Tn1403 IR and an incomplete 30-bp Tn1403 IR, is made up of partial skeleton of Tn1403, a class I integron harboring bla OXA-10, and a Tn5563a transposon. Moreover, one tnfxB3-tmexC3.2-tmexD3b-toprJ1b cluster was found in the plasmid and another one in the the chromosome. Furthermore, plasmid PLA28.4 could be conjugated to P. aeruginosa PAO1, with high fitness cost. Discussion: A multidrug-resistant plasmid carrying tmexCD3-toprJ1b and two novel transposons carrying bla VIM-2 and bla OXA-10 -resistant genes was found in hospital sewage, increasing the risk of transmission of antibiotic-resistant genes. These finding highlight the necessary of controlling the development and spread of medication resistance requires continuous monitoring and management of resistant microorganisms in hospital sewage.

Understanding drug nanocarrier and blood-brain barrier interaction based on a microfluidic microphysiological model.

As many nanoparticles (NPs) have been exploited as drug carriers to overcome the resistance of the blood-brain barrier (BBB), reliable in vitro BBB models are urgently needed to help researchers to comprehensively understand drug nanocarrier-BBB interaction during penetration, which can prompt pre-clinical nanodrug exploitation. Herein, we developed a microfluidic microphysiological model, allowing the analysis of BBB homeostasis and NP penetration. We found that the BBB penetrability of gold nanoparticles (AuNPs) was size- and modification-dependent, which might be caused by a distinct transendocytosis pathway. Notably, transferrin-modified 13 nm AuNPs held the strongest BBB penetrability and induced the slightest BBB dysfunction, while bare 80 nm and 120 nm AuNPs showed opposite results. Moreover, further analysis of the protein corona showed that PEGylation reduced the protein absorption, and some proteins facilitated the BBB penetration of NPs. The developed microphysiological model provides a powerful tool for understanding the drug nanocarrier-BBB interaction, which is vital for exploiting high-efficiency and biocompatible nanodrugs.

Function analysis of choline binding domains (CBDs) of LytA, LytC and CbpD in biofilm formation of Streptococcus pneumoniae.

Biofilm formation is an important strategy for the colonization of Streptococcus pneumoniae, which can increase the capacity to evade antibiotic and host immune stress. Extracellular choline-binding proteins (CBPs) are required for successful biofilm formation, but the function of extracellular CBPs in the process of biofilm formation is not fully understood. In this study, we tend to analyze the functions of LytA, LytC and CbpD in biofilm formation by in vitro studies with their choline-binding domains (CBDs). Biofilm formation of S. pneumoniae was enhanced when cultured in medium supplemented with CBD-C and CBD-D. Parallel assays with ChBp-Is (choline binding repeats with different C-terminal tails) and character analysis of CBDs reveal a higher isoelectric point (pI) is related to promotion of biofilm formation. Phenotype characterization of biofilms revel CBD-C and CBD-D function differently, CBD-C promoting the formation of membrane-like structures and CBD-D promoting the formation of regular reticular structures. Gene expression analysis reveals membrane transport pathways are influenced with the binding of CBDs, among which the phosphate uptake and PTS of galactose pathways are both up-regulated under conditions with CBDs. Further, extracellular substances detection revealed that extracellular proteins increased with CBD-A and CBD-D, exhibiting as increase in extracellular high molecular weight proteins. Extracellular DNA increased under CBD-A but decreased under CBD-C and CBD-D; Extracellular phosphate increased under CBD-C. These support the alterations in membrane transport pathways, and reveal diverse reactions to extracellular protein, DNA and phosphate of these three CBDs. Overall, our results indicated extracellular CBP participate in biofilm formation by affecting surface charge and membrane transport pathways of pneumococcal cells, as well as promoting reactions to extracellular substances.

Subinhibitory antibiotic concentrations promote the horizontal transfer of plasmid-borne resistance genes from Klebsiellae pneumoniae to Escherichia coli.

