pubs.acs.org/acssensors Article Toward Rapid Detection of Viable Bacteria in Whole Blood for Early Sepsis Diagnostics and Susceptibility Testing Sharath Narayana Iyengar,⊥ Jiri Dietvorst,⊥ Amparo Ferrer-Vilanova, Gonzalo Guirado, Xavier Muñoz-Berbel,* and Aman Russom* Cite This: ACS Sens. 2021, 6, 3357−3366 Read Online ACCESS Metrics & More Article Recommendations * sı Supporting Information ABSTRACT: Sepsis is a serious bloodstream infection where the immunity of the host body is compromised, leading to organ failure and death of the patient. In early sepsis, the concentration of bacteria is very low and the time of diagnosis is very critical since mortality increases exponentially with every hour after infection. Common culture-based methods fail in fast bacteria determination, while recent rapid diagnostic methods are expensive and prone to false positives. In this work, we present a sepsis kit for fast detection of bacteria in whole blood, here achieved by combining selective cell lysis and a sensitive colorimetric approach detecting as low as 103 CFU/mL bacteria in less than 5 h. Homemade selective cell lysis buffer (combination of saponin and sodium cholate) allows fast processing of whole blood in 5 min while maintaining bacteria alive (100% viability). After filtration, retained bacteria on filter paper are incubated under constant illumination with the electrochromic precursors, i.e., ferricyanide and ferric ammonium citrate. Viable bacteria metabolically reduce iron(III) complexes, initiating a photocatalytic cascade toward Prussian blue formation. As a proof of concept, we combine this method with antibiotic susceptibility testing to determine the minimum inhibitory concentration (MIC) using two antibiotics (ampicillin and gentamicin). Although this kit is used to demonstrate its applicability to sepsis, this approach is expected to impact other key sectors such as hygiene evaluation, microbial contaminated food/beverage, or UTI, among others. KEYWORDS: sepsis, bacteria, E. coli, selective cell lysis, Prussian blue, colorimetric, blood S epsis is a serious medical condition characterized by a whole-body inflammatory state due to bloodstream infection, with Escherichia coli (E. coli) and Staphylococcus sp. 24 and 72 h before appropriate antibiotics could be prescribed to the patients.5−7 Better performances are obtained with sepsis diagnosis kits (Staph) as the most leading causes of infection.1 In sepsis, currently available in the market, e.g., IRIDICA, SeptiFast, bacteria reach the bloodstream from a local region of infection SeptiTest, or U-them. These kits combine lysis buffers, for fast and spread, leading to organ dysfunction and, in the most blood sample pretreatment and DNA extraction, with PCR analysis, enabling the detection of bacterial DNA, and thus severe conditions, to the patient’s death.2 According to the diagnosing sepsis within 4 and 8 h. The main limitations of 2018 statistics from the World Health Organization (WHO), these kits are their low sensitivity and specificity due to the about 30 million people are affected by sepsis worldwide, background from the human DNA and the impossibility to including 3 million newborn kids and 1.2 million children. distinguish between DNA coming from live or dead bacteria.8 Regarding mortality, 6 million people die of sepsis in the world Moreover, since bacteria are lysed to extract DNA, it is not every year, 500 thousand of them are newborn kids, and this possible to perform antimicrobial susceptibility tests and condition is also responsible for 1 in 10 maternal deaths. This medical prescriptions are based on genetic information, e.g., high mortality is mostly associated with the difficulty of the presence of resistance genes. Although some technologies diagnosing sepsis at its early stages: initial bacterial concentration does not exceed 100 colony forming units Received: June 9, 2021 (CFU)/mL3 and every hour of delay in the diagnostic and Accepted: August 9, 2021 treatment increases the mortality of patients up to 10%.4 These Published: August 19, 2021 fast diagnostic requirements are not attainable with traditional golden standards, i.e., cell culture and polymerase chain reaction (PCR) amplification methods, which require between © 2021 The Authors. Published by American Chemical Society https://doi.org/10.1021/acssensors.1c01219 3357 ACS Sens. 2021, 6, 3357−3366 ACS Sensors pubs.acs.org/acssensors Article Figure 1. Schematic showing the working principle of selective isolation and detection of bacteria from whole blood. The method involves three steps. In the first step, lysis buffer is mixed with the whole blood sample (v/v) and incubated for 5 min with continuous mixing. The lysis buffer selectively ruptures blood cells keeping the bacteria completely viable. In step 2, the ruptured blood cells are filtered out through a simple filtration system. In step 3, bacteria captured on the filter paper are dipped into a culture medium with PB precursors. Filter paper