Larvae Infection Model Research Articles

Molecular characterization of colistin-resistant Klebsiella pneumoniae isolates and their conjugative mcr-carrying plasmids

BackgroundThis study aimed to characterize colistin-resistant K. pneumoniae (CoRKp) strains isolated from patients with urinary tract infections and bacteremia between 1999 and 2022 at a tertiary teaching hospital in Taiwan. MethodsA total of 1,966K. pneumoniae isolates were collected, among which 21 strains were identified as CoRKp. The antimicrobial susceptibility of these CoRKp strains to 19 antibiotics was assessed. The genome characteristics of 21 CoRKp strains were determined by Nanopore-Illumina hybrid whole genome sequencing. Additionally, conjugation assays were conducted to determine the transferability of plasmids carrying mcr genes to K. pneumoniae ATCC BAA-1706 and E. coli C600. The larvae infection model was used to analyze the differences in virulence between transconjugants and recipient strains. ResultsAmong the 21 CoRKp, 12 were multidrug-resistant, and four were extensively drug-resistant. The distribution of sequence types (STs) and K types among the CoRKp strains was quite diverse, and ST307 (5 strains) and K64 (3 strains) dominated in CoRKp. The insertion elements IS903B and ISVsa5, were found to inactivate mgrB of 1 and 2 CoRKp isolates, respectively. Moreover, 1, 4, 6, and 1 missense mutations of PhoQ, PmrA, PmrB, and MgrB, were identified in 21 CoRKp. Only two isolates SC-KP169 and SC-KP585 carried mcr-1 and mcr-8, respectively. The plasmid pSC-KP169-1 could be transferred inter- and intra-genus and contributed to the virulence of K. pneumoniae to larvae. In contrast, the plasmid pSC-KP585-1 could be transferred to E. coli but could not affect its virulence to larvae. ConclusionsWe identified 21 CoRKp from 1,966 isolates and found a conjugative plasmid carrying mcr-1 gene that contributed to the virulence of K. pneumoniae to larvae.

Anti-virulence potential of iclaprim, a novel folic acid synthesis inhibitor, against Staphylococcus aureus

Infections caused by Staphylococcus aureus pose a significant global public problem. Therefore, new antibiotics and therapeutic strategies are needed to combat this pathogen. This investigation delves into the effects of iclaprim, a newly discovered inhibitor of folic acid synthesis, on S. aureus virulence. The phenotypic and genotypic effects of iclaprim were thoroughly examined in relation to virulence factors, biofilm formation, and dispersal, as well as partial virulence-encoding genes associated with exoproteins, adherence, and regulation in S. aureus MW2, N315, and ATCC 25923. Then, the in vivo effectiveness of iclaprim on S. aureus pathogenicity was explored by a Galleria mellonella larvae infection model. The use of iclaprim at sub-inhibitory concentrations (sub-MICs) resulted in a reduction of α-hemolysin (Hla) production and a differential effect on the activity of coagulase in S. aureus strains. The results of biofilm formation and eradication assay showed that iclaprim was highly effective in depolymerizing the mature biofilm of S. aureus strains at concentrations of 1 MIC or greater, however, inhibited the biofilm-forming ability of only strains N315 and ATCC 25923 at sub-MICs. Interestingly, treatment of strains with sub-MICs of iclaprim resulted in significant stimulation or suppression of most virulence-encoding genes expression. Iclaprim did not affect the production of δ-hemolysin or staphylococcal protein A (SpA), nor did it impact the total activity of proteases, nucleases, and lipases. In vivo testing showed that sub-MICs of iclaprim significantly improves infected larvae survival. The present study offered valuable insights towards a better understating of the influence of iclaprim on different strains of S. aureus. The findings suggest that iclaprim may have potential as an anti-virulence and antibiofilm agent, thus potentially mitigating the pathogenicity of S. aureus and improving clinical outcomes associated with infections caused by this pathogen.Key points• Iclaprim effectively inhibits α-hemolysin production and biofilm formation in a strain-dependent manner and was an excellent depolymerizing agent of mature biofilm• Iclaprim affected the mRNA expression of virulence-encoding genes associated with exoproteins, adherence, and regulation• In vivo study in G. mellonella larvae challenged with S. aureus exhibited that iclaprim improves larvae survival