Horizontal gene transfer plays an important role in the spread of antibiotic resistance, in which plasmid-mediated conjugation transfer is the most important mechanism. While sub-minimal inhibitory concentrations (sub-MIC) of antibiotics could promote conjugation frequency, the mechanism by which sub-MIC levels of antibiotics affect conjugation frequency is not clear. Here, we used Klebsiella pneumoniae SW1780 carrying the multi-drug resistance plasmid pSW1780-KPC as the donor strain, to investigate the effects of sub-MICs of meropenem (MEM), ciprofloxacin (CIP), cefotaxime (CTX), and amikacin (AK) on conjugational transfer of pSW1780-KPC from SW1780 to Escherichia coli J53. Our results showed that the transfer frequencies increased significantly by treating SW1780 strain with sub-MIC levels of MEM, CIP, CTX and AK. Transfer frequencies at sub-MIC conditions in a Galleria mellonella were significantly higher than in vitro. To investigate gene expression and metabolic effects, RT-qPCR and LC-MS-based metabolome sequencing were performed. Transcript levels of T4SS genes virB1, virB2, virB4, virB8, and conjugation-related genes traB, traK, traE, and traL were significantly upregulated by exposure to sub-MICs of MEM, CIP, CTX, and AK. Metabolome sequencing revealed nine differentially regulated metabolites. Our findings are an early warning for a wide assessment of the roles of sub-MIC levels of antibiotics in the spread of antibiotic resistance.

The relationship between the number of stenotic coronary arteries and the gut microbiome in coronary heart disease patients.

An increasing number of studies have shown that the gut microbiome plays an important role in the development of coronary heart disease (CHD). However, there are no clear studies on the relationship between the gut microbiome and the number of stenotic coronary arteries. To clarify whether the gut microbiome is associated with the number of stenotic coronary arteries in CHD, we performed the 16S rRNA gene sequencing for the V3-V4 region in the gut microbiota from 9 healthy controls (C) and 36 CHD patients, which including 25 CHD patients with multivessel (MV) lesion and 11 CHD patients with single-vessel (SV) lesion. It showed that the abundance of the genus Escherichia-Shigella was significantly increased in the MV and SV groups compared with C group, while the abundance of the genera Subdoligranulum and Collinsella was significantly decreased. Biomarkers based on three gut microbiotas (Escherichia-Shigella, Subdoligranulum, and Collinsella) and three plasma metabolites(left atrial diameter (LA), low density lipoprotein (LDL), and total bile acids (TBA)) were able to distinguish CHD patients with different numbers of stenotic coronary arteries. Functional prediction of the gut microbiome was performed based on the Kyoto Encyclopedia of Genes and Genomes (KEGG) database. The results showed that the gut microbial function of MV and SV group patients was richer than C group in betaine biosynthesis and unsaturated fatty acid biosynthesis, in the contrast less than C group in sphingolipid metabolism and primary bile acid biosynthesis. In summary, our study showed that the composition and function of the gut microbiome changed significantly from healthy controls to CHD patients with different numbers of coronary lesions.

Identification of the BolA Protein Reveals a Novel Virulence Factor in K. pneumoniae That Contributes to Survival in Host.

BolA has been characterized as an important transcriptional regulator, which is induced in the stationary phase of growth and is often associated with bacterial virulence. This study was initiated to elucidate the role of the BolA in the virulence of K. pneumoniae. Using a mouse infection model, we revealed bolA mutant strain yielded significantly decreased bacterial loads in the liver, spleen, lung, and kidney, and failed to form liver abscesses. Gene deletion demonstrated that the bolA was required for siderophore production, biofilm formation, and adhesion to human colon cancer epithelial cells HCT116. Quantitative reverse transcriptase PCR (RT-qPCR) indicated that BolA could impact the expression of pulK, pulF, pulE, clpV, vgrG, entE, relA, and spoT genes on a genome-wide scale, which are related to type II secretion system (T2SS), type VI secretion system (T6SS), guanosine tetraphosphate (ppGpp), and siderophore synthesis and contribute to fitness in the host. Furthermore, the metabolome analysis showed that the deletion of the bolA gene led to decreased pools of five metabolites: biotin, spermine, cadaverine, guanosine, and flavin adenine dinucleotide, all of which are involved in pathways related to virulence and stress resistance. Taken together, we provided evidence that BolA was a significant virulence factor in the ability of K. pneumoniae to survive, and this was an important step in progress to an understanding of the pathways underlying bacterial virulence. IMPORTANCE BolA has been characterized as an important transcriptional regulator, which is induced in the stationary phase of growth and affects different pathways directly associated with bacterial virulence. Here, we unraveled the role of BolA in several phenotypes associated with the process of cell morphology, siderophore production, biofilm formation, cell adhesion, tissue colonization, and liver abscess. We also uncovered the importance of BolA for the success of K. pneumoniae infection and provided new clues to the pathogenesis strategies of this organism. This work constitutes a relevant step toward an understanding of the role of BolA protein as a master regulator and virulence factor. Therefore, this study is of great importance for understanding the pathways underlying K. pneumoniae virulence and may contribute to public health care applications.

