Novel-detection Methods For Gram-positive Bacteria Utilizing Daptomycin Affinity Mechanism

As one of the leading causes of illness and death,infectious diseases impose severe threat to public health and safety,particularly in most developing countries.Statistically,various bacterial infections are responsible for around one quarter of global mortality.Gram-positive bacteria are the main pathogens responsible to clinical infection,among which staphylococcal infection is the most common.Staphylococcus can lead to many fatal diseases,such as pneumonia,endocarditis,osteomyelitis,bacteremia and so on.Therefore,it is of great importance to develop rapid,simple and sensitive methods for the detection of Gram-positive bacteria.In this study,three facile methods were developed for Gram-positive bacterial detection based on the affinity between daptomycin and bacterial cell membrane.?1?Antibiotic-affinity strategy for bioluminescent detection of viable Gram-positive bacteria using daptomycin as recognition agentA bioluminescent method was proposed for rapid detection of viable Gram-positive bacteria based on a novel antibiotic-affinity strategy on a magnetic beads?MBs?platform.Daptomycin,a highly efficient lipopeptide antibiotic for Gram-positive bacteria,was used as a recognition agent to functionalize MBs.The daptomycin-functionalized MBs showed high capture and concentration efficiency for Gram-positive bacteria due to the strong binding between daptomycin and bacterial cell membrane in the presence of Ca²⁺ion.The captured bacteria were lysed by hexadecyl trimethyl ammonium bromide solution,followed by a bioluminescent detection of the released intracellular adenosine triphosphate?ATP?.Four Gram-positive bacteria,including Staphylococcus aureus?S.aureus?,Streptococcus mutans,Bacillus subtilis and Staphylococcus epidermidis,were detected as model bacteria by this method.Under the optimal conditions,the bacteria can be detected within a linear range of 1.0×10²-3.0×10⁶ CFU mL^-1,with a detection limit of 33 CFU mL^-1.The whole detection procedure could be completed within 20 min.Gram-negative bacteria and dead Gram-positive bacteria showed negligible interference to the detection of viable Gram-positive bacteria.The proposed method was successfully applied to quantify the amount of viable Gram-positive bacteria in cheese,milk,lake water,human urine and physiological saline injection with acceptable recovery values ranging from 75.0%to 120.0%.The strategy possessed some advantages such as high sensitivity,short assay time and simple operation,thus showed great promise for food hygiene,environment monitoring,clinical diagnosis and drug safety.?2?Daptomycin-functionalized magnetic beads integrated with lysostaphin for bioluminescent detection of StaphylococcusA novel antibiotic-affinity strategy was proposed to detect Staphylococcus based on the strong binding between daptomycin and Gram-positive bacteria cell membrane,as well as the specific lytic action of lysostaphin to Staphylococcus.Daptomycin functionalized MBs were adopted to capture and separate Staphylococcus from sample matrix.Afterwards,lysostaphin was used as the specific lysate for Staphylococcus,which can hydrolyze pentaglycine cross-bridges of peptidoglycan in the cell wall of Staphylococcus.Then Staphylococcus can be detected by collecting the bioluminescent signal of the released intracellular ATP of the captured Staphylococcus.S.aureus was used as the model bacteria to investigate the feasibility of this method for the detection of Staphylococcus.Under the optimized conditions,S.aureus can be detected within the linear range of 5.0×10²-5.0×10⁶ CFU mL^-1 within 20 min,with a detection limit of3.8×10² CFU mL^-1.Some Gram-positive bacteria and Gram-negative bacteria,including Micrococcus luteus,Bacillus subtilis,Enterococcus faecium,Escherichia coli,Salmonella typhimurium and Shigella dysenteriae,all showed negligible interference to S.aureus detection.This method was also successfully applied for the detection of S.aureus in milk and physiological saline injection,indicating its potential practical application.?3?A dual-recognition fluorescent protocol for the detection of S.aureusA dual-recognition method was developed for the detection of S.aureus utilizing the binding capability of daptomycin and immunoglobulin G?IgG?to S.aureus.Pig IgG was adopted as the first recognition agent to capture S.aureus based on the fact that the Fc region of IgG can specifically bind with protein A on the surface of S.aureus.Daptomycin can insert into bacterial cell membrane in the presence of Ca²⁺ion,leading to strong binding with the captured S.aureus.Moreover,daptomycin still remained its binding and antibacterial activity after being functionalized by fluorescein isothiocyanate.Therefore,fluorescein isothiocyanate functionalized daptomycin was adopted as the second recognition agent and the signal tracer for the sensing of S.aureus.Under the optimized conditions,S.aureus can be detected within the linear range of 5.0×10³-5.0×10⁸ CFU mL^-1 within 90 min,with a detection limit of 3.6×10³ CFU mL^-1.The proposed protocol can exclude the interference from Gram-negative bacteria and other Gram-positive bacteria.This method was also successfully applied for the detection of S.aureus in lake water and physiological saline injection with acceptable recovery values ranging from87.0%to 120.0%.