Enhancing bactericidal activities of ciprofloxacin by targeting the trans-translation system that is involved in stress responses in Klebsiella pneumoniae.

Although the trans-translation system is a promising target for antcibiotic development, its antibacterial mechanism in Klebsiella pneumoniae (KP) is unclear. Considering that tmRNA was the core component of trans-translation, this study firstly investigated phenotypic changes caused by various environmental stresses in KP lacking trans-translation activities (tmRNA-deleted), and then aimed to evaluate antibacterial activities of the trans-translation-targeting antibiotic combination (tobramycin/ciprofloxacin) in clinical KP isolates based on inhibition activities of aminoglycosides against trans-translation. We found that the tmRNA-deleted strain P4325/ΔssrA was significantly more susceptible than the wild-type KP strain P4325 under environments with hypertonicity (0.5 and 1 M NaCl), hydrogen peroxide (40 mM), and UV irradiation. No significant differences in biofilm formation and survivals under human serum were observed between P4325/ΔssrA and P4325. tmRNA deletion caused twofold lower MIC values for aminoglycosides. As for the membrane permeability, tmRNA deletion increased ethidium bromide (EtBr) uptake of KP in the presence or absence of verapamil and carbonyl cyanide-m-chlorophenylhydrazone (CCCP), decreased EtBr uptake in presence of reserpine in P4325/ΔssrA, and reduced EtBr efflux in P4325/ΔssrA in the presence of CCCP. The time-kill curve and in vitro experiments revealed significant bactericidal activities of the tmRNA-targeting aminoglycoside-based antibiotic combination (tobramycin/ciprofloxacin). Thus, the corresponding tmRNA-targeting antibiotic combinations (aminoglycoside-based) might be effective and promising treatment options against multi-drug resistant KP.

Murepavadin promotes the killing efficacies of aminoglycoside antibiotics against Pseudomonas aeruginosa by enhancing membrane potential.

Murepavadin is a peptidomimetic that specifically targets the lipopolysaccharide transport protein LptD of Pseudomonas aeruginosa. Here, we found that murepavadin enhances the bactericidal efficacies of tobramycin and amikacin. We further demonstrated that murepavadin enhances bacterial respiration activity and subsequent membrane potential, which promotes intracellular uptake of aminoglycoside antibiotics. In addition, the murepavadin-amikacin combination displayed a synergistic bactericidal effect in a murine pneumonia model.

Prediction and validation of enzymatic degradation of aflatoxin M1: Genomics and proteomics analysis of Bacillus pumilus E-1-1-1 enzymes.

Aflatoxins are a class of highly toxic mycotoxins. Aflatoxin M1 (AFM1) is hydroxylated metabolite of aflatoxin B1, having comparable toxicity, which is more commonly found in milk. In this study, the whole genome sequencing of Bacillus pumilus E-1-1-1 isolated from feces of 38 kinds of animals, having aflatoxin M1 degradation ability was conducted. Bacterial genome sequencing indicated that a total of 3445 sequences were finally annotated on 23 different cluster of orthologous groups (COG) categories. Then, the potential AFM1 degradation proteins were verified by proteomics; the properties of these proteins were further explored, including protein molecular weight, hydrophobicity, secondary structure prediction, and three-dimensional structures. Bacterial genome sequencing combined with proteomics showed that eight genes were the most capable of degrading AFM1 including three catalases, one superoxide dismutase, and four peroxidases to clone. These eight genes with AFM1 degrading capacity were successfully expressed. These results indicated that AFM1 can be degraded by Bacillus pumilus E-1-1-1 protein and the most degrading proteins were oxidoreductases.

Neutrophil-mediated delivery of the combination of colistin and azithromycin for the treatment of bacterial infection.