containing viable bacteria turns blue, visible to the naked eye. Experimental results comparing the effect of lysis buffer on RBCs before and after exposure to lysis buffer and decreased viscosity of blood due to lysis buffer rupture are shown below step 1. Scale bar: 10 μm. for viable bacteria isolation are now being developed based on microfluidic chip to capture CD-64 and CD-69 and to detect microfiltration, they suffer from blood clogging and low them within 2 h, providing results that are significantly throughput.9 On the other hand, many strategies based on different between healthy and sepsis patient samples. Klouche electrochromic metabolic indicators are available commercially et al. measured the levels of the CD-14 subtype (presepsin) as or being developed to distinguish between live and dead a biomarker to diagnose community-acquired pneumonia in 44 bacteria.10 These bioassays are based on the use of a redox ICU patients.20 These studies are highly interesting but rely on molecule that is reduced due to bacterial metabolism changing the specificity of these biomarkers for sepsis. Phua et al., for its color or resulting in the production of a fluorescent example, reported that patients showing systemic inflammatory compound, e.g., Presto Blue or Alamar Blue. As a result, the response syndrome had negative culture tests, raising the presence of live bacteria can be detected after 10−15 h of question of the true influence of infection for the observed incubation. inflammatory response.22,23 Other mimickers of sepsis can be Alternative promising technologies are now being developed due to tissue injury, thyroid storm, or inflammatory disorder, based on different detection strategies. Chu et al.,11 developed among others.23−25 In addition, the increased levels of IL-6, a colorimetric sensor for the detection of low concentrations PCT, and CRP biomarkers have also been reported in the early (5−300 CFU/mL) of eight strains of bacteria using tryptic soy and later stages of the current viral pandemic COVID-19 and broth (TSB) media. Although high sensitivity is demonstrated, in patients with hypoxemia.26,27 In addition, high to moderate this method has two important drawbacks, namely, (i) the levels of these biomarkers in the blood are needed for effective need for sample pretreatment when using real blood samples detection,28 which indicates the need for patient samples who (ii) and the duration of the assay, which expands up to 24 h.11 are in the later stage of sepsis. It has also been found that one- This long incubation time is the most limiting aspect of these third of the cases in ICU patients with pneumonia had viral bioassays for their implementation as routine techniques. infections found by PCR assays.23,29 Thus, this approach Other approaches are based on the detection of biomarkers in makes it impossible to differentiate between a viral and blood for early sepsis diagnosis. Rios-Toro et al.,12 Buchegger bacterial infection and does not provide any information on and Preininger,13 and Kemmler et al.14 demonstrated that antibiotic susceptibility, where the clinicians have to still interleukin-6 (IL-6), C-reactive protein (CRP), and procalci- depend on blood cultures to prescribe a specific antibiotic. tonin (PCT) may be good candidates for diagnosing severe Thus, there is an urgent need for efficient methods to diagnose sepsis and septic shock cases, the latter developing a point-of- sepsis in a very short time, here attained by selective isolation care (POC) device for sepsis detection. Min et al., on the other of viable bacteria from whole blood and their rapid detection hand, demonstrated that another potential biomarker, IL-3, with a highly sensitive photocatalytic colorimetric approach. could be detected with high sensitivity and specificity using a This kit for sepsis diagnosis combines two reagents. On the magneto-electrochemical sensor.15 Ghonge et al.16 and Hassan one hand, the composition of the lysis buffer is adjusted to et al.17 measured CD-64 biomarkers in whole blood using a ensure fast and efficient rupture of blood mammalian cell microfluidic biochip with integrated smartphone imaging in 50 membranes while preserving the bacterial cells’ integrity and min, and Zhang et al.18 and Zhou et al.19 also developed a viability. On the other hand, a metabolic indicator was 3358 https://doi.org/10.1021/acssensors.1c01219 ACS Sens. 2021, 6, 3357−3366 ACS Sensors pubs.acs.org/acssensors Article implemented for sensitive live bacteria detection, which also bacterial isolation and detection directly from the whole blood allowed fast antibiotic susceptibility testing. Bacteria detection sample, observable as a change of color to the naked eye in a is achieved through a photocatalytic approach based on a few hours (<5 h for an initial change). cyanotype reaction where the metabolic production of Prussian Effect of Lysis Buffer on Lymphocytes and Platelets. blue (PB) is used to detect the presence of live bacteria.21 The To evaluate the effect of the lysis buffer on other blood cell starting iron donors for PB formation are a specific fractions, lymphocytes and platelets were isolated using the concentration mixture of potassium ferricyanide and ferric Ficoll density gradient-based separation method and analyzed ammonium citrate. Bacteria readily reduce ferricyanide to independently (Figure 2A). Due to the difference in density, a ferrocyanide. 