Discovery of quaternized pyridine-thiazole-ruthenium complexes as potent anti-Staphylococcus aureus agents

Quaternization of ruthenium complexes may be a promising strategy for the development of new antibiotics. In response to the increasing bacterial resistance, we integrated the quaternary amine structure into the design of ruthenium complexes and evaluated their antibacterial activity. All the ruthenium complexes showed good antibacterial activity against the tested Staphylococcus aureus (S. aureus). Ru-8 was the most effective antibacterial agent that displayed excellent antibacterial activity against S. aureus (MIC = 0.78–1.56 μg/mL). In vitro experiments showed that all nine ruthenium complexes had low hemolytic toxicity to rabbit erythrocytes. Notably, Ru-8 was found to disrupt bacterial cell membranes, alter their permeability, and induce ROS production in bacteria, all the above leading to the death of bacteria without inducing drug resistance. To further explore the antibacterial activity of Ru-8in vivo, we established a mouse skin wound infection model and a G. mellonella larvae infection model. Ru-8 exhibited significant antibacterial efficacy against S. aureus in vivo and low toxicity to mouse tissues. The Ru-8 showed low toxicity to Raw264.7 cells (mouse monocyte macrophage leukemia cells). This study indicates that the ruthenium complex ruthenium quaternary was a promising strategy for the development of new antibacterial agents.

Suppressing the virulence factors of Candida auris with baicalein through multifaceted mechanisms.

Candida auris, a rapidly spreading multi-drug-resistant fungus, is causing lethal infections under certain conditions globally. Baicalin (BE), an active ingredient extracted from the dried root of Scutellaria baicalensis Georgi, exhibits antifungal activity. However, studies have shown the distinctive advantages of Traditional Chinese medicine in combating fungal infections, while the effect of BE, an active ingredient extracted from the dried roots of Scutellaria baicalensis Georgi, on C. auris, remains unknown. Therefore, this study aims to evaluate the potential of BE as an antifungal agent against the emerging multidrug-resistant C. auris. Various assays and models, including microbroth dilution, time growth curve analysis, spot assays, adhesion tests, flocculation test, cell surface hydrophobicity assay, hydrolase activity assays, XTT assay, violet crystal assay, scanning electron microscope (SEM), confocal laser scanning microscope (CLSM), flow cytometry, Live/dead fluorescent staining, reactive oxygen species (ROS), cell wall assay, aggregation assay, porcine skin model, Galleria mellonella larvae (G. mellonella larvae) infection model, and reverse transcription-quantitative polymerase chain reaction (RT-PCR) were utilized to investigate how baicalein suppresses C. auris through possible multifaceted mechanisms. The findings indicate that BE strongly inhibited C. auris growth, adhesion, and biofilm formation. It also effectively reduced drug resistance and aggregation by disrupting the cell membrane and cell wall while reducing colonization and invasion of the host. Transcriptome analysis showed significant modulation in gene expression related to different virulence factors post-BE treatment. In conclusion, BE exhibits significant effectiveness against C. auris, suggesting its potential as a viable treatment option due to its multifaceted suppression mechanisms.

Development of a transcription factor decoy-nanocarrier system as a successful inhibitor of Enterococcus faecalis virulence in vitro and in vivo

Enterococcus faecalis is a troublesome nosocomial pathogen that acquired resistance to most available antimicrobial agents. Antivirulence agents represent an unconventional treatment approach. Here, transcription factor decoy (TFD)-loaded cationic liposomes (TLL) were developed as an inhibitor of the Fsr quorum-sensing system and its associated virulence traits, in E. faecalis. The consensus sequence of the FsrA binding site was found conserved among 651 E. faecalis annotated genomes. The TFD was synthesized as an 82 bp DNA duplex, containing the conserved binding sequence, and loaded onto cationic liposomes. The optimum loading capacity, mean particle size, and zeta potential of the TLL were characterized. The developed TLL lacked any effect on E. faecalis growth and significantly inhibited the in vitro production of the proteolytic enzymes controlled by the Fsr system; gelatinase and serine protease, in a concentration-dependent manner. This inhibition was accompanied by a significant reduction in the transcription levels of FsrA-regulated genes (fsrB, gelE, and sprE). The developed TLL were safe as evidenced by the nonhemolytic effect on human RBCs and the negligible cytotoxicity on human skin fibroblast cells. Moreover, in the larvae infection model, TLL displayed a significant abolish in the mortality rates of Galleria mellonella larvae infected with E. faecalis. In conclusion, the developed TLL offer a new safe strategy for combating E. faecalis infection through the inhibition of quorum-sensing-mediated virulence; providing a platform for the development of similar agents to combat many other pathogens.

