Brucella abortus (B. abortus) is one of the primary causative agents of brucellosis, posing potential threats to livestock production and public health security. This study aims to screen specific M13 phages targeting the bacteria and to establish a phage-functionalized electrochemical biosensor, thereby validating its detection performance and application potential in actual sample testing.

Using B. abortus as a target, biopanning of a phage display peptide library was performed, combined with negative screening to enrich candidate M13 phage clones. Indirect ELISA was employed to assess the affinity and specificity of each clone. The phages were immobilized onto a gold electrode surface, with bovine serum albumin used to block non-specific sites to construct an electrochemical biosensor. Differential pulse voltammetry (DPV) current response values were employed as an indicator to optimize phage titer, phage incubation time, and sample incubation time. The DPV current response values were measured for concentrations ranging from 1. 0×10³ to 1. 0×10⁷ CFU/mL. A four-parameter logistic function was used to fit the standard curve, and the limit of detection (LOD) was calculated. Staphylococcus aureus, Staphylococcus epidermidis, Acinetobacter baumannii, and Escherichia coli were selected as negative controls to evaluate the specificity of the sensor. The same concentration of B. abortus samples was repeatedly detected 10 times, and the relative standard deviation (RSD) was calculated to assess repeatability. The sensor was stored at 4℃, and B. abortus was detected at different time points to calculate the RSD and evaluate the stability. B. abortus bacterial suspensions were prepared as spiked serum samples using fetal bovine serum as the matrix. SPF female BALB/c mice (6 to 8 weeks old, weighing 16 to 18 g) were randomly divided into a control group and an infected group, with 5 animals in each group. The infection group was intraperitoneally inoculated with B. abortus, and the blank group was inoculated with the same volume of PBS. Mouse spleen samples were collected. Both the constructed electrochemical biosensor and plate counting method were used to detect the above 2 types of samples. The recovery rate was calculated to evaluate the detection accuracy of the sensor in actual samples.

Through biopanning and indirect ELISA validation, M13 phage clone P-1 capable of specifically binding to B. abortus was screened, and its binding affinity to B. abortus was superior to that of other control strains. The optimal construction conditions for the sensor were as follows: phage titer of 1. 0×10¹¹ PFU/mL, phage incubation time of 2 h, and sample incubation time of 1 h. The fitting equation for this sensor within the bacterial concentration range of 1. 0×10³ to 1. 0×10⁷ CFU/mL is I/μA = 81. 13 + $\frac{(184.08-81.13)}{{\left(1+\frac{{\log }_{10}\mathrm{C}}{5.01}\right)}^{8.68}}\left(R^2=0.9999\right)$, with an LOD of 157 CFU/mL. The sensor demonstrated favorable detection specificity (P<0. 0001) and repeatability (RSD=2. 70%), with stable performance (RSD=2. 37%). The recovery rate of B. abortus detected by the sensor in fetal bovine serum samples ranged from 98. 2% to 106. 0%, while the bacterial load in spleen samples from infected mouse model was detected as 1. 23×10³ CFU/mL, and the accuracy of this method was consistent with the gold standard plate culture method.

A specific M13 phage clone, P-1, targeting B. abortus was identified through screening, and this clone was used to construct an electrochemical biosensor capable of quantitatively detecting the target bacterium.

To prepare a high performance electrochemical immunosensor for detecting Brucella abortus (B. abortus).

Prussian blue (PB), multi walled carbon nanotubes (MWCNTs) and gold nanoparticles (GNPs) (PB-MWCNTs-GNPs) nanocomposites were prepared, and appropriate antibody was used to construct the immunosensor for detecting B. abortus samples. The optimal conditions were clarified by examining the key factors in sensor construction, and then the performance of the sensor was evaluated.

The optimal construction conditions were determined as follows: the ratio of MWCNTs-PB was 1 ∶5, the drying temperature was 37 ℃, the pH value of buffer system was 7.5, and the incubation time of antibody and sample was 1 h and 30 min, respectively. B. abortus exhibited a good linear relationship, when ranging from 10 to 1×10⁵ CFU/mL. The sensor had good anti-interference ability, repeatability, stability and high accuracy.

Our prepared PB-MWCNTs GNPs nanomaterials modified electrochemical immunosensor for detecting B. abortus is easy to prepare, has good performance, and can provide reference for the early clinical diagnosis of brucellosis.

To investigate anti-biofilm activity and mechanisms of emodin against methicillin-resistant Staphylococcus aureus (MRSA) in vitro.

Microbroth dilution was explored to determine minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of emodin against MRSA252 strain and clinical isolations. Inhibitory curve were draw to estimate dynamic bacteriostatic effect. Crystal violet staining was used to investigate the inhibitory effect of emodin against MRSA biofilm. The effect of emodin on the morphology of MRSA biofilm was observed by scanning electron microscopy (SEM) and laser scanning confocal microscopy (LSCM). The expression of intercellular adhesion (ica) operon was detected by fluorescence quantitative PCR.

The MICs of emodin against different strains of MRSA ranged from 2 to 8 μg/mL, while MBCs were from 4 to 16 μg/mL. Emodin obviously inhibited the formation of biofilm in a dose-dependent manner. Besides, it destroyed biofilm structure and increased the ratio of dead bacteria under biofilm. Fluorescence quantitative PCR showed that emodin significantly down-regulated the expression of ica operon.

Emodin can inhibit MRSA biofilm in vitro, and may restrain the formation of bacterial biofilm by down-regulating the expression of ica operon.