30−32 However, this reduced form cannot spontaneously react with ferric ammonium citrate at the provided dilution. The bond between iron and citrate cannot be spontaneously broken by the presence of ferrocyanide or bacteria but requires light activation. Our previous work has shown that using simply visible light illumination, provided by an artificial light source, the iron molecules uncouple from the citrate, freeing it for Prussian blue formation.31,33,34 If incubated with selected concentrations of antibiotics, bacterial susceptibility and/or resistance could also be determined with the same protocol. The sepsis kit is tested in the bacterial suspension of E. coli ATCC 25922 and Staphylococcus capitis (Staph) in a culture medium and blood. The interference of the components of the lysis buffer, the culture medium, and Figure 2. Effect of lysis buffer on WBCs and platelets. Ficoll density the blood in bacterial detection is studied. The kit is finally gradient separation was performed on whole blood to separate blood validated for susceptibility testing in spiked blood samples. from its components. Isolated WBCs and platelets were stained using ■ calcein dye and an anti-CD-61 antibody, respectively, comparing RESULTS AND DISCUSSION them before and after treatment with lysis buffer. WBCs and platelets were completely ruptured when treated with lysis buffer after 5 min. Working Principle of the Sepsis Kit. The method for Scale bar:100 μm. isolating and detecting bacteria in whole blood involves two main stages: first, the selective lysis of blood cells by keeping the bacteria intact and viable, and second, the detection of Ficoll layer was formed between RBCs (collected at the viable bacteria using a photochemical amplification reaction bottom) and white blood cells (WBCs), while platelets and producing intense blue color PB molecules. The working plasma were collected at the top layer. Isolated pure WBCs and principle is illustrated in Figure 1 and summarized in three platelets were exposed to the lysis buffer for 5 min. For main steps. In the first one, whole blood samples containing visualization, WBCs and platelets were stained separately using bacteria were mixed with lysis buffer containing saponin and calcein green AM dye and fluorescent-labeled anti-CD61 sodium cholate and incubated for 5 min with continuous antibodies, respectively. The effect of lysis buffer was evident stirring. The optimal concentration of lysis buffer was by fluorescence microscopy, where intact cells resulted in an determined by evaluating the effect of different volume ratios intense green (for WBCs) or red (for platelets) color. As of the lysis buffer mixture on whole blood samples (v/v). A shown in Figure 2B, cell fluorescence disappeared, resulting ratio of blood to lysis buffer of 1:10 (v/v) was found optimal, from complete lysis of WBCs and platelets after 5 min of aiming to lyse red blood cells (RBCs) with minimal dilution incubation. (see the Supporting Information, Figure S1). Regarding the Effect of Lysis Buffer on Bacterial Viability. After incubation time, the effect of lysis buffer and its components verifying that among the lysis buffer components, only sodium over time on RBCs was evaluated by optical microscopy cholate may compromise bacterial proliferation (Figure S3), (Figure S2). RBCs treated with lysis buffer were completely the effect of lysis buffer on Gram-negative bacteria E. coli ruptured after 5 min, as shown in the representative images ATCC 25922 and Gram-positive bacteria S. capitis (Staph) was included in Figure 1 (images below step 1). Lysed samples evaluated. High bacterial concentrations (106 CFU/mL) were presented a less intense red color and lower viscosity due to spiked into whole blood and exposed to lysis buffer for 5, 30, the rupture of most RBCs initially present, being optimal for and 60 min (Figure 3). After exposure, bacteria were grown further microbial analysis. under optimal conditions overnight (17 h) in a culture In the second step, previously lysed blood samples were medium and the OD at 600 nm was then determined as a filtered out using a 0.45 μm cellulose filter, and the filter holder semiquantitative measurement of bacterial proliferation. The setup is shown in Figure 1. Intact bacteria were captured on following samples were analyzed for completeness of the the filtering membrane in a fast and simple process not experiment: (i) an LB medium alone (“LB”) and the LB requiring more than 1 min. After that, in step 3, the filter with medium mixed with lysis buffer (“LB + Lysis”) were used