Synergistic bactericidal activity of a novel dual β-lactam combination against methicillin-resistant Staphylococcus aureus.

MRSA is a major cause of hospital-acquired and community-acquired infections. Treatment options for MRSA are limited because of the rapid development of β-lactam resistance. Combining antibiotics offers an affordable, time-saving, viable and efficient approach for developing novel antimicrobial therapies. Both amoxicillin and cefdinir are oral β-lactams with indications for a wide range of bacterial infections and mild side effects. This study aimed to investigate the in vitro and in vivo efficacy of combining these two β-lactams against MRSA strains. Fourteen representative prevalent MRSA strains with diverse sequence types (STs) were tested with a combination of amoxicillin and cefdinir, using chequerboard and time-kill assays. The Galleria mellonella larvae infection model was used to evaluate the in vivo efficacy of this dual combination against the community-acquired MRSA (CA-MRSA) strain USA300 and the hospital-acquired MRSA (HA-MRSA) strain COL. The chequerboard assay revealed a synergistic activity of the dual amoxicillin/cefdinir combination against all tested MRSA strains, with fractional inhibitory concentration index (FICI) values below 0.5 and at least a 4-fold reduction in the MICs of both antibiotics. Time-kill assays demonstrated synergistic bactericidal activity of this dual combination against the MRSA strain USA300 and strain COL. Moreover, in vivo studies showed that the administration of amoxicillin/cefdinir combination to G. mellonella larvae infected with MRSA strains significantly improved the survival rate up to 82%, which was comparable to the efficacy of vancomycin. In vitro and in vivo studies indicate that the dual combination of amoxicillin/cefdinir demonstrates a synergistic bactericidal efficacy against MRSA strains of various STs. Further research is needed to explore its potential as a treatment option for MRSA infections.

Low toxicity contributes to Sporothrix globosa invade the skin of patients in low-epidemic areas of China.

This study aims to assess the clinical characteristics of sporotrichosis in low-endemic areas of China, including the prevalence geography, genotypic traits of patients, clinical manifestations, and strain virulence and drug sensitivities. The objective is to improve the currently used clinical management strategies for sporotrichosis. Retrospective data were collected from patients diagnosed with sporotrichosis through fungal culture identification. The isolates from purified cultures underwent identification using CAL (Calmodulin) gene sequencing. Virulence of each strain was assessed using a Galleria mellonella (G. mellonella) larvae infection model. Invitro susceptibility testing against commonly used clinical antifungal agents for sporotrichosis was conducted following CLSI criteria. In our low-endemic region for sporotrichosis, the majority of cases (23) were observed in middle-aged and elderly women with a history of trauma, with a higher incidence during winter and spring. All clinical isolates were identified as Sporothrix globosa (S. globosa). The G. mellonella larvae infection model indicated independent and dose-dependent virulence among strains, with varying toxicity levels demonstrated by the degree of melanization of the G. mellonella. Surprisingly, lymphocutaneous types caused by S. globosa exhibited lower invitro virulence but were more common in affected skin. In addition, all S.globosa strains displayed high resistances to fluconazole, while remaining highly susceptible to terbinafine, itraconazole and amphotericin B. Given the predominance of elderly women engaged in agricultural labour in our region, which is a low-epidemic areas, they should be considered as crucial targets for sporotrichosis monitoring. S. globosa appears to be the sole causative agent locally. However, varying degrees of melanization in larvae were observed among these isolates, indicating a divergence in their virulence. Itraconazole, terbinafine and amphotericin B remain viable first-line antifungal options for treating S.globosa infection.

