Antiparasitic nitazoxanide potentiates colistin against colistin-resistant Acinetobacter baumannii and Escherichia coli in vitro and in vivo.

IMPORTANCE: Colistin is used as a last resort in many infections caused by multidrug-resistant Gram-negative bacteria; however, colistin-resistant (COL-R) is on the rise. Hence, it is critical to develop new antimicrobial strategies to overcome COL-R. We found that nitazoxanide (NTZ) combined with colistin showed notable synergetic antibacterial activity. These findings suggest that the NTZ/colistin combination may provide an effective alternative route to combat COL-R A. baumannii and COL-R Escherichia coli infections.

Clinical impact of the type VI secretion system on clinical characteristics, virulence and prognosis of Acinetobacter baumannii during bloodstream infection.

The type VI secretion system (T6SS) has been regarded as a late-model virulence factor widely distributed in Acinetobacter baumannii (A. baumannii). This study aimed to elucidate the clinical manifestations, the genetic background and microbiological characteristics of A. baumannii isolates causing bloodstream infection (BSI), and assessed the impact of T6SS carrying state on the clinical course. In this study, Clinical samples of A. baumannii causing BSI were collected from a teaching hospital in China from 2016 to 2020 and a retrospective cohort was conducted. Experimental strains were categorized into T6SS positive and negative groups through PCR targeting on hcp gene. The antimicrobials sensitivity test, virulence genes, biofilm formation ability, serum resistance of A. baumannii strains and Galleria mellonella infection model were investigated. Independent risk factors for T6SS+ A. baumannii BSI and Kaplan-Meier curve through follow-up survey were analyzed. A total of 182 A. baumannii strains were isolated from patients with BSI during 5 years and the medical records of all patients were retrospectively reviewed. The proportion of T6SS+ isolates was 62.64% (114/182), which exhibited significantly higher resistance rates of commonly used antibacterial drugs compared to T6SS- group. We found that T6SS+ A. baumannii strains had significantly weaker biofilm formation ability compared to T6SS- A. baumannii. Despite no difference in the positivity rate of tested virulence genes in two groups, T6SS+ strains exhibited higher resistance to the serum and increased virulence in vivo compared to T6SS- strains, indicating that T6SS is likely to enhance the survival and invasive capabilities of A. baumannii in vivo. Indwelling catheter, respiratory diseases, ICU history, white blood cell count and percentage of neutrophils increasing were independent risk factors for T6SS+ A. baumannii BSI. At last, the Kaplan-Meier curve confirmed a higher mortality rate associated with T6SS+ A. baumannii BSI, suggesting that the presence of T6SS may serve as a prognostic factor for mortality. In conclusion, our study revealed that T6SS+ A. baumannii exhibited distinct clinical features, characterized by high antimicrobial resistance and enhanced virulence, providing valuable insights for clinical treatment considerations.

Characteristics of rare ST463 carbapenem-resistant Pseudomonas aeruginosa clinical isolates from blood.

OBJECTIVES: This study aimed to elucidate resistance to carbapenems and fluoroquinolones, the transmission mechanism of blaKPC-2, and the virulence characteristics of a Pseudomonas aeruginosa strain (TL3773) isolated in East China. METHODS: The virulence and resistance mechanisms of TL3773 were investigated by whole genome sequencing (WGS), comparative genomic analysis, conjugation experiments, and virulence assays. RESULTS: This study isolated carbapenem-resistant P. aeruginosa from blood resistant to carbapenems. The patient's clinical data showed poor prognosis compounded by multiple sites of infection. WGS showed that TL3773 carried aph (3')-IIb, blaPAO, blaOXA-486, fosA, catB7, and two crpP resistance genes on chromosome, and the carbapenem resistance gene blaKPC-2 on plasmid. We identified a novel crpP gene named TL3773-crpP2. Cloning experiments proved that TL3773-crpP2 was not the primary cause of fluoroquinolone resistance in TL3773. GyrA and ParC mutations may confer fluoroquinolone resistance. The blaKPC-2 genetic environment was IS26-TnpR-ISKpn27-blaKPC-2-ISKpn6-IS26-Tn3-IS26, potentially mediating the transmission of blaKPC-2 in P. aeruginosa. The overall virulence of TL3773 was lower than that of PAO1. However, the pyocyanin and biofilm formation of TL3773 was higher than that of PAO1. WGS further indicated that TL3773 was less virulent than PAO1. Phylogenetic analysis showed that TL3773 was most similar to the P. aeruginosa isolate ZYPA29 from Hangzhou, China. These observations further indicate that ST463 P. aeruginosa is rapidly spreading. CONCLUSIONS: The threat of ST463 P. aeruginosa harbouring blaKPC-2 is emergent and may pose a threat to human health. More extensive surveillance and effective actions are urgently needed to control its further spread.