Isolation and Characterization of vB_kpnM_17-11, a Novel Phage Efficient Against Carbapenem-Resistant Klebsiella pneumoniae.

Phages and phage-encoded proteins exhibit promising prospects in the treatment of Carbapenem-Resistant Klebsiella pneumoniae (CRKP) infections. In this study, a novel Klebsiella pneumoniae phage vB_kpnM_17-11 was isolated and identified by using a CRKP host. vB_kpnM_17-11 has an icosahedral head and a retractable tail. The latent and exponential phases were 30 and 60 minutes, respectively; the burst size was 31.7 PFU/cell and the optimal MOI was 0.001. vB_kpnM_17-11 remained stable in a wide range of pH (4-8) and temperature (4-40°C). The genome of vB_kpnM_17-11 is 165,894 bp, double-stranded DNA (dsDNA), containing 275 Open Reading Frames (ORFs). It belongs to the family of Myoviridae, order Caudovirales, and has a close evolutionary relationship with Klebsiella phage PKO111. Sequence analysis showed that the 4530 bp orf022 of vB_kpnM_17-11 encodes a putative depolymerase. In vitro testing demonstrated that vB_kpnM_17-11 can decrease the number of K. pneumoniae by 105-fold. In a mouse model of infection, phage administration improved survival and reduced the number of K. pneumoniae in the abdominal cavity by 104-fold. In conclusion, vB_kpnM_17-11 showed excellent in vitro and in vivo performance against K. pneumoniae infection and constitutes a promising candidate for the development of phage therapy against CRKP.

Comparison of Two Distinct Subpopulations of Klebsiella pneumoniae ST16 Co-Occurring in a Single Patient.

The higher resistance rate to ceftazidime-avibactam (CZA) is mainly related to carbapenem resistance, especially New Delhi metallo-ß-lactamase (NDM). The CZA-susceptible Klebsiella pneumoniae (K191663) and the later CZA-resistant isolates (K191724, K191725, K191773) co-producing NDM-4 and OXA-181 were obtained from the same hospitalized patient returning from Vietnam. Our study aims to elucidate the diversity of K. pneumoniae ST16 through comparative analysis of whole-genome sequencing (WGS) data and identify the potential evolution of plasmids by sequencing longitudinal clinical isolates during antibiotic treatment. Firstly, multilocus sequence typing analysis and phylogenic analysis suggested that these strains belong to the two lineages of K. pneumoniae ST16. Surprisingly, the CZA-resistant strains were closely related to K. pneumoniae ST16 described in South Korea, instead of the blaNDM-4- or blaOXA-181-carrying ST16 reported in Vietnam. Secondly, blaNDM-4, blaTEM-1B, and rmtB co-existed on a self-conjugative IncFII(Yp)-like plasmid, which played a significant role in CZA resistance. It could transfer into the recipient Escherichia coli J53 at high frequency, indicating the risk of mobile carbapenemases. In addition, the loss of 12-kbp fragment occurred in blaNDM-4-positive isolate (K191773), which was likely caused by insertion sequence-mediated homologous recombination. Last but not least, as a repressor of acrAB operon system, acrR was truncated by a frameshift mutation in K191663. Thus, our study provided baseline information for monitoring the occurrence and development of bacterial resistance. IMPORTANCE As a leading health care-acquired infection pathogen, Klebsiella pneumoniae is threatening a large number of inpatients due to its diverse antibiotic resistance and virulence factors. Heretofore, with a growing number of reports about the coexistence of several carbapenemases in carbapenem-resistant K. pneumoniae (CRKP), epidemiologic surveillance has been strengthened. Nevertheless, the nosocomial outbreaks by CRKP ST16 are gradually increasing worldwide. Our study provides a deeper insight into the diversification of clinical isolates of CRKP ST16 in China. In addition, the comparison analysis of resistant plasmids may reveal the transmission of carbapenemase-encoding genes. Furthermore, our study also highlights the importance of longitudinal specimen collection and continuous monitoring during the treatment, which play a crucial role in understanding the development of antibiotic resistance and the evolution of resistance plasmids.

Co-occurrence of Klebsiella variicola and Klebsiella pneumoniae Both Carrying bla KPC from a Respiratory Intensive Care Unit Patient.