as viable bacteria was dipped into culture media containing the negative controls (negative controls 1 and 2, respectively); (ii) precursors of the photochemical reaction (PB solution), i.e., bacteria spiked in the LB medium (“LB + E. coli” or “LB + ferric citrate and ferricyanide, and incubated at 37 °C with Staph”) and bacteria exposed to lysis buffer (“E. coli + Lysis” or exposure to continuous visible light irradiation. Bacterial “Staph + Lysis”) were used as positive controls (positive proliferation over time turned both the filter paper and the controls 1 and 2, respectively); and bacteria spiked in whole solution into an intense blue color visible to the naked eye, blood and exposed to lysis buffer (“Blood + E. coli + Lysis” or which results from the metabolic production of PB molecules. “Blood + Staph + Lysis”) were used as the sample. Results This simple method provided a quick and selective way for presented in graphs (Figure 3A,B) show no significant 3359 https://doi.org/10.1021/acssensors.1c01219 ACS Sens. 2021, 6, 3357−3366 ACS Sensors pubs.acs.org/acssensors Article since 100% viability was obtained considering the bacterial sample in the LB medium as proliferation reference. It is important to note here that even when sodium cholate alone presented important bactericidal activity, the correct combi- nation of reagents in the final lysis buffer composition did not compromise bacterial viability, even when incubating the samples for long times of up to 1 h. This opens the possibility to expand the incubation time when necessary. Optimization of Viable Bacteria Detection through the Photochemical Formation of PB. Detection of viable bacteria captured on the filter paper (step 2 in Figure 1) was achieved using the photochemical reaction, which resulted in the formation of PB molecules. Briefly, bacteria were incubated in a solution containing the PB precursors ammonium ferric citrate and potassium hexacyanoferrate. Viable bacteria metabolically reduce iron(III) complexes, initiating a photo- catalytic cascade toward PB formation under constant illumination. Constant illumination was necessary for PB formation since it induced the photoactivation of ferric citrate and the release of free iron ions. The reaction of metabolically reduced iron(III) complexes with the photocatalytically released free iron ions resulted in the formation of PB Figure 3. Effect of lysis buffer on the viability of bacteria. OD values molecules after short exposure times (<5 h). Before performing were measured for different cases with two negative and positive the experiments with blood, initial studies in bacterial culture controls (n = 3). These controls were compared with the sample media were conducted to optimize the bacterial detection (bacteria in blood and Lysis) for different lysis buffer exposure times protocol and to establish the assay conditions, e.g., sensitivity (5, 30, and 60 min). (A,B) Graphs showing the OD measurements for E. coli and Staph in separate experiments, respectively. Similar OD and limit of detection. E. coli samples containing 1000 CFU/ values for the sample and positive controls for both E. coli and Staph mL were prepared in bacterial culture media and analyzed show that 100% bacterial viability can be retained up to 1 h of lysis following the protocol described in Figure 1, by detecting the buffer exposure. presence of bacteria on the filter paper and in the medium as an intense color change. For quantification, the OD of the differences (p > 0.05) between positive controls 1 and 2 and medium was measured at 600 and 720 nm (Figure 4A), the samples (n = 3) for all lysis buffer exposure times (5, 30, and latter corresponding to the absorption wavelength of PB 60 min). Negative controls were significantly different than molecules. In addition, images of the filter papers dipped in PB positive ones and samples (p > 0.001) since no E. coli or Staph solution were captured after 17 h of incubation (Figure 4B). proliferation was recorded in negative controls, confirming no Filter papers processed with samples “MH + PB” and “MH + contamination of these controls. It may be concluded that the lysis buffer” dipped in PB solution were used as negative lysis buffer produced the selective disruption of the eukaryotic controls. Filter processed with samples having E. coli in MH cell membrane, eliminating blood cell interferences in future dipped in MH media (“MH + E. coli in MH”) was used as a blood analysis, but without compromising bacterial integrity positive control of bacterial proliferation. As E. coli proliferated, Figure 4. Detection of E. coli captured on filter paper after the selective cell lysis step using PB color formation. (A) Graph showing the OD values of PB solution-containing filter papers processed with negative controls (“MH in PB” and “MH + Lysis in PB”) and samples (“MH + E. coli in PB” and “MH + E. coli + lysis buffer in PB”) at 600 and 720 nm. “MH+ E. coli in MH” was used as a positive control. Similar OD values between the samples again showed no influence of lysis buffer on E. coli viability. (B) Prussian blue color was observed on the filter paper that was processed with samples containing E. coli, showing the need for bacterial viability for PB color formation (n = 3). 