Resveratrol Increases Sensitivity of Clinical Colistin-Resistant Pseudomonas aeruginosa to Colistin In Vitro and In Vivo.

Infections caused by colistin-resistant P. aeruginosa strains pose a serious threat to public health. It is therefore urgent to find new strategies to deal with these bacterial infections. We aimed to investigate the efficacy and mechanisms of the colistin/resveratrol combination in eradicating colistin-resistant P. aeruginosa isolates and their biofilms both in vitro and in vivo. The results revealed that six clinically isolated colistin-resistant P. aeruginosa strains were multidrug resistant (MDR) strains, and resveratrol showed no antimicrobial activity against eight P. aeruginosa strains. Checkerboard assay and time-kill assays indicated that the combination therapy of resveratrol and colistin indicated a remarkable synergistic effect in vitro, and biofilm assays and SEM indicated synergistic antibiofilm activity. Furthermore, this combination could efficiently eliminate MDR bacteria in a murine infection model and improve the survival rate of Galleria mellonella. Fluorescence analysis, ALP, and ß-galactosidase activity test results indicated that the colistin/resveratrol combination increased the membrane permeability of bacteria. In conclusion, our results may provide an efficient alternative pathway against colistin-resistant P. aeruginosa infections. IMPORTANCE P. aeruginosa is a ubiquitous Gram-negative opportunistic pathogen associated with a wide array of life-threatening acute and chronic infections. However, the improper and excessive use of antibiotics has contributed to the increasing emergence of multidrug-resistant (MDR) P. aeruginosa, even colistin-resistant strains, which presents a major challenge to clinical anti-infection treatment. Resveratrol, a naturally occurring polyphenolic antioxidant, can effectively slow down or avoid the occurrence and development of bacterial resistance and is expected to offer a promising strategy to overcome bacterial infections. In this study, colistin/resveratrol combination could synergistically damage the bacterial cell membrane, thereby inducing cell lysis while addressing the emergence of drug resistance. Moreover, this combination therapy may provide an efficient alternative pathway to combat the colistin-resistant P. aeruginosa in clinical practice.

Evaluation of resazurin microplate method for rapid detection of vancomycin and linezolid resistance in Enterococcus faecalis and Enterococcus faecium clinical isolates.

BACKGROUND: Vancomycin and linezolid resistance among enterococci is an increasing problem due to a lack of alternative antibiotics. Early identification of vancomycin-resistant and linezolid-resistant strains can help prevent the spread of resistance to these antibiotics. Hence, early, rapid and accurate detection of vancomycin and linezolid resistance is critical. OBJECTIVES: The resazurin microplate method (RMM) was developed for detecting vancomycin and linezolid susceptibility among Enterococcus faecalis (E. faecalis) and Enterococcus faecium (E. faecium) clinical isolates, and its performance was further evaluated. METHODS: A total of 209 non-duplicate clinical isolates and three strains from the faeces of domestic animals, including 142 E. faecalis (71 linezolid non-susceptible and 71 linezolid susceptible) and 70 E. faecium (23 vancomycin non-susceptible, 23 vancomycin susceptible, 12 linezolid non-susceptible and 12 linezolid susceptible), were tested using RMM. RESULTS: The susceptibility of E. faecium to vancomycin was detected within 5 h, with high susceptibility (23/23) and specificity (23/23). The susceptibility of E. faecalis and E. faecium to linezolid was detected within 4 h, with specificities of 98.59% and 100% and susceptibilities of 94.37% and 58.33% for E. faecalis and E. faecium, respectively. CONCLUSIONS: RMM had a good positive predictive value for the detection of vancomycin-non-susceptible E. faecium and linezolid-non-susceptible E. faecalis. It thus has the potential to become an alternative method for the rapid screening of these resistant pathogens in clinical practice.

Effects of aerobactin-encoding gene iucB and regulator of mucoid phenotype rmpA on the virulence of Klebsiella pneumoniae causing liver abscess.