Novel treatment strategies are in urgent need to deal with the rapid development of antibiotic-resistant superbugs. Combination therapies and targeted drug delivery have been exploited to promote treatment efficacies. In this study, we loaded neutrophils with azithromycin and colistin to combine the advantages of antibiotic combinations, targeted delivery, and immunomodulatory effect of azithromycin to treat infections caused by Gram-negative pathogens. Delivery of colistin into neutrophils was mediated by fusogenic liposome, while azithromycin was directly taken up by neutrophils. Neutrophils loaded with the drugs maintained the abilitity to generate reactive oxygen species and migrate. In vitro assays demonstrated enhanced bactericidal activity against multidrug-resistant pathogens and reduced inflammatory cytokine production by the drug-loaded neutrophils. A single intravenous administration of the drug-loaded neutrophils effectively protected mice from Pseudomonas aeruginosa infection in an acute pneumonia model. This study provides a potential effective therapeutic approach for the treatment of bacterial infections.

Development of Resistance to Eravacycline by Klebsiella pneumoniae and Collateral Sensitivity-Guided Design of Combination Therapies.

The evolution of bacterial antibiotic resistance is exhausting the list of currently used antibiotics and endangers those in the pipeline. The combination of antibiotics is a promising strategy that may suppress resistance development and/or achieve synergistic therapeutic effects. Eravacycline is a newly approved antibiotic that is effective against a variety of multidrug-resistant (MDR) pathogens. However, the evolution of resistance to eravacycline and strategies to suppress the evolution remain unexplored. Here, we demonstrated that a carbapenem-resistant Klebsiella pneumoniae clinical isolate quickly developed resistance to eravacycline, which is mainly caused by mutations in the gene encoding the Lon protease. The evolved resistant mutants display collateral sensitivities to ß-lactam/ß-lactamase inhibitor (BLBLI) combinations aztreonam/avibactam and ceftazidime-avibactam. Proteomic analysis revealed upregulation of the multidrug efflux system AcrA-AcrB-TolC and porin proteins OmpA and OmpU, which contributed to the increased resistance to eravacycline and susceptibility to BLBLIs, respectively. The combination of eravacycline with aztreonam/avibactam or ceftazidime-avibactam suppresses resistance development. We further demonstrated that eravacycline-resistant mutants evolved from an NDM-1-containing K. pneumoniae strain display collateral sensitivity to aztreonam/avibactam, and the combination of eravacycline with aztreonam/avibactam suppresses resistance development. In addition, the combination of eravacycline with aztreonam/avibactam or ceftazidime-avibactam displayed synergistic therapeutic effects in a murine cutaneous abscess model. Overall, our results revealed mechanisms of resistance to eravacycline and collateral sensitivities to BLBLIs and provided promising antibiotic combinations in the treatment of multidrug-resistant K. pneumoniae infections. IMPORTANCE The increasing bacterial antibiotic resistance is a serious threat to global public health, which demands novel antimicrobial medicines and treatment strategies. Eravacycline is a newly approved antibiotic that belongs to the tetracycline antibiotics. Here, we found that a multidrug-resistant Klebsiella pneumoniae clinical isolate rapidly developed resistance to eravacycline and the evolved resistant mutants displayed collateral sensitivity to antibiotics aztreonam/avibactam and ceftazidime-avibactam. We demonstrated that the combination of eravacycline with aztreonam/avibactam or ceftazidime-avibactam repressed resistance development and improved the treatment efficacies. We also elucidated the mechanisms that contribute to the increased resistance to eravacycline and susceptibility to aztreonam/avibactam and ceftazidime-avibactam. This work demonstrated the mechanisms of antibiotic resistance and collateral sensitivity and provided a new therapeutically option for effective antibiotic combinations.

Antifouling BaTiO3/PVDF piezoelectric membrane for ultrafiltration of oily bilge water.

Barium titanate/polyvinylidene fluoride (BaTiO3/PVDF) piezoelectric membrane was successfully prepared and generated in-situ vibrations to reduce membrane fouling by applying alternating current (AC) signal for oily bilge water ultrafiltration. The effect of in-situ vibration on membrane fouling was investigated through changing in the excitation alternating voltage and its frequency, pH, crossflow rate. The results indicated that the piezoelectric membrane by applying AC signal remarkably alleviated the membrane fouling for bilge water ultrafiltration. The membrane fouling decreased with increasing the AC signal voltage. The final steady-state permeate flux from the piezoelectric membrane for bilge water ultrafiltration increased with the AC signal voltage, raising it by up to 63.4% at AC signal voltage of 20 V compared to that of the membrane without applying AC voltage. The high permeate flux was obtained at the resonant frequency of 220 kHz. During the 50-h ultrafiltration of bilge water with the piezoelectric membrane excited at 220 kHz and 15 V, the permeate flux from the membrane was stable. The oil concentration in outflow from the piezoelectric membrane was below 14 ppm, which met the discharged level required by IMO convention. The total organic carbon removal rate in bilge water was over 94%.