3360 https://doi.org/10.1021/acssensors.1c01219 ACS Sens. 2021, 6, 3357−3366 ACS Sensors pubs.acs.org/acssensors Article Figure 5. Dependence of time for PB color formation on bacterial concentration. The graph shows the OD values of the PB solution measured for four different concentrations of E. coli (108, 106, 103, and 102 CFU/mL), spiked into MH media in separate experiments (n = 3). Filtration was performed and PB color formation on the filter paper over four different time points (t = 3, 5, 11, and 17 h) was examined. It was observed that filter papers processed with E. coli concentrations of 108, 106, and 103 CFU/mL showed blue color formation between t = 3 and 5 h. For a lower E. coli concentration (102 CFU/mL), the time of PB color formation on the filter paper increased from 5 to 11 h. blue color formation was observed in both the filter paper shown in Figure 5. The filter papers were imaged for all the (containing viable bacteria) and the solution (as bacteria also cases at the corresponding time points. For each of the cases, grew in the solution) of the samples containing bacteria in PB three experimental sets were performed (n = 3) and a precursor solution. Due to its high intensity, the color representative paper sample is imaged in Figure 5. Taking the formation in the filter was even evident with the naked eye. negative control sample as a reference, significant differences in OD values (n = 3) for filters processed with “MH + E. coli” and the OD values were already obtained after 3 h of incubation by “MH + E. coli + lysis buffer” were not significantly different, samples containing 106 (p < 0.05) and 108 CFU/mL (p < confirming that the lysis buffer did not influence E. coli viability 0.001). Samples containing 103 CFU/mL (p < 0.05) were and proliferation (Figure 3). It is important to note that the significantly different after 5 h of incubation, while samples negative controls showed low background OD and no blue containing 102 CFU/mL required 11 h to be significantly color formation on the filter paper (Figure 3B), highlighting different than the negative controls (p < 0.001).Thus, the time the need for viable bacteria for PB color formation. necessary to detect bacteria in the sample with this method As a final remark, the MH media used in this study may be depended on the initial bacterial concentration, and concen- substituted by conventional LB media since nonspecific trations above 103 CFU/mL could be detected within the first interactions were observed between components of the PB 3 h of incubation, while samples containing 102 CFU/mL precursor solution, LB media, and lysis buffer, as demonstrated require more than 5 h. in Figure S4 in the Supporting Information. The protocol was Sepsis Detection in Bacterial-Spiked Whole Blood not only valid for Gram-negative bacteria but also produced Samples. A known concentration (i.e., 103 CFU/mL) of E. positive responses in the case of Gram-positive bacteria (e.g., coli and Staph was spiked into whole blood in separate Staphylococcus; see Figure 6) and even mixtures of Gram- experiments, and the capacity of the assay to detect bacteria in positive and Gram-negative bacteria (see the Supporting blood was evaluated by following the protocol described in Information, Figure S5). This result confirmed the wide Figure 1. The graph in Figure 6A shows the OD quantification applicability of the assay that is sensitive to different bacterial of the PB solution at 600 and 720 nm, measured for the strains and even complex bacterial mixtures. This opens the positive control (“MH + E. coli”), negative controls (“MH”, possibility to detect sepsis early without long genotypic studies. “MH + Lysis”, and “Blood + Lysis + MH”), and the sample Evaluation of the Sensitivity and Response Time of (“Blood + E. coli + Lysis”) with n = 3. Additionally, the the Sepsis Kit. As with all metabolic processes, the corresponding images of the filter papers dipped in the PB photochemically catalyzed production of PB by bacterial solution were also acquired after 17 h (Figure 6B). No activity was a kinetic process depending on the concentration significant differences were obtained when comparing the OD and metabolic activity of the culture. The time needed for the measurements at 600 nm for positive controls and samples (p formation of PB color based on bacterial concentration was > 0.05), which confirmed the low influence of blood and lysis studied by spiking different concentrations of E. coli (108 to 102 buffer on bacterial viability. Higher OD values at 720 nm and CFU/mL) in MH media and analyzing them with the protocol PB color formation were observed only on the filter papers detailed in Figure 1. “MH + lysis buffer” was used as a