This study aimed to analyze the influence of the main aerobactin-encoding gene iucB and the regulator of mucoid phenotype rmpA on the virulence of Klebsiella pneumoniae causing liver abscess. In addition, the possible regulatory effects of the main encoding gene iucB on the regulator of mucoid phenotype rmpA were explored, thus providing novel strategies for the prevention and control of hypervirulent K. pneumoniae (hvKp) causing liver abscess. The virulence-related genes iucB and rmpA of K. pneumoniae were detected by PCR. iucB and rmpA were cloned into K. pneumoniae strain by using plasmid pET28b as vector. Quantitative real-time PCR (RT-qPCR) was employed to detect the relative expression of rmpA gene in K. pneumoniae. We investigated the potential effects of aerobactin coding gene iucB and regulator of mucoid phenotype rmpA on the virulence of K. pneumoniae by establishing the Galleria mellonella infection model. Capsule quantitative experiment was conducted to investigate the impact of aerobactin-encoding gene iucB on the modulation of regulator of mucoid phenotype rmpA. The results of the G. mellonella infection model indicated that iucB gene could significantly enhance the virulence of K. pneumoniae, but the presence of rmpA gene did not markedly affect the virulence of K. pneumoniae. RT-qPCR showed that iucB inhibited the expression of rmpA gene. Quantitative capsulation experiments showed that the presence of rmpA gene could not increase the capsulation production of K. pneumoniae. The main encoding gene of aerobactin, namely iucB, could substantially enhance the virulence of K. pneumoniae. The gene iucB might be involved in the biosynthesis of the capsular polysaccharide through an unknown mechanism instead of the gene rmpA. Overall, these findings provide important theoretical support for the treatment of infections caused by hvKp.

Clinical characteristics, tolerance mechanisms, and molecular epidemiology of reduced susceptibility to chlorhexidine among Pseudomonas aeruginosa isolated from a teaching hospital in China.

Chlorhexidine is used widely to prevent the spread of bacteria in the hospital environment. However, bacteria are increasingly becoming tolerant to chlorhexidine. Here we investigated clinical characteristics, tolerance mechanisms, and molecular epidemiology of chlorhexidine-tolerant Pseudomonas aeruginosa. According to the proposed epidemiological cut-off value to determine chlorhexidine tolerance (50 µg/mL) in P. aeruginosa, 32 chlorhexidine-tolerant isolates were detected from 294 P. aeruginosa isolates, which accounted for 10.9%. Our results indicated MICs of chlorhexidine-tolerant strains were 64 µg/mL. Patient's data showed chlorhexidine tolerance was associated with following factors: hospital length of stay, ICU admission, length of stay in ICU, invasive procedure, duration of mechanical ventilation, chlorhexidine usage, and occurrence of nosocomial pneumonia. Tolerance mechanisms were analyzed by efflux pump inhibition test, qRT-PCR, and serial passage experiment. Increased expression of efflux pump genes mexA, mexC, mexE and mexX, and decreased expression of oprD were observed in chlorhexidine-tolerant and chlorhexidine-induced strains, which suggested that hyperexpression of Mex-Opr efflux pump was the main mechanism. Moreover, serial passage experiment found chlorhexidine-induced strains showed decreased susceptibility to tested antibiotics, which illustrated that long-term exposure of P. aeruginosa to chlorhexidine could result in multidrug-resistant (MDR) or cross-resistance phenotypes. MLST and PFGE analysis demonstrated the homology of 32 chlorhexidine-tolerant strains was low and no obvious clonal transmission was observed. We comprehensively investigated the development and molecular mechanisms of chlorhexidine-tolerant P. aeruginosa, which revealed that the control and surveillance of chlorhexidine tolerance should be more strict. Moreover, it seems to make sense to avoid the continuous or unreasonable application of chlorhexidine in hospital settings.

Acquisition of Daptomycin Resistance by Enterococcus faecium Confers Collateral Sensitivity to Glycopeptides.