A Retrospective Observation of Treatment Outcomes Using Decitabine-Combined Standard Conditioning Regimens Before Transplantation in Patients With Relapsed or Refractory Acute Myeloid Leukemia.

Hypomethylating agents, decitabine (DAC) and azacitidine, can act as prophylactic and pre-emptive approaches after allogeneic hematopoietic stem cell transplantation (allo-HSCT) and a non-intensive bridging approach before allo-HSCT. However, they are rarely used as a part of conditioning regimens in patients with relapsed or refractory acute myeloid leukemia (AML). This retrospectively study included a total of 65 patients (median, 37; range, 13-63) with relapsed or refractory AML who were treated by allo-HSCT after myeloablative conditioning regimens without or with DAC (high-dose DAC schedule, 75 mg/m2 on day -9 and 50 mg/m2 on day -8; low-dose DAC schedule, 25 mg/m2/day on day -10 to -8). DAC exerted no impact on hematopoietic reconstitution. However, patients who were treated with the high-dose DAC schedule had significantly higher incidence of overall survival (OS, 50.0%) and leukemia-free survival (LFS, 35.0%), and lower incidence of relapse (41.1%) and grade II-IV acute graft versus host disease (aGVHD, 10.0%) at 3 years, when compared with those treated with standard conditioning regimens or with the low-dose DAC schedule. In conclusion, high-dose DAC combined with standard conditioning regimens before allo-HSCT is feasible and efficient and might improve outcomes of patients with relapsed or refractory AML, which provides a potential approach to treat these patients.

Synergistic bactericidal activities of tobramycin with ciprofloxacin and azithromycin against Klebsiella pneumoniae.

Trans-translation is a unique bacterial ribosome rescue system that plays important roles in the tolerance to environmental stresses. It is composed of an ssrA-encoded tmRNA and a protein SmpB. In this study, we examined the role of trans-translation in antibiotic tolerance in Klebsiella pneumoniae and explored whether the inhibition of this mechanism could enhance the bactericidal activities of antibiotics. We found that deletion of the ssrA gene reduced the survival of K. pneumoniae after treatment with kanamycin, tobramycin, azithromycin, and ciprofloxacin, indicating an important role of the trans-translation in bacterial antibiotic tolerance. By using a modified ssrA gene with a 6×His tag we demonstrated that tobramycin suppressed the azithromycin and ciprofloxacin-elicited activation of trans-translation. The results were further confirmed with a trans-translation reporter system that is composed of a normal mCherry gene and a gfp gene without the stop codon. Compared to each individual antibiotic, combination of tobramycin with azithromycin or ciprofloxacin synergistically enhanced the killing activities against planktonic K. pneumoniae cells and improved bacterial clearance in a murine cutaneous abscess infection model. In addition, the combination of tobramycin and ciprofloxacin increased the bactericidal activities against biofilm-associated cells. Overall, our results suggest that the combination of tobramycin with azithromycin or ciprofloxacin is a promising strategy in combating K. pneumoniae infections.

Evaluation of TaqMan Array card (TAC) for the detection of 28 respiratory pathogens.

