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Introduction: Antibiotic resistance in bacterial species constitutes a growing problem in the clinical management of infections. Not only does it limit therapeutic options, but application of ineffective antibiotics allows resistant species to progress prior to prescribing more effective treatment to patients. Methicillin resistance in Staphylococcus aureus is a major problem in clinical infections as it is the most common hospital acquired infection. Methods: We developed a photoacoustic flow cytometer using engineered bacteriophage as probes for rapid determination of methicillin resistance in Staphylococcus aureus with thirteen clinical samples obtained from keratitis patients. This method irradiates cells under flow with 532 nm laser light and selectively generates acoustic waves in labeled bacterial cells, thus enabling detection and enumeration of them. Staphylococcus aureus isolates were classified from culture isolation as either methicillin resistant or susceptible using cefoxitin disk diffusion testing. The photoacoustic method enumerates bacterial cells before and after treatment with antibiotics. Decreasing counts of bacteria after treatment indicate susceptible strains. We quantified the bacterial cells in the treated and untreated samples. Results: Using k-means clustering on the data, we achieved 100% concordance with the classification of Staphylococcus aureus resistance using culture. Discussion: Photoacoustics can be used to differentiate methicillin resistant and susceptible strains of bacteria from ocular infections. This method may be generalized to other bacterial species using appropriate bacteriophages and testing for resistance using other antibiotics.
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Photoacoustic flow cytometry is a method to detect rare analytes in fluids. We developed photoacoustic flow cytometry to detect pathological cells in body fluids, such as circulating tumor cells or bacteria in blood. In order to induce specific optical absorption in bacteria, we use modified bacteriophage that precisely target bacterial species or subspecies for rapid identification. In order to reduce detection variability and to halt the lytic lifescycle that results in lysis of the bacteria, we attached dyed latex microspheres to the tail fibers of bacteriophage that retained the bacterial recognition binding sites. We tested these microsphere complexes using Salmonella enterica (Salmonella) and Escherichia coli (E. coli) bacteria and found robust and specific detection of targeted bacteria. In our work we used LT2, a strain of Salmonella, against K12, a strain of E. coli. Using Det7, a bacteriophage that binds to LT2 and not to K12, we detected an average of 109.3±9.0 of LT2 versus 2.0±1.7 of K12 using red microspheres and 86.7±13.2 of LT2 versus 0.3±0.6 of K12 using blue microspheres. These results confirmed our ability to selectively detect bacterial species using photoacoustic flow cytometry.
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OBJECTIVES: Bacteremia is a serious and potentially lethal condition. Staphylococcus aureus is a leading cause of bacteremia and methicillin-resistant S. aureus (MRSA) accounts for more than a third of the cases. Compared to methicillin-sensitive S. aureus, MRSA is more than twice as likely to be fatal. Furthermore, subpopulations of seemingly isogenic bacteria may exhibit a range of susceptibilities, often called heterogenous resistance. These heterogeneous antibiotic-resistant infections are often misdiagnosed as hospital-acquired secondary infections because there are no clinically used tests that can differentiate between homogeneous and heterogeneous antibiotic resistance. We describe the development and proof of concept of rapid bacterial identification using photoacoustic flow cytometry and labeled bacteriophages with the characterization and differentiation of heterogeneous antibiotic-resistant bacterial infections. METHODS: In photoacoustic flow cytometry, pulsed laser light is delivered to a sample flowing past a focused transducer and particles that absorb laser light create an acoustic response. Optically labeled bacteriophage are added to a bacterial mixture that flows through the photoacoustic chamber. The presence of target bacteria is determined by bound labeled phage which are detected photoacoustically. Incubation of bacterial samples in the presence and absence of the antibiotic daptomycin creates a difference in bacterial cell numbers that is quantified using photoacoustic flow cytometry. RESULTS: Four clinical isolates were tested in the presence and absence of daptomycin. Photoacoustic events for each isolate were recorded and compared to growth curves. Samples treated with daptomycin fell into three categories: resistant, susceptible, and heterogeneous resistant. CONCLUSIONS: Here we show a method to determine the presence of bacteria as a marker for bloodstream infection level and antibiotic sensitivity in less than 4 hours. Additionally, these results show an ability to identify heterogeneous resistant strains that are often misidentified.
Assuntos
Bacteriemia , Infecção Hospitalar , Daptomicina , Staphylococcus aureus Resistente à Meticilina , Infecções Estafilocócicas , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Bacteriemia/diagnóstico , Bacteriemia/tratamento farmacológico , Bacteriemia/microbiologia , Daptomicina/uso terapêutico , Humanos , Testes de Sensibilidade Microbiana , Infecções Estafilocócicas/diagnóstico , Infecções Estafilocócicas/tratamento farmacológico , Infecções Estafilocócicas/microbiologia , Staphylococcus aureusRESUMO
Early detection of cancer has been a goal of cancer research in general and melanoma research in particular (Birnbaum et al., Lancet Glob Health 6:e885-e893, 2018; Alendar et al., Bosnian J Basic Med Sci 9:77-80, 2009). Early detection of metastasis has been targeted as pivotal to increasing survival rates (Menezes et al., Adv Cancer Res 132:1-44, 2016). Melanoma, though curable in its early stages, has a dramatic decrease in survival rates once metastasis has occurred (Sharma et al., Biotechnol Adv 36:1063-1078, 2018). The transition to metastasis is not well understood and is an area of increasing interest. Metastasis is always premeditated by the shedding of circulating tumor cells (CTCs) from the primary tumor. The ability to isolate rare CTCs from the bloodstream has led to a host of new targets and therapies for cancer (Micalizzi et al., Genes Dev 31:1827-1840, 2017). Detection of CTCs also allows for disease progression to be tracked in real time while eliminating the need to wait for additional tumors to grow. Using a photoacoustic flowmeter, in which we induce ultrasonic responses from circulating melanoma cells (CMCs), we identify and quantify these cells in order to track disease progression. Additionally, these CMCs are captured and isolated allowing for future analysis such as RNA-Seq or microarray analysis.