Daptomycin is a last-line antibiotic used in the treatment of multidrug-resistant Enterococcus faecium infections. Alarmingly, daptomycin-resistant E. faecium isolates have emerged. In this study, we investigated the evolution and mechanisms of daptomycin resistance in clinical E. faecium isolates and the corresponding acquisition of collateral sensitivity (CS) as an evolutionary trade-off. We evolved daptomycin resistance in six daptomycin-susceptible E. faecium isolates to obtain daptomycin-resistant mutants. The six E. faecium strains successfully acquired high-level resistance to daptomycin in vitro, but this led to fitness costs in terms of growth, in vitro competition, and virulence. Mutations in liaFSR, yycFG, and cls; increased surface positive charge; thicker cell walls; and elevated expression of dltABCD and tagGH were observed in daptomycin-resistant mutants. Surprisingly, we observed the emergence of CS in SC1762 isolates after the induction of daptomycin resistance. Compared with parental strains, the SC1174-D strain (i.e., daptomycin-resistant mutant of SC1174; non-CS) showed significantly upregulated expression of the vanA gene cluster. However, in SC1762-D (i.e., daptomycin-resistant mutant of SC1762), all vanA cluster genes except the vanX gene were obviously downregulated. Further in silico analyses revealed that an IS1216E-based composite transposon was generated in SC1762-D, and it disrupted the vanH gene, likely affecting the structure and expression of the vanA gene cluster and resulting in resensitization to glycopeptides. Overall, this study reports a novel form of CS between daptomycin and glycopeptides in E. faecium. Further, it provides a valuable foundation for developing effective regimens and sequential combinations of daptomycin and glycopeptides against E. faecium.

Cluster Differences in Antibiotic Resistance, Biofilm Formation, Mobility, and Virulence of Clinical Enterobacter cloacae Complex.

Due to the lack of research on the characteristics of different clusters of Enterobacter cloacae complex (ECC), this study aimed to characterize and explore the differences among species of the ECC. An analysis based on hsp60 showed that Enterobacter hormaechei was predominant in ECC. Interestingly, the antibiotic resistance rates of clusters were different, among which E. hormaechei subsp. steigerwaltii (cluster VIII) and Enterobacter cloacae IX (cluster IX) possessed high resistant rates to ciprofloxacin and levofloxacin, but cluster II (Enterobacter kobei) had low resistant rates. Cluster II exhibited a strong biofilm formation ability. Different motility and protease production ability were shown for distinct clusters. A PCR analysis showed that clusters I, III, VI, VIII, and IX carried more virulence genes, while cluster II had fewer. Clusters I, VIII, and IX with high pathogenicity were evaluated using the Galleria mellonella infection model. Thus, the characteristics of resistance, biofilm-forming ability, mobility, and virulence differed among the clusters. The strains were divided into 12 subgroups based on hsp60. The main clusters of ECC clinical strains were I, II, III, VI, VIII, and IX, among which IX, VIII, and I were predominant with high resistance and pathogenicity, and cluster II (E. kobei) was a special taxon with a strong biofilm formation ability under nutrient deficiency, but was associated with low resistance, virulence, and pathogenicity. Hence, clinical classification methods to identify ECC subgroups are an urgent requirement to guide the treatment of clinical infections.

Antimicrobial peptide WAM-1: a promising antibacterial and anti-inflammatory drug against carbapenem-resistant Klebsiella pneumoniae.

BACKGROUND: The emergence and spread of carbapenem-resistant Klebsiella pneumoniae (CRKP) pose a threat to public health. Antimicrobial peptides provide a new treatment option for CRKP infections. OBJECTIVES: We studied antibacterial activities of WAM-1 against CRKP in vitro and in vivo and explored its possible mechanism. We verified safety and factors affecting antibacterial effect. Furthermore, anti-inflammatory effects were investigated. METHODS: We selected eight CRKP and eight carbapenem-susceptible K. pneumoniae to explore the antibacterial activity of WAM-1 by broth microdilution (BMD). The possible mechanism was investigated by alkaline phosphatase leakage and propidium iodide (PI). We evaluated safety of WAM-1 by cytotoxicity and haemolysis and effects of temperature and serum on the antibacterial activity. We investigated in vivo efficacy of WAM-1 by the Galleria mellonella infection model. We investigated the effect of WAM-1 on TNF-α. RESULTS: BMD showed that WAM-1 had a good antibacterial effect with MICs of 2-4 mg/L and MBCs of 4-8 mg/L. RT-qPCR showed that WAM-1 could inhibit the expression of TNF-α. The cytotoxicity and haemolysis test proved that WAM-1 had certain potential application in vivo. Alkaline phosphatase leakage and PI fluorescence showed that WAM-1 was highly likely to exert an antibacterial effect by destroying bacterial membrane. The G. mellonella infection model suggested that WAM-1 may have a good therapeutic effect in vivo. Temperature had little effect on the activity of WAM-1. Serum, however, reduced WAM-1 activity. CONCLUSIONS: WAM-1 has good antibacterial effect and potential anti-inflammatory effect on infection caused by CRKP.