BACKGROUND: Respiratory infections are a serious threat to human health. So, rapid detection of all respiratory pathogens can facilitate prompt treatment and prevent the deterioration of respiratory disease. Previously published primers and probes of the TaqMan array card (TAC) for respiratory pathogens are not sensitive to Chinese clinical specimens. This study aimed to develop and improve the TAC assay to detect 28 respiratory viral and bacterial pathogens in a Chinese population. METHODS: To improve the sensitivity, we redesigned the primers and probes, and labeled the probes with minor groove binders. The amplification efficiency, sensitivity, and specificity of the primers and probes were determined using target-gene containing standard plasmids. The detection performance of the TAC was evaluated on 754 clinical specimens and the results were compared with those from conventional methods. RESULTS: The performance of the TAC assay was evaluated using 754 clinical throat swab samples and the results were compared with those from gold-standard methods. The sensitivity and specificity were 95.4 and 96.6%, respectively. The lowest detection limit of the TAC was 10 to 100 copies/µL. CONCLUSIONS: TAC is an efficient, accurate, and high-throughput approach to detecting multiple respiratory pathogens simultaneously and is a promising tool for the identification of pathogen outbreaks.

Sorption of tri-n-butyl phosphate and tris(2-chloroethyl) phosphate on polyethylene and polyvinyl chloride microplastics in seawater.

Microplastics and organophosphate esters are ubiquitous pollutants in the marine environment. However, their interactions are poorly understood. In the present study, the sorptions of tri-n-butyl phosphate (TnBP) and tris(2-chloroethyl) phosphate (TCEP) on polyethylene (PE) and polyvinyl chloride (PVC) microplastics in seawater were investigated. Results indicated that the first-order kinetic model and pseudo-second-order were suitable to describe PE and PVC microparticles for the adsorption of the two organophosphate esters, respectively. The adsorption capacity increased with the decrease in particle size. The highest adsorption capacity appeared at 15 °C. The equilibrium isotherms data for the adsorption of the two organophosphate esters on PVC and PE microplastics were best fitted with Freundlich isotherm model and Langmuir isotherm model, respectively. The pore-filling mechanism involved in the adsorption of TnBP and TCEP on PVC microplastics and the monolayer coverage was the predominant mechanism for the adsorption of TnBP and TCEP on PE microplastics.

Combination of Azithromycin and Gentamicin for Efficient Treatment of Pseudomonas aeruginosa Infections.

BACKGROUND: Trans-translation is a ribosome rescue system that plays an important role in bacterial tolerance to environmental stresses. It is absent in animals, making it a potential treatment target. However, its role in antibiotic tolerance in Pseudomonas aeruginosa remains unknown. METHODS: The role and activity of trans-translation during antibiotic treatment were examined with a trans-translation-deficient strain and a genetically modified trans-translation component gene, respectively. In vitro assays and murine infection models were used to examine the effects of suppression of trans-translation. RESULTS: We found that the trans-translation system plays an essential role in P. aeruginosa tolerance to azithromycin and multiple aminoglycoside antibiotics. We further demonstrated that gentamicin could suppress the azithromycin-induced activation of trans-translation. Compared with each antibiotic individually, gentamicin and azithromycin combined increased the killing efficacy against planktonic and biofilm-associated P. aeruginosa cells, including a reference strain PA14 and its isogenic carbapenem-resistance oprD mutant, the mucoid strain FRD1, and multiple clinical isolates. Furthermore, the gentamicin-azithromycin resulted in improved bacterial clearance in murine acute pneumonia, biofilm implant, and cutaneous abscess infection models. CONCLUSIONS: Combination treatment with gentamicin and azithromycin is a promising strategy in combating P. aeruginosa infections.

Prevalence and risk factors of human pegivirus type 1 infection in hematopoietic stem cell transplantation patients.

OBJECTIVES: To investigate the prevalence, risk factors, and genotypes of human pegivirus type 1 (HPgV-1) in hematopoietic stem cell transplantation (HSCT) patients. METHODS: One hundred and eighty-eight HSCT patients and 694 healthy blood donors were investigated retrospectively, including their demographic information and HPgV-1 infection status. RESULTS: When compared with healthy blood donors, a significantly higher HPgV-1 prevalence (18.6% vs. 2.3%) and a high risk of HPgV-1 infection (odds ratio 9.7) were observed in HSCT patients (p<0.05). The number of transfusions in patients with RNA test conversions (negative to positive) was significantly higher than the number in patients without conversions (negative to negative) (median 10 vs. 1) (p<0.05). Although HPgV-1 infection is independent of age, sex, blood type, hepatitis B virus infection, hepatitis C virus infection, marriage status, and type of hematological malignancy (p>0.05), race might be a risk factor for infection (p<0.05). The great majority (95.7%) of HPgV-1-positive patients were infected with genotype 3. CONCLUSIONS: HPgV-1 is highly prevalent in HSCT patients, and blood transfusions can significantly increase the risk of HPgV-1 infection. Thus, HPgV-1 screening is recommended in HSCT patients to reduce the potential impact of infection on survival, as well as in their blood and stem cell donors to reduce the risk of infection after transfusions, unless the beneficial effects of HPgV-1 infection in immunocompromised patients are clearly confirmed.