Galleria mellonella in vitro model for chromoblastomycosis shows large differences in virulence between isolates

BackgroundChromoblastomycosis is the World Health Organization (WHO)-recognized fungal implantation disease that eventually leads to severe mutilation. Cladophialophora carrionii (C. carrionii) is one of the agents. However, the pathogenesis of C. carrionii is not fully investigated yet.MethodsWe investigated the pathogenic potential of the fungus in a Galleria mellonella (G. mellonella) larvae infection model. Six strains of C. carrionii, and three of its environmental relative C. yegresii were tested. The G. mellonella model was also applied to determine antifungal efficacy of amphotericin B, itraconazole, voriconazole, posaconazole, and terbinafine.ResultsAll strains were able to infect the larvae, but virulence potentials were strain-specific and showed no correlation with clinical background of the respective isolate. Survival of larvae also varied with infection dose, and with growth speed and melanization of the fungus. Posaconazole and voriconazole exhibited best activity against Cladophialophora, followed by itraconazole and terbinafine, while limited efficacy was seen for amphotericin B.ConclusionInfection behavior deviates significantly between strains. In vitro antifungal susceptibility of tested strains only partly explained the limited treatment efficacy in vivo.

Emergence of an ST1934:KL121 hypervirulent Klebsiella pneumoniae carrying a novel virulence-resistance hybrid plasmid with chromosomal integration of ICEKp1.

To identify the phenotypic and genomic characteristics of K. pneumoniae KP43 from bloodstream infection. KP43 was resistant to ticarcillin and tetracycline and was hypervirulent in the Galleria mellonella larvae infection model, positive for string test, and possessed high-level macrophage killing resistance. The hypervirulence phenotype was associated with the chromosome integration of ICEKp1 carrying iroBCDN-iroP, rmpADC, and peg-344, and a novel plasmid pKP43_vir_amr harboring iutAiucABCD. pKP43_vir_amr was an IncFIBκ/FII virulence-resistance hybrid conjugative plasmid which also carried antibiotic resistance genes. The emergence of such a strain and the spread of the novel virulence-resistance plasmid might pose a potential threat to public health.

Caspofungin enhances the potency of rifampin against Gram-negative bacteria.

Developing antibiotic adjuvants is an effective strategy to combat antimicrobial resistance (AMR). The envelope of Gram-negative bacteria (GNB) is a barrier to prevent the entry of antibiotics, making it an attractive target for novel antibiotic and adjuvant development. In this study, we identified Caspofungin acetate (CAS) as an antibiotic adjuvant against GNB in the repurposing screen of 3,158 FDA-approved drugs. Checkerboard assays suggested that CAS could enhance the antimicrobial activity of rifampin or colistin against various GNB strains in vitro, Moreover, Galleria mellonella larvae infection model also indicated that CAS significantly potentiated the efficacy of rifampin against multidrug-resistant Escherichia coli 72 strain in vivo. Most importantly, resistance development assay showed that CAS was less susceptible to accelerating the resistance development of drug-sensitive strain E. coli MG1655. Functional studies and RNA-seq analysis confirmed that the mechanisms by which CAS enhanced the antimicrobial activities of antibiotics were involved in permeabilizing the bacterial cell envelope, disrupting proton motive force and inhibiting bacterial biofilm formation. Additionally, it has been found that PgaC is the CAS target and enzymatic assay has confirmed the inhibition activity. Our results illustrate the feasibility of CAS as an antibiotic adjuvant against GNB, which is an alternative strategy of anti-infection.