Evolution of tet(A) variant mediating tigecycline resistance in KPC-2-producing Klebsiella pneumoniae during tigecycline treatment.

OBJECTIVES: This study investigated the underlying mechanism of the evolution of tigecycline resistance during treatment in a patient infected with Klebsiella pneumoniae harbouring blaKPC-2. METHODS: A total of seven clonal K. pneumoniae strains were continuously isolated from a patient during hospitalisation. Antimicrobial resistance in the strains was determined by antimicrobial susceptibility testing. Multilocus sequence typing (MLST) and pulsed-field gel electrophoresis (PFGE) were performed to explore the homology of the isolates. Whole-genome shotgun (WGS) analysis and cloning experiments were used to investigate the underlying mechanism of the evolution of tigecycline resistance. RESULTS: All of the isolates had a minimum inhibitory concentration (MIC) for tigecycline of 4 µg/mL, except strain FK6768 that had a MIC of 32 µg/mL. Carbapenem-resistant K. pneumoniae strains (FK6614, FK6768 and FK6809) were consecutively isolated from faeces at different times. Antimicrobial susceptibility testing indicated that tigecycline resistance increased in FK6768 and subsequently decreased in FK6809, which attracted our attention. WGS and further bioinformatics analysis showed a homology for the three faecal isolates of >99%. The blaKPC-2 carbapenemase gene and a tet(A) mutation were found in tigecycline-resistant isolate FK6768. Subsequent cloning experiments confirmed the contribution of a tet(A) variant to reduced tigecycline susceptibility. CONCLUSION: Here we report a K. pneumoniae isolate carrying both tet(A) mutation and the blaKPC-2 gene, which led to increased tigecycline resistance during tigecycline treatment. This is the first report describing tigecycline resistance of K. pneumoniae first increasing and subsequently decreasing in vivo.

Combining Colistin with Furanone C-30 Rescues Colistin Resistance of Gram-Negative Bacteria in Vitro and in Vivo.

The spread of multidrug-resistant (MDR) Gram-negative bacteria (GNB) has led to serious public health problems worldwide. Colistin, as a "last resort" for the treatment of MDR bacterial infections, has been used significantly in recent years and has led to the continuous emergence of colistin-resistant strains. In this study, we aimed to investigate the synergistic effect on the antimicrobial and antibiofilm activities of a colistin/furanone C-30 combination against colistin-resistant GNB in vitro and in vivo. According to antimicrobial resistance profiles, most of the colistin-resistant strains we collected showed MDR phenotypes. The checkerboard method and time-kill curve showed that the combination with furanone C-30 increases the antibacterial activity of colistin significantly. In addition, the furanone C-30/colistin combination can not only inhibit the formation of bacterial biofilm but also has a better eradication effect on preformed mature biofilms. The result of scanning electron microscopy (SEM) demonstrated that the furanone C-30/colistin combination led to a significant reduction in the number of cells in biofilms. Furthermore, furanone C-30 at 50 µg/ml did not cause any additional toxicity to RAW264.7 cells according to a cytotoxicity assay. In in vivo infection experiments, the furanone C-30/colistin combination increased the survival rate of infected Galleria mellonella larvae as well as decreased the microbial load in a mouse thigh infection model. The synergistic effect of the furanone C-30/colistin combination against colistin-resistant GNB is encouraging, and this work may shed light on a new therapeutic approach to combat colistin-resistant pathogens. IMPORTANCE Colistin is among the few antibiotics effective against multidrug-resistant Gram-negative bacteria (GNB) clinical isolates. However, colistin-resistant GNB strains have emerged in recent years. Therefore, the combination of colistin and nonantibacterial drugs has attracted much attention. In this study, the furanone C-30/colistin combination showed good antibacterial and antibiofilm activity in vitro and in vivo. In addition, increased membrane permeability leads to the synergistic effect of the furanone C-30/colistin combination. Because of the low cytotoxicity of furanone C-30, this combination has good application prospects in clinical anti-infective therapy. This finding might shed light on the discovery of combination therapy for infections caused by colistin-resistant GNB pathogens.

Anti-leukaemia effects of leonurine in vitro and in vivo.