Comparison of rRNA-based and DNA-based nucleic acid amplifications for detection of Chlamydia trachomatis, Neisseria gonorrhoeae, and Ureaplasma urealyticum in urogenital swabs.

BACKGROUND: Nucleic acid amplification tests (NAAT) are well-accepted in diagnosis and surveillance of sexually infectious pathogens worldwide. However, performance differences between a RNA-based NAAT and DNA-based NAAT are rarely reported. This study compares the performances of the RNA-based SAT (simultaneous amplification and testing) assay and the DNA-based quantitative real-time polymerase chain reaction (qPCR) assay. METHODS: A total of 123 urogenital swabs were collected from outpatients with suspected genital infections in our hospital. Chlamydia trachomatis (CT), Neisseria gonorrhoeae (NG), and Ureaplasma urealyticum (UU) in these swabs were simultaneously tested by SAT and qPCR. Any swabs were positive in the qPCR assay were further verified by following cloning and sequencing. All statistical analysis was performed using the SPSS software. RESULTS: When the concentrations of CT, NG, or UU were more than 1 × 103 copies/ml, 100% agreements between SAT and qPCR were observed regardless of the pathogen. No discrepancy was found. However, the sensitivity of SAT is significantly higher than qPCR in samples with concentration less than 1 × 103 copies/ml. When tested by SAT and qPCR, 57.14 and 28.57% were positive for CT, 46.15% and 0 were positive for NG, 80% and 0 were positive for UU, respectively. CONCLUSIONS: The SAT assay has better agreements and higher sensitivities when compared with the qPCR assay, and thus could be a better choice for screening, diagnosis, and surveillance of sexually transmitted diseases, especially for CT and NG.

Detection of pathogenic microorganisms from bloodstream infection specimens using TaqMan array card technology.

Bloodstream infections (BSIs) are often life-threatening, and rapid identification is critical. Here, we developed a TaqMan array card (TAC) assay to detect pathogens in BSI specimens. The TAC included 30 primer/probe pairs targeting 27 species and 3 controls. Reverse transcription and 0.1% blue dextran 2000 increased the TAC assay efficiency. The primer/probe pairs had a limit of detection of 100-102 CFU/mL and a specificity of 100%. For whole blood specimens, the TAC assay showed a sensitivity and specificity of 79.4% and 99.69%, respectively. For blood culture samples, the TAC assay showed a sensitivity and specificity of 100% and 99.67%, respectively. The TAC assay could be a promising method for early detection of bloodstream infection.

Development and application of a real-time polymerase chain reaction assay for detection of a novel gut bacteriophage (crAssphage).

crAssphage is a novel and by far the most abundant bacteriophage in human gut. This bacteriophage might modulate gut microbiota balance so as to be involved in some diseases like obesity, diabetes, metabolic disorders, hypertension, and cancer. Therefore, a rapid and reliable detection and quantification method for crAssphage is essential for studying its molecular epidemiology and pathogenicity in human diseases. The primers-probes set for the quantitative real-time PCR assay was designed based on the DNA polymerase gene (ORF00018) of crAssphage. The sensitivity and specificity, as well as comparison testing with the conventional PCR and sequencing were evaluated. The assay could specifically detect crAssphage, and no cross-reactions with other gut microbes were observed. The detection limit was 15.6 copies/µL of clinical samples (46.8 copies/reaction). When using clinical samples, the assay showed higher ability to detect samples with low viral DNA copies and had an agreement of 93.33% when compared with the conventional PCR amplification and sequencing. The established real-time PCR assay is a sensitive, specific, and repeatable method for quantitatively detecting crAssphage, and thus is a very useful tool for investigating the molecular epidemiology, dynamics, and pathogenicity of crAssphage in human diseases.