OBJECTIVE: The aim of this study was to use whole-genome sequencing to characterize Klebsiella pneumoniae SKp2F and Klebsiella variicola SKv2E, both carrying bla KPC, co-isolated from the same sputum specimen. METHODS: Antimicrobial susceptibility testing was performed using microbroth dilution. Biofilm formation was determined by crystal violet staining and virulence was measured by a serum killing assay. Whole-genome sequencing of SKp2F and SKv2E was performed using an Illumina sequencer and the genetic characteristics were analyzed by computer. RESULTS: SKp2F and SKv2E were sensitive only to tigecycline and polymyxin among the tested antibiotics. The biofilm-forming ability of SKv2E is stronger than that of SKp2F. The grades of serum resistance of SKp2F and SKv2E are 4 and 3. MLST analysis of the 6,115,610 bp and 5,403,687 bp of SKv2E and SKp2F showed associations with ST1615 and ST631, respectively. SKv2E carried 13 resistance genes (bla KPC-2, bla TEM-1A, bla LEN17, aadA16, arr-3, qnrB4, oqxA/B, dfrA27, sul1, tetD, fosA, qacEΔ1) and SKp2F carried 23 (bla KPC-2, bla CTX-M-3, bla TEM-1B, bla CTX-M-65, bla SHV-27, aac(6')-IIa, rmtB, arr-3, aph(3')-Ia, aadA16, qnrS1, aac(6')-Ib-cr, qnrB91, oqxA/B, mph(A), tet(A), fosA, dfrA27, and two copies of qacEΔ1-sul1). Most of them were carried by various mobile genetic elements, such as IncFIB(K)/IncFII(K)/IncFII(Yp), IncFII(K) plasmid, Tn6338, and In469. Both SKv2E and SKp2F carried a large number of virulence factors, including type 1 and 3 fimbriae, capsule, aerobactin (iutA), ent siderophore (entABCDEFS, fepABCDGfes), and salmochelin (iroE/iroEN). SKv2E also carried type IV pili (pilW), fimbrial adherence (steB, stfD), and capsule biosynthesis gene (glf). CONCLUSION: bla KPC-2-carrying K. variicola and K. pneumoniae, which carried multiple resistance genes, virulence factors, and highly similar mobile genetic elements, were identified from the same specimen, indicating that clinical samples may carry multiple bacteria. We should avoid misidentification, and bear in mind that resistance genes carrying mobile genetic elements can be transmitted or integrated between bacteria in the same host.

Whole-Genomic Analysis of NDM-5-Producing Enterobacteriaceae Recovered from an Urban River in China.

PURPOSE: Three NDM-5-producing Enterobacteriaceae (Escherichia coli, Klebsiella pneumoniae, and Citrobacter braakii, one each) were isolated during a screening study for the presence of carbapenemase-producing Enterobacteriaceae (CPE) strains in urban rivers in China. The aim of the present study was to characterize these NDM-5-producing isolates by using whole-genome analysis. METHODS: In vitro susceptibility testing was performed using the broth microdilution method. Conjugation assay was carried out to investigate the transferability of bla NDM-5-harboring plasmids. Whole-genome sequencing was performed using an Illumina HiSeq combined with the PacBio RSII system. The genetic characteristics of the bla NDM-5-harboring plasmids were analyzed. Antimicrobial resistance genes and virulence genes were identified from the genome sequences. Phylogenetic analysis was performed based on core genome. RESULTS: Antimicrobial susceptibility testing showed that all three isolates were resistant to carbapenems, cephalosporins, quinolones, and aminoglycosides, and susceptible to colistin. Whole-genome sequencing showed that each isolate carried multiple antibiotic resistance genes mediating multidrug resistance, and harbored numerous virulence genes, some of which were located on plasmids. In these isolates, bla NDM-5 was carried by an IncX3 plasmid in K. pneumoniae and C. braakii, and on an IncR/IncX1 plasmid in E. coli. Conjugation experiments showed that these bla NDM-5-haboring plasmids were successfully transferred to E. coli J53. Phylogenetic analysis revealed that E. coli SCLZR49 was present in a cluster with isolates of different origin, K. pneumoniae SCLZR50 was mainly clustered with clinical isolates, and C. braakii SCLZR53 had closely genetic relationship with environmental isolates. CONCLUSION: This study revealed contamination of the urban river ecosystems by clinically significant carbapenemase gene bla NDM-5, raising the possibility of plasmid transmission into the environmental from humans and highlighting the need for a constant surveillance of CPE in the environment under the "One-Health" perspective.