The present study was conducted to explore the anti-acute myeloid leukaemia (AML) effects of leonurine. HL-60 and U-937 cells were used to assess the antileukaemia effect of leonurine in vitro, and HL-60 and U-937 xenograft nude mice were used to evaluate its antitumour effect in vivo. Leonurine inhibited the proliferation of HL-60 and U-937 cells in a time- and dose-dependent manner. Moreover, leonurine therapy prevented the growth of tumours in both xenograft animal models. Leonurine could induce apoptosis in HL-60 and U-937 cells. The cytotoxic effects of leonurine on HL-60 and U-937 cells were associated with an increased ratio of Bax/Bcl-2, activation of caspase-3, caspase-8 and caspase-9, and increased expression of cytochrome c in the cytoplasm. Leonurine inhibited activation of the PI3K/Akt pathway in HL-60 and U-937 cells by lowering the phosphorylation levels of PI3K and Akt. Our results indicate that leonurine is a potential anti-AML agent, and this activity may be associated with its repression of the phosphorylation of PI3K and Akt.

Molecular Mechanisms and Epidemiology of Carbapenem-Resistant Enterobacter cloacae Complex Isolated from Chinese Patients During 2004-2018.

BACKGROUND: The emergence and spread of carbapenem-resistant Enterobacter cloacae complex (ECC) have posed a serious threat to human health worldwide. This study aimed to investigate the molecular mechanism of carbapenem resistance and its prevalence among ECC in China. METHODS: A total of 1314 ECC clinical isolates were collected from the First Affiliated Hospital of Wenzhou Medical University from 2004 to 2018. Sensitivity to antibiotics was determined using the agar dilution method. The production of carbapenemases and the prevalence of resistance-associated genes were investigated using PCR. The expression of outer membrane porin (OMP) genes (ompC/ompF) and cephalosporinase gene ampC was analyzed by quantitative real-time PCR. The effect of efflux pump mechanism on carbapenem resistance was tested. ECC was typed by multilocus sequence typing (MLST). RESULTS: In this study, 113 carbapenem-nonsusceptible ECC strains were identified. The prevalence rates of carbapenemase genes bla KPC-2 and bla NDM were 12.4% (14/113) and 17.7% (20/113), and that of the extended-spectrum ß-lactamase (ESBL) genes bla CTX-M, bla TEM, and bla SHV were 28.3% (32/113), 27.4% (31/113), and 14.2% (16/113), respectively. Among 67 carbapenem-nonsusceptible ECC isolates producing non-carbapenemase, low expression of ompC/ompF and overexpression of ampC were found in 46 and 40 strains, respectively. In addition, the carbapenem resistance was related to the overexpression of the efflux pump in the study. Finally, the 113 carbapenem-nonsusceptible ECC strains were categorized into 39 different sequence types using MLST. CONCLUSION: Carbapenem-nonsusceptible ECC strains producing non-carbapenemase were predominant. The low expression of OMP with the overexpression of cephalosporinase or production of ESBLs and overexpression of efflux pump might contribute to the resistance to carbapenem for carbapenem-nonsusceptible ECC strains producing non-carbapenemase. The bla NDM and bla KPC comprised the principal resistance mechanism of carbapenemase-producing ECC in the hospital, causing a threat to public health. Therefore, monitoring programs to prevent the emergence and further spread of antibiotic resistance are urgently needed.

In vitro Activity of Meropenem-Vaborbactam versus Other Antibiotics Against Carbapenem-Resistant Escherichia coli from Southeastern China.

PURPOSE: This study aimed to evaluate the in vitro activity of meropenem-vaborbactam (MVB) against a collection of carbapenem-resistant Escherichia coli (CREC) isolates and to compare the activity with other antibiotics with regard to different separation sites, carbapenem-resistant mechanisms, and sequence types (STs). METHODS: A total of 58 CREC strains were used as the experimental strains from the First Affiliated Hospital of Wenzhou Medical University in southeastern China. The minimum inhibitory concentrations of MVB, ceftazidime-avibactam, and tigecycline against all the experimental strains were determined by the microdilution broth method. RESULTS: MVB exhibited higher antimicrobial activity (83% susceptibility) than that of other antibiotics, except for colistin and tigecycline. The susceptibility of CREC strains towards MVB varied with regard to carbapenem-resistant mechanisms and STs, especially in Klebsiella pneumoniae carbapenemase (KPC)-positive isolates and ST8 isolates. CONCLUSION: MVB exhibited considerably high activity against KPC-producing and ST8 CREC isolates. It has the great potential to be an alternative for the treatment of infections caused by CREC after determining the type of carbapenemase, the susceptibility to MVB and/or STs.