Eubacterium moniliforme Bacteremia in a Woman with Fractures.

E. moniliforme infections in humans have not been reported previously. We firstly described blood-stream infections caused by E. moniliforme in an elder woman with fractures of her left thigh. This study highlights the strategies to detect this anaerobic pathogen and the importance of investigating its molecular epidemiology in humans.

Clonal replacement of epidemic KPC-producing Klebsiella pneumoniae in a hospital in China.

BACKGROUND: Klebsiella pneumoniae is a frequent nosocomial pathogen causing difficult-to-treat infections worldwide. The prevalence of Klebsiella pneumoniae carbapenemase (KPC)-producing Klebsiella pneumoniae (KPC-KP) is increasing in China. The aim of this study was to investigate the molecular epidemiology of KPC-KP in a nosocomial outbreak. METHODS: Fifty-four KPC-KP isolates were consecutively collected between November 2013 and August 2014 during a KPC-KP outbreak in a tertiary care hospital in Beijing, China. Antimicrobial susceptibility was determined by agar dilution. Carbapenemase, extended-spectrum ß-lactamase, 16S rRNA methylase, AmpC ß-lactamase, and plasmid-mediated quinolone resistance determinants were detected by PCR amplification. The genetic relatedness of isolates was analyzed by pulsed-field gel electrophoresis and multi-locus sequence typing. RESULTS: All isolates belonged to ST11 except one isolate which was identified as a new sequence type (ST2040). PFGE profile of genomic DNA revealed seven clusters, of which cluster A and C dominated the KPC-KP outbreak and cluster A was replaced by cluster C during the outbreak. PFGE of genomic DNA, S1-PFGE of plasmids, replicon typing, and drug resistant characteristics showed that clonal spread occurred during the outbreak. When compared with isolates within cluster A, all isolates in cluster C harbored rmtB and showed higher level of resistance to cefepime, amikacin, tobramycin, and tigecycline. CONCLUSION: We reported a nosocomial outbreak of KPC-KP with clonal replacement and a new sequence type (ST2040) of KP. High degree of awareness and surveillance of KPC-KP should be given to avoid potential outbreaks, especially in ICU wards.

Sequencing of blaIMP-Carrying IncN2 Plasmids, and Comparative Genomics of IncN2 Plasmids Harboring Class 1 Integrons.

This work presents the complete nucleotide sequences of p0801-IMP from Klebsiella pneumoniae, p7121-IMP from K. oxytoca, and p17285-IMP from Citrobacter freundii, which are recovered from three different cases of nosocomial infection. These three plasmids represent the first fully sequenced blaIMP-carrying IncN2 plasmids. Further comparative genomics analysis of all the five integron-carrying IncN2 plasmids p0801-IMP, p7121-IMP, p17285-IMP, pJIE137, and p34983-59.134kb indicates that they possess conserved IncN2 backbones with limited genetic variations with respect to gene content and organization. Four class 1 integrons (blaIMP-1-carrying In1223 in p0801-IMP/p7121-IMP, blaIMP-8-carrying In655 in p17285-IMP, In27 in pJIE137, and In1130 in p34983-59.134kb), two insertion sequence-based transposition units (ISEcp1-orfRA1-14 in p17285-IMP, and ISEcp1-blaCTX-M-62-Δorf477-orfRA1-14 in pJIE137), and a novel Tn1696-related transposon Tn6325 carrying In1130 in p34983-59.134kb are indentified in the plasmid accessory regions. In1223 and In655 represent ancestral Tn402-associated integrons, while In27 and In1130 belong to complex class 1 integrons. The relatively small IncN2 backbones are able to integrate different mobile elements which carry various resistance markers, promoting the accumulation and spread of antimicrobial resistance genes among enterobacterial species.

Complete Genome Sequence of Serratia rubidaea Isolated in China.

We report here the first complete genome of Serratia rubidaea, isolated from a patient in China.