negative corresponding to positive controls and samples, again control. For each E. coli concentration, bacterial growth was confirming the need for viable E. coli to produce a detectable determined by measuring the OD values at 600 nm (n = 3) of PB color formation. No statistically significant differences (p > the PB solution at four time points (3, 5, 11, and 17 h), as 0.05) were observed between most of the negative controls, 3361 https://doi.org/10.1021/acssensors.1c01219 ACS Sens. 2021, 6, 3357−3366 ACS Sensors pubs.acs.org/acssensors Article Figure 6. Detection of bacteria in spiked whole blood samples using PB color formation. A known concentration of bacteria (103 CFU/mL) was spiked into whole blood and the protocol described in Figure 1 was performed. (A) Bar graph showing the OD values of the PB solution (into which the filter papers were dipped) measured after 17 h of incubation with E. coli (n = 3). Similar OD values of the PB solution on the filter paper processed with the positive control (“MH + E. coli”) and sample (“Blood + E. coli + Lysis”) showed no influence of blood and lysis buffer on bacteria viability. (B) Images of the filter papers taken after 17 h of incubation showed blue color formation on the filter papers processed with the positive control and sample having viable E. coli (n = 3). (C, D) OD values and the corresponding filter paper images for Staph are shown. Only those samples containing Staph dipped in PB solution showed PB formation. Figure 7. Antibiotic susceptibility testing using PB color formation. The lysis buffer-based sample preparation method combined with detection using PB formation was used to perform antibiotic susceptibility testing using different concentrations of ampicillin and gentamicin for E. coli ATCC 25922. Images of the PB color formation on the filter paper and the corresponding OD values measured after 17 h of incubation are shown in the graph. For both antibiotics, it was observed that the OD values and the intensity of blue color formation on the filter paper decreased with an increase in antibiotic concentration (n = 3). (A, B) MIC of ampicillin and gentamicin was determined to be 16 and 8 mg/L, respectively, which agreed with the MIC range provided by the EUCAST. 3362 https://doi.org/10.1021/acssensors.1c01219 ACS Sens. 2021, 6, 3357−3366 ACS Sensors pubs.acs.org/acssensors Article and the small difference between the “MH” and “MH + Lysis” when comparing new approaches with those established by the samples, which was statistically significant (p < 0.05), may be EUCAST, and even the EUCAST states that an MIC value attributed to the absorption/scattering of components of the differing 2-fold from the standard method should be lysis buffer. Similar results were obtained in the case of blood considered reliable. In this case, the cyanotype-based reaction samples spiked with Gram-positive bacteria, i.e., Staph, as was two orders of magnitude more sensitive than broth shown in Figure 6C,D (OD values and filter paper images, microdilution, being able to detect 103 CFU/mL within 3−4 h respectively). These results confirmed the low or null cross- of the reaction. Due to this enhanced sensitivity, the MIC reactivity between the components of the precursor solution values provided by this assay were slightly higher than those and those present in the blood samples. tabulated, although very close to the 2-fold limit stablished by Antibiotic Susceptibility Testing. Due to the severity of the EUCAST. The current method may be additionally blood infections, wide-spectrum antibiotics are prescribed as improved by quantifying blue color formation with spectro- the first-choice treatment for sepsis before any genotypic or scopic systems instead of by visual inspection, as performed in phenotypic analysis is performed. This allows fast decision- the current work. making with a high probability of failure by, e.g., the presence As a proof of principle, the experiment was repeated with a of resistant bacteria or due to inappropriate selection of low concentration of E. coli (103 CFU/mL) and the MIC for antibiotics. One way to solve this issue is by performing fast ampicillin was determined to be 16 mg/L as shown in Figure antibiotic susceptibility testing directly in blood samples, S7. Although the measurements were performed after 17 h, the without culture or purification of the bacteria. This is possible initial blue color formation on the filter paper was already with the protocol detailed in Figure 1, by incubating viable observed after 3 h of incubation, thus having the possibility to blood bacteria captured in the filter paper with the antibiotic provide MIC in a much shorter time. and performing a susceptibility test in less than 5 h. The This result demonstrates the applicability of this selective minimum inhibitory concentration (MIC) was determined by cell lysis and photochemical reaction for fast antibiotic following the protocol provided by the European Committee susceptibility testing