Improved pharmacokinetics and enhanced efficacy of the vancomycin derivative FU002 using a liposomal nanocarrier

Antibiotic resistance still represents a global health concern which diminishes the pool of effective antibiotics. With the vancomycin derivative FU002, we recently reported a highly potent substance active against Gram-positive bacteria with the potential to overcome vancomycin resistance. However, the translation of its excellent antimicrobial activity into clinical efficiency could be hampered by its rapid elimination from the blood stream. To improve its pharmacokinetics, we encapsulated FU002 in PEGylated liposomes. For PEG-liposomal FU002, no relevant cytotoxicity on liver, kidney and red blood cells was observed. Studies in Wistar rats revealed a significantly prolonged blood circulation of the liposomal antibiotic. In microdilution assays it could be demonstrated that encapsulation does not diminish the antimicrobial activity against staphylococci and enterococci. Highlighting its great potency, liposomal FU002 exhibited a superior therapeutic efficacy when compared to the free form in a Galleria mellonella larvae infection model.

The streptococcal phase-variable type I restriction modification system SsuCC20p dictates the methylome of Streptococcus suis impacting the transcriptome and virulence in a zebrafish larvae infection model.

Phase variation allows a single strain to produce phenotypic diverse subpopulations. Phase-variable restriction modification (RM) systems are systems that allow for such phase variation via epigenetic regulation of gene expression levels. The phase-variable RM system SsuCC20p was found in multiple streptococcal species and was acquired by an emerging zoonotic lineage of Streptococcus suis. We show that the phase variability of SsuCC20p is dependent on a recombinase encoded within the SsuCC20p locus. We characterized the genome methylation profiles of the different phases of SsuCC20p and demonstrated the consequential impact on the transcriptome and virulence in a zebrafish infection model. Acquiring mobile genetic elements containing epigenetic regulatory systems, like phase-variable RM systems, enables bacterial pathogens to produce diverse phenotypic subpopulations that are better adapted to specific (host) environments encountered during infection.

Pathogenetic detection, retrospective and pathogenicity analysis of a fatal case of Vibrio vulnificus in Shenzhen, China

We report a 36-year-old male patient died of V. vulnificus-induced septicaemia and multiple organ failure syndrome after oyster consumption at a restaurant. We isolated and identified V. vulnificus vv16015 from the patient’s blood sample and antibiotic susceptibility tests indicated sensitivity to all 21 antibiotics. Oyster samples were subsequently collected from the restaurant’s supplier and three strains of V. vulnificus were isolated. Whole genome sequencing and analysis revealed vv16015 to be distantly related to these strains and confirmed that V. vulnificus contamination was present in the seafood of the restaurant and supplier. Using a Galleria mellonella larvae infection model, the virulence of vv16015 was determined to be higher than that of comparison strains isolated from a surviving patient (vv15018) and an oyster (vv220015). The human and environment distribution of V. vulnificus in Shenzhen is sporadic and heterogeneous, and vv16015 is highly virulent compared to other strains.

Tyramine, one quorum sensing inhibitor, reduces pathogenicity and restores tetracycline susceptibility in Burkholderia cenocepacia

Burkholderia cenocepacia is an opportunistic respiratory pathogen of particular relevance to patients with cystic fibrosis (CF), primarily regulating its biological functions and virulence factors through two quorum sensing (QS) systems (CepI/R and CciI/R). The highly persistent incidence of multidrug resistant Burkholderia cenocepacia poses a global threat to public health. In this study, we investigated the effects of tyramine, one biogenic amine, on the QS systems of Burkholderia cenocepacia. Genetic and biochemical analyses revealed that tyramine inhibited the production of N-hexanoyl-homoserine (AHL) signaling molecules (C8-HSL and C6-HSL) by blocking the CepI/R and CciI/R systems. As a result, the inhibition of QS systems leads to reduced production of various virulence factors, such as biofilm formation, extracellular polysaccharides, lipase, and swarming motility. Notably, as a potential quorum sensing inhibitor, tyramine exhibits low toxicity in vivo in Galleria mellonella larvae and is well characterized by Lipinski’s five rules. It also shows high gastrointestinal absorption and the ability to cross the blood–brain barrier according to SwissADME database and ProTox-II server. Additionally, tyramine was found to enhance the efficacy of tetracycline in reducing the infectivity of Burkholderia cenocepacia in Galleria mellonella larvae infection model. Therefore, tyramine could be a promising candidate for combination therapy with traditional antimicrobials to improve their effectiveness against Burkholderia cenocepacia.

Within-Host Resistance and Virulence Evolution of a Hypervirulent Carbapenem-Resistant Klebsiella pneumoniae ST11 Under Antibiotic Pressure.

