Disclosed herein are methods and compositions for antimicrobial quantification and functional measurement. In one aspect, a method for quantifying antimicrobial comprises: obtaining a biological sample from a patient receiving an antimicrobial; incubating the biological sample with a reference microbial strain and a fluorophore for detecting cell lesion; measuring a first signal of fluorescent intensity in the incubated biological sample using flow cytometry; and comparing the first signal to a calibrating curve previously generated for the antimicrobial, thereby quantifying the antimicrobial present in the biological sample.

Methods for determining bacterial identity and susceptibility or resistance to antibiotic or antimicrobial agents are provided. In one embodiment, the bacteria is cultured in the presence or absence or the antibiotic agent to generate a plurality of primary cultures, which are then cultured in the presence or absence of transforming phages to generate a first secondary culture that comprise transformed bacteria that have been treated with the antibiotic agent and a second secondary culture that comprises transformed bacteria that have not been treated with the antibiotic agent. The recombinant phages are specific to the bacteria and comprise a heterologous marker. The susceptibility or resistance of the bacteria to the antibiotic or antimicrobial agent is determined by comparing a level or activity of the marker in the first and second secondary cultures.

Methods for determining bacterial identity and susceptibility or resistance to antibiotic or antimicrobial agents are provided. In one embodiment, the bacteria is cultured in the presence or absence or the antibiotic agent to generate a plurality of primary cultures, which are then cultured in the presence or absence of transforming phages to generate a first secondary culture that comprise transformed bacteria that have been treated with the antibiotic agent and a second secondary culture that comprises transformed bacteria that have not been treated with the antibiotic agent. The recombinant phages are specific to the bacteria and comprise a heterologous marker. The susceptibility or resistance of the bacteria to the antibiotic or antimicrobial agent is determined by comparing a level or activity of the marker in the first and second secondary cultures.

Detecting single bacterial cells in a sample includes collecting, from a sample provided to an imaging apparatus, a multiplicity of images of the sample over a length of time; assessing a trajectory of each bacterial cell in the sample; and assessing, based on the trajectory of each bacterial cell in the sample, a number of bacterial cell divisions that occur in the sample during the length of time.

Detecting single bacterial cells in a sample includes collecting, from a sample provided to an imaging apparatus, a multiplicity of images of the sample over a length of time; assessing a trajectory of each bacterial cell in the sample; and assessing, based on the trajectory of each bacterial cell in the sample, a number of bacterial cell divisions that occur in the sample during the length of time.

Methods for determining the susceptibility or resistance of bacteria to antibiotic agents are provided. In one embodiment, the methods include culturing the bacteria in the presence or absence or the antimicrobial agent to generate a primary culture which is then cultured in the presence or absence of transforming phages. The recombinant phages are specific to the bacteria and comprise a heterologous marker (e.g., a nucleic acid that is expressible as a detectable product such as an RNA or a protein). The susceptibility or resistance of the bacteria to the antimicrobial agent may be determined by assaying the culture for the presence or absence of the heterologous marker, wherein a reduction in the level or activity of the marker in the culture compared to the level or activity of the marker in a comparative culture indicates that the bacteria is sensitive to the antibiotic agent.

Methods for determining the susceptibility or resistance of bacteria to antibiotic agents are provided. In one embodiment, the methods include culturing the bacteria in the presence or absence or the antimicrobial agent to generate a primary culture which is then cultured in the presence or absence of transforming phages. The recombinant phages are specific to the bacteria and comprise a heterologous marker (e.g., a nucleic acid that is expressible as a detectable product such as an RNA or a protein). The susceptibility or resistance of the bacteria to the antimicrobial agent may be determined by assaying the culture for the presence or absence of the heterologous marker, wherein a reduction in the level or activity of the marker in the culture compared to the level or activity of the marker in a comparative culture indicates that the bacteria is sensitive to the antibiotic agent.

A system and method for automated testing a sample of a body fluid for the presence of bacteria is described. The system includes a fluid handling device, Incubator, flow cytometer, at least a processor, and a memory configuring the at least a processor to distribute a portion of the plurality of fluid samples within a well plate to at least a first well, divide the portion of the plurality of fluid samples from the at least a first well into at least two wells including a T.sub.0 well and a T.sub.1 well, obtain a T.sub.0 enumerative baseline bacterial value at time T.sub.0, culture the fluid samples in the T.sub.1 well using the incubator, obtain a T.sub.1 enumerative control bacterial value at time T.sub.1, and determine a presence of bacteria as a function of the T.sub.0 enumerative baseline bacterial value and the T.sub.1 enumerative control bacterial value.

A system and method for automated testing a sample of a body fluid for the presence of bacteria is described. The system includes a fluid handling device, Incubator, flow cytometer, at least a processor, and a memory configuring the at least a processor to distribute a portion of the plurality of fluid samples within a well plate to at least a first well, divide the portion of the plurality of fluid samples from the at least a first well into at least two wells including a T.sub.0 well and a T.sub.1 well, obtain a T.sub.0 enumerative baseline bacterial value at time T.sub.0, culture the fluid samples in the T.sub.1 well using the incubator, obtain a T.sub.1 enumerative control bacterial value at time T.sub.1, and determine a presence of bacteria as a function of the T.sub.0 enumerative baseline bacterial value and the T.sub.1 enumerative control bacterial value.

A method of performing antimicrobial susceptibility testing (AST) on a sample uses a reader device that mounts on a mobile phone having a camera. A microtiter plate containing wells preloaded with the bacteria-containing sample, growth medium, and drugs of differing concentrations is loaded into the reader device. The wells are illuminated using an array of illumination sources contained in the reader device. Images of the wells are acquired with the camera of the mobile phone. In one embodiment, the images are transmitted to a separate computing device for processing to classify each well as turbid or not turbid and generating MIC values and a susceptibility characterization for each drug in the panel based on the turbidity classification of the array of wells. The MIC values and the susceptibility characterizations for each drug are transmitted or returned to the mobile phone for display thereon.