Antibacterial and Anti-biofilm Efficacy of Chinese Dragon's Blood Against Staphylococcus aureus Isolated From Infected Wounds.

Chinese dragon's blood (CDB), a characteristic red resin, is an important traditional Chinese medicine (TCM), and empiric therapy of infected wounds with CDB is performed in clinical settings. For the first time, we herein report the antibacterial and anti-biofilm efficacy of CDB against Staphylococcus aureus (S. aureus). Antimicrobial susceptibility testing, growth curve assay, time-kill curve assay, crystal violet biofilm assay, scanning electron microscope (SEM) analysis, cell membrane tests, and quantitative real-time polymerase chain reaction (qRT-PCR) were used for this purpose. The results suggested that the minimum inhibitory concentration (MIC) values of CDB against S. aureus ranged from 32 to 128 µg/mL. Growth curves and time-kill curves confirmed that CDB could inhibit the growth of S. aureus. The biofilm formation ability and the expression levels of saeR, saeS, and hla of S. aureus in the presence and absence of CDB were statistically significant (P < 0.01). The results of SEM analysis and cell membrane tests revealed that exposure to CDB had some destructive effects on S. aureus cells. In conclusion, CDB exhibits positive antibacterial activity against S. aureus. Moreover, CDB could reduce the biofilm formation and the virulence factors of S. aureus by downregulating the expression levels of saeR, saeS, and hla genes. These findings indicated that CDB has immense potential to serve as a viable alternative for the treatment of infected wounds caused by S. aureus in clinical settings.

Synergistic Activity and Biofilm Formation Effect of Colistin Combined with PFK-158 Against Colistin-Resistant Gram-Negative Bacteria.

PURPOSE: The emergence of colistin resistance among Gram-negative bacteria (GNB) poses a serious public health threat. Therefore, it is necessary to enhance the antibacterial activity of colistin through the combination with other drugs. In this study, we demonstrated the synergistic activity and the possible synergy mechanism of colistin with PFK-158 against colistin-resistant GNB, including non-fermenting bacteria and Enterobacteriaceae. PATIENTS AND METHODS: Thirty-one colistin-resistant GNB, including Pseudomonas aeruginosa (n = 9), Acinetobacter baumannii (n = 5), Escherichia coli (n = 8) and Klebsiella pneumoniae (n = 9), were collected as the experimental strains and the minimum inhibitory concentrations (MICs) of colistin, other routine antimicrobial agents and PFK-158 against all strains were determined by the broth microdilution method. The synergistic activity of colistin with PFK-158 was assessed by the checkerboard assay and time-kill assay. The biofilm formation assay and scanning electron microscopy were used to demonstrate the biofilm formation effect of colistin with PFK-158 against colistin-resistant GNB. RESULTS: The results of the checkerboard assay showed that when colistin was used in combination with PFK-158, synergistic activity was observed against the 31 colistin-resistant GNB. The time-kill assay presented a significant killing activity of colistin with PFK-158 against the 9 colistin-resistant GNB selected randomly, including Pseudomonas aeruginosa (n = 6), Acinetobacter baumannii (n = 1), Escherichia coli (n = 1), and Klebsiella pneumoniae (n = 1). The biofilm formation assay and scanning electron microscopjihy showed that colistin with PFK-158 can effectively suppress the formation of biofilm and reduce the cell arrangement density of biofilm against most experimental strains. CONCLUSION: The results of the performed experiments suggest that the combination of colistin and PFK-158 may be a potential new choice as a new antibiofilm group for the treatment of infections caused by the colistin-resistant GNB.

The prevalence and functional characteristics of CrpP-like in Pseudomonas aeruginosa isolates from China.

Modifying enzyme-CrpP and its variants reduced the MICs of fluoroquinolones in Pseudomonas aeruginosa. This study investigated the dissemination and functional characteristics of CrpP-like in P. aeruginosa from China. The positive rate of crpP-like genes in 228 P. aeruginosa was 25.4% (58/228), and 6 new crpP-like genes were determined. Transformation experiments showed that CrpP-like had a low effect on CIP and LEV susceptibility. The genetic of crpP-positive was diverse. Furthermore, the mean expression level of crpP was no statistical difference between fluoroquinolone-susceptible and -resistant group (P > 0.05). CrpP-like may not play a significant role in fluoroquinolone resistance in P. aeruginosa.