Hypervirulent carbapenem-resistant Klebsiella pneumoniae (hv-CRKP) has recently aroused an extremely severe health challenge and public concern. However, the underlying mechanisms of fitness costs that accompany antibiotic resistance acquisition remain largely unexplored. Here, we report a hv-CRKP-associated fatal infection and reveal a reduction in virulence due to the acquisition of aminoglycoside resistance. The bacterial identification, antimicrobial susceptibility, hypermucoviscosity, virulence factors, MLST and serotypes were profiled.The clonal homology and plasmid acquisition among hv-CRKP strains were detected by XbaI and S1-PFGE. The virulence potential of the strains was evaluated using Galleria mellonella larvae infection model, serum resistance assay, capsular polysaccharide quantification, and biofilm formation assay. Genomic variations were identified using whole-genome sequencing (WGS). Four K. pneumoniae carbapenemase (KPC)-producing CRKP strains were consecutively isolated from an 86-year-old patient with severe pneumonia. Whole-genome sequencing (WGS) showed that all four hv-CRKP strains belonged to the ST11-KL64 clone. PFGE analysis revealed that the four ST11-KL64 hv-CRKP strains could be grouped into the same PFGE type. Under the pressure of antibiotics, the antimicrobial resistance of the strains increased and the virulence potential decreased. Further sequencing, using the Nanopore platform, was performed on three representative isolates (WYKP586, WYKP589, and WYKP594). Genomic analysis showed that the plasmids of these three strains underwent a large number of breaks and recombination events under antibiotic pressure. We found that as aminoglycoside resistance emerged via acquisition of the rmtB gene, the hypermucoviscosity and virulence of the strains decreased because of internal mutations in the rmpA and rmpA2 genes. This study shows that ST11-KL64 hv-CRKP can further evolve to acquire aminoglycoside resistance accompanied by decreased virulence to adapt to antibiotic pressure in the host.

Emergence of colistin-heteroresistant and carbapenem-resistant hypervirulent Klebsiella pneumoniae

To investigate the clinical emergence of colistin-heteroresistant, hypervirulent, and multidrug-resistant Klebsiella pneumoniae, and characterize the underlying molecular mechanisms. The population analysis profiles (PAPs) method was used to detect colistin heteroresistance. The time-killing assay was used to examine the effect of colistin on carbapenem-resistant Klebsiella pneumoniae (CRKP) in vitro. Galleria mellonella larvae infection model was used to test the potential virulence. qRT-PCR assay was conducted to compare the expression levels of efflux pump genes. Next and third-generation sequencing were conducted to analyse the genomic features. Two colistin-heteroresistant isolates were detected from a multi-center carbapenem-resistant Enterobacterales (CRE) surveillance study in China, which exhibited similar survival rates as the K2 hypervirulent reference strain ATCC 43816 in a G. mellonella larvae model. The two isolates belonged to ST11, harbouring the iucABCD, iutA, iroBCD, and rpmA2 hypervirulent genes and pLVPK-like virulence plasmids. Colistin showed a weak effect on the heteroresistant strains in vitro. The efflux pump genes acrA, acrB, tolC, oqxA, and oqxB were upregulated in this subpopulation compared to the parental strains. This study showed the clinical emergence of colistin-heteroresistant, hypervirulent, and multidrug-resistant Klebsiella pneumoniae. AcrAB-TolC and OqxAB efflux overexpression were involved in mediating colistin heteroresistance.

Metal-ruthenium complex based on dipyridylamine group as membrane-active antibacterial agent effectively decrease the development of drug-resistance on Staphylococcus aureus