directly in blood samples, being able to on Antimicrobial Susceptibility Testing (EUCAST).35 MIC determine the most suitable treatment in sepsis in a few hours, was selected for the concentration of the antibiotics at which even when not knowing the specific bacterial strain responsible low or null PB color formation was observed. Two antibiotics for the infection. were tested in this case, ampicillin and gentamicin, and the experimental values were compared with those tabulated by the EUCAST for E. coli ATCC 25922, i.e., 2−8 mg/L for ■ CONCLUSIONS In this paper, we present a novel sepsis kit, where a selective ampicillin and 0.25−1 mg/L for gentamicin. Experimentally, a cell lysis-based sample preparation method is combined with a known concentration of E. coli (108 CFU/mL, recommended highly sensitive photochemical reaction capable of reporting McFarland 0.5 standard concentration) was spiked into whole the presence of bacterial infection in blood samples within 5 h blood and the protocol described in Figure 1 was followed. for 103 CFU/mL bacterial concentration through a simple Different concentrations of ampicillin (0−16 mg/L) and color change. We demonstrate this by initially characterizing gentamicin (0−8 mg/L) were added to the PB precursor the selective cell lysis buffer to specifically rupture blood cells solution during incubation (step 3 of Figure 1). “MH + Lysis”, in 5 to 10 min, without compromising bacterial integrity and “MH + E. coli”, and “Blood + E. coli + Lysis” were used as the activity (i.e,. 100% viable). Using a simple syringe-based negative control (NC), positive control (0 mg/L), and sample, filtration setup, viable bacteria are retained in a filter paper, respectively. In addition to taking the images of the filter paper, while lysed blood cells are completely washed out, not OD values of the PB solution were also acquired at 600 nm interfering with the subsequent analysis. Filter papers after 17 h of incubation by comparison with standard MIC containing blood bacteria are then incubated in the PB protocols. The bar graph in Figure 7 shows the average value precursor solution in an MH medium under continuous and standard deviation of the measured OD values of three irradiation. This incubation starts a photocatalytic and experimental sets (n = 3) for each antibiotic concentration metabolically mediated chemical reaction, resulting in the under study. Filter images in the figure correspond to formation of intense blue PB molecules only when living and representative samples. As expected, OD values for the positive metabolically active bacteria are retained in the filter (negative control and sample decreased when increasing antibiotic control samples without bacteria remain white). The detection concentration (Figure 7A,B). No significant differences were process is sensitive to both Gram-negative and Gram-positive obtained when comparing absorbance values at 600 and 720 bacteria and even to a mixture of different bacterial strains. nm, as illustrated in the Supporting Information (Figure S6 for Furthermore, as a proof of concept, we show that this ampicillin and Figure S8 for gentamicin), confirming that both method can also be used to perform antibiotic susceptibility wavelengths are equally valid to report on bacterial activity testing by exposing the filter paper having bacteria to different through this assay. In agreement with previous results, the concentrations of antibiotics, which was tested using ampicillin intensity of blue color formation on the filter paper also and gentamicin, and providing similar results as gold standards decreased with the increasing antibiotic concentration, after 3 h of incubation. The current plating method needs 2 to signifying the loss of bacterial viability and metabolic activity 3 days from sample preparation to detect bacteria in sepsis at high antibiotic concentrations. Considering both OD and blood samples. As a short time to diagnose sepsis is very blue color formation, MIC values for ampicillin and gentamicin crucial, we believe that this new method has great potential in of 16 and 8 mg/L, respectively, were obtained. These values the clinical settings to quickly select positive patient samples were slightly higher than those provided by the EUCAST, from the negative ones, which helps in prescribing appropriate which are in ranges of 2−8 and 0.25−1 mg/L for ampicillin antibiotics, thus reducing antibiotic abuse. Furthermore, this and gentamicin, respectively. As already reported by Mouton et simple syringe-based sample preparation to color change-based al.,36 a small difference in the MIC magnitude can be obtained bacterial detection has very high potential in resource-limited 3363 https://doi.org/10.1021/acssensors.1c01219 ACS Sens. 2021, 6, 3357−3366 ACS Sensors pubs.acs.org/acssensors Article settings toward sepsis diagnostics, as it is easy to use and does color formation at 600 and 720 nm, respectively. Here, 720 nm is the not demand the need for expensive