Resistance Profiles and Biological Characteristics of Rifampicin-Resistant Staphylococcus aureus Small-Colony Variants.

BACKGROUND: Staphylococcus aureus (S. aureus) is a major contributor to nosocomial and community-acquired infections. S. aureus small colony variants (SCVs) which changed in relevant phenotype have made more limited and difficult for therapeutic options against S. aureus infections increasingly. Rifampicin is considered as the "last-resort" antibiotic against S. aureus. Our study investigated resistance profiles and biological characteristics of rifampicin-resistant S. aureus SCVs. METHODS: We collected S. aureus SCVs that were selected from 41 rifampicin-resistant clinical isolates. Then, biological characteristics, resistance spectrum, and rifampicin resistance mechanisms of tested S. aureus SCVs and corresponding parental strains were investigated by classic microbiological methods, agar dilution method, polymerase chain reaction (PCR). Moreover, the fitness cost of S. aureus SCVs, including growth, biofilm formation ability, and virulence profile, was also determined by bacterial growth curve assay, biofilm formation assay, and Galleria mellonella infection model. RESULTS: There were three S. aureus SCVs (JP310 SCVs, JP1450 SCVs, JP1486 SCVs) that were selected from 41 rifampicin-resistant S. aureus. S. aureus SCVs colonies were tiny, with decreased pigmentation, and the hemolysis circle was not obvious compared with corresponding parental strains. And SCVs could not be restored to normal-colony phenotype after hemin, menaquinone, or thymidine supplementation. Different rpoB mutations occurred in JP1486 SCVs. Antimicrobial susceptibility testing revealed MICs of SCVs were higher than corresponding parental strains. Besides, the growth ability and virulence of SCVs were lower, and biofilm formation ability of which increased compared with parental strains. CONCLUSION: S. aureus SCVs share the rifampicin resistance mechanisms with parental strains, although there were some differences in the position of rpoB mutations. Moreover, we found that the biological characteristics of SCVs were significantly different from corresponding parental strains. In contrast, decreased susceptibility to other antibiotics of SCVs was observed during phenotype switch. Furthermore, SCVs incur the fitness cost.

Long non­coding RNA LINC00460 serves as a potential biomarker and oncogene via regulation of the miR­320b/PBX3 axis in acute myeloid leukemia.

Long non­coding RNA 00460 (LINC00460) has been reported to be involved in the tumorigenesis of various cancer types. However, the function of LINC00460 in acute myeloid leukemia (AML) remains elusive. Therefore, the present study aimed to investigate the role of LINC00460 in AML. The expression of LINC00460 in the serum of 80 diagnosed patients with AML and 67 healthy controls was measured via reverse transcription­quantitative polymerase chain reaction, and the results were compared with clinical features and patient outcomes. The expression of LINC00460 in 45 patients with cytogenetically normal­AML (CN­AML) was also assayed. Receiver operating characteristic (ROC) curves were generated to evaluate the sensitivity and specificity of serum LINC00460. In addition, the effects of LINC00460 on the viability, cell cycle distribution and apoptosis of AML cells were investigated. Bioinformatics tools were used to identify the possible mechanisms of how LINC00460 affects AML cells. It was found that the expression of LINC00460 was significantly upregulated in the serum of patients with AML and those with CN­AML. Higher expression of serum LINC00460 was positively associated with French­American­British classification and cytogenetics. Furthermore, ROC curve analyses demonstrated that serum LINC00460 could differentiate patients with AML from healthy individuals with an area under the curve of 0.8488 (95% CI, 0.7697­0.9279). The serum LINC00460 expression was also significantly decreased when the patients achieved complete remission. Kaplan­Meier analysis indicated that patients with high serum LINC00460 expression had a shorter overall survival time compared with the low serum LINC00460 expression group. Knockdown of LINC00460 inhibited viability, while inducing cell cycle arrest and apoptosis in AML cells. LINC00460 was also a decoy of microRNA (miR)­320b, which can further inhibit the expression of PBX homeobox 3 (PBX3). Collectively, the results suggested that LINC00460 may be applied as a potential diagnostic and prognostic biomarker for patients with AML. It was identified that LINC00460 may exert its effects, at least partly, via the miR­320b/PBX3 axis in AML.