Staphylococcus aureus (S. aureus), one of the Gram-positive bacteria, is easily to develop drug-resistance. Drug-resistant S. aureus infection leads to high morbidity and mortality. The complexes, namely [Ru(dpa)2(PSPIP)](PF6)2 (Ru1), [Ru(dpa)2(TSPIP)](PF6)2 (Ru2), and [Ru(dpa)2(TBPIP)](PF6)2 (Ru3), were synthesized using 2, 2′-dipyridylamine as an auxiliary ligand and three main ligands PSPIP, TSPIP, TBPIP. In vitro studies demonstrated that the Ru1–3 exhibited excellent antibacterial activity against S. aureus while showing low hemolytic toxicity to rabbit red blood cells. Notably, Ru3 was found to disrupt the bacterial cell membrane and alter its permeability through fluorescence staining and scanning electron microscopy (SEM) analysis. Furthermore, Ru3 displayed low toxicity in G. mellonella Larvae. Ru3 exhibited good activity against S. aureus in G. mellonella Larvae infection model and mouse skin infection model.To some extent, Ru3 inhibited biofilm formation on S. aureus as well as hemolytic toxin production, thereby attenuating the development of drug resistance without cross-resistance with other antibiotics. In addition, complex Ru3 exhibited a synergistic effect when combined with antibiotics amikacin, kanamycin, tobramycin and chloramphenicol, making it a valuable antibiotics adjuvant.

Ru-Based Organometallic Agents Bearing Phenyl Hydroxide: Synthesis and Antibacterial Mechanism Study against Staphylococcus aureus.

The development of antimicrobial agents with novel model of actions is a promising strategy to combat multiple resistant bacteria. Here, three ruthenium-based complexes, which acted as potential antimicrobial agents, were synthesized and characterized. Importantly, three complexes all showed strong bactericidal potency against Staphylococcus aureus. In particular, the most active one has a MIC of 6.25 μg/mL. Mechanistic studies indicated that ruthenium complex killed S. aureus by releasing ROS and damaging the integrity of bacterial cell membrane. In addition, the most active complex not only could inhibit the biofilm formation and hemolytic toxin secretion of S. aureus, but also serve as a potential antimicrobial adjuvant as well, which showed synergistic effects with eight traditional antibiotics. Finally, both G. mellonella larva infection model and mouse skin infection model all demonstrated that ruthenium complex also showed significant efficacy against S. aureus in vivo. In summary, our study suggested that ruthenium-based complexes bearing a phenyl hydroxide are promising antimicrobial agents for combating S. aureus.

Genome-based characterization of conjugative IncHI1B plasmid carrying carbapenemase genes blaVIM-1, blaIMP-23, and truncated blaOXA-256 in 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 (≤1μg/mL), polymyxin B (0.25μg/mL), colistin (0.25μg/mL), eravacycline (0.25μg/mL), cefepime/zidebactam (1μg/mL), omadacycline (4μg/mL), and tigecycline (0.5μg/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.

Species identification, antibiotic resistance, and virulence in Enterobacter cloacae complex clinical isolates from South Korea.

This study aimed to identify the species of Enterobacter cloacae complex (ECC) isolates and compare the genotype, antibiotic resistance, and virulence among them. A total of 183 ECC isolates were collected from patients in eight hospitals in South Korea. Based on partial sequences of hsp60 and phylogenetic analysis, all ECC isolates were identified as nine species and six subspecies. Enterobacter hormaechei was the predominant species (47.0%), followed by Enterobacter kobei, Enterobacter asburiae, Enterobacter ludiwigii, and Enterobacter roggenkampii. Multilocus sequence typing analysis revealed that dissemination was not limited to a few clones, but E. hormaechei subsp. xiangfangensis, E. hormaechei subsp. steigerwaltii, and E. ludwigii formed large clonal complexes. Antibiotic resistance rates were different between the ECC species. In particular, E. asburiae, E. kobei, E. roggenkampii, and E. cloacae isolates were highly resistant to colistin, whereas most E. hormaechei and E. ludwigii isolates were susceptible to colistin. Virulence was evaluated through serum bactericidal assay and the Galleria mellonella larvae infection model. Consistency in the results between the serum resistance and the G. mellonella larvae infection assay was observed. Serum bactericidal assay showed that E. hormaechei, E. kobei, and E. ludwigii were significantly more virulent than E. asburiae and E. roggenkampii. In this study, we identified the predominant ECC species in South Korea and observed the differences in antibiotic resistance and virulence between the species. Our findings suggest that correct species identification, as well as continuous monitoring is crucial in clinical settings.