equipment. wavelength specific to the absorbance of PB.31,34 Captured bacteria on ■ the filter paper were detected by colorimetric analysis. After the filtration step, filter papers were dipped into 5 mL of a solution METHODS containing 625 μL each of 2.5 mM ammonium ferric citrate and 0.625 Lysis Buffer. Lysis buffer was prepared by mixing sodium cholate mM potassium hexacyanoferrate in MH media (referred to as PB hydrate (MW: 430.55 g, Sigma-Aldrich) and saponin (1.015−1.020 solution). Two negative controls and two samples were used in the g/mL at 20 °C, Sigma-Aldrich). Concentrations of 4% sodium cholate experiment to demonstrate the formation of PB color in the presence and 2% saponin were dissolved in 1X phosphate-buffered saline (PBS of bacteria. Negative controls included (1) MH media and (2) MH tablets, pH: −7.2 to 7.6, Sigma-Aldrich) separately. Equal volumes of media mixed with lysis buffer (1:10 v/v). The two samples included each were mixed to obtain a final pH of 6.5, which was used as a lysis were (3) E. coli in MH and (4) E. coli in MH exposed to the lysis buffer for the sample preparation method. The final concentration in buffer. Filter papers were dipped into PB solution at 37 °C for 17 h the lysis buffer is 1% saponin and 2% sodium cholate. (overnight) and continuously exposed to light. After overnight Prussian Blue. Colorimetric detection was done using Prussian culture, OD measurements were performed at 600 and 720 nm. blue color formation. Ammonium ferric citrate (2.5 mM; iron content Blood Samples Analysis. For the blood sample analysis, fresh of 16.5 to 18.5%, Sigma-Aldrich) and potassium hexacyanoferrate blood samples were collected from healthy blood donors from the (0.625 mM; MW = 329.24 g/mol) were prepared using Mueller− blood center GeBlod, Stockholm, Sweden, in EDTA tubes. Experi- Hinton (MH) media. The final solution was adjusted to a pH of 6.5. ments were performed on the day of blood collection. No other blood Antibiotics. Ampicillin powder (MW = 349.4 g/mol) and pretreatment step was performed. One milliliter of the whole blood gentamicin sulfate salt white powder (potency: 600 μg of gentamicin was mixed with lysis buffer and incubated for 5 min at room per mg) were ordered from Sigma-Aldrich. temperature with continuous stirring. Different ratios of whole blood Culture Media. Luria−Bertani (LB) broth miller (pH range: 6.8 to lysis buffer volume were tried, ranging from 1:1 to 1:10 (v/v). to 7.2) and MH broth 2 (final pH: 7.3. ± 0.2 at 25 °C) were ordered Antibiotic Susceptibility Testing. Different concentrations of from Sigma-Aldrich. For all the experiments with MH media, the pH antibiotics ampicillin and gentamicin were used in the experiments. A of the final MH liquid broth was adjusted to 6.5 using 0.1 M HCl. stock solution (5.12 mg/mL) was prepared by dissolving the Characterization and Optimization of Lysis Buffer Activity. antibiotic powder in Milli-Q water. The desired concentrations of For characterizing the lysis buffer, a known concentration of E. coli antibiotics were selected using the EUCAST (European Committee ATCC 25922 strain was spiked into LB media and was mixed with on Antimicrobial susceptibility testing) method. The required lysis buffer (1:10 v/v). The mixture was filtered using a 10 mL B.D concentration of antibiotics was further obtained by diluting the plastic Luer-lock syringe by placing a 0.45 μm nitrocellulose filter stock using MH media. After capturing the bacteria on the filter paper paper (MF: Millipore membrane filter mixed with cellulose esters, using the process explained in Figure 1, the filter paper was dipped Sigma-Aldrich) with 25 mm diameter in a filter holder (easy-pressure into a solution containing 2.5 mL of antibiotics and 625 μL each of syringe filter holder, 25 mm, Pall Laboratory). Translucent poly- 2.5 mM ammonium ferric citrate and 0.625 mM potassium ethylene terephthalate (PET) filters (SABEU plastics and membrane hexacyanoferrate and made up to a total volume of 5 mL using technology) with 0.4 μm pore size with 25 mm diameter were used MH media. This setup was finally incubated in the presence of a light for PET-based experiments. Filter papers with captured E. coli were source in a 37 °C incubator overnight. dipped into a freshly autoclaved LB/MH broth and were incubated at Data and Statistical Analysis. Statistical analysis was performed 37 °C overnight (17 h). After overnight culture, optical density (OD) using GraphPad Prism 6 software (RRID:SCR_002798; GraphPad measurements were performed using a spectrophotometer (Spec- Software, La Jolla, CA, USA). All results presented in the article are tromax M series, molecular devices) at OD = 600 nm. expressed as mean ± standard deviation. One- or two-way ANOVA Ficoll Separation. Standard Ficoll density gradient separation was followed by Bonferroni’s post-hoc correction was, respectively, used performed on 1 mL of whole blood to separate blood cells from its to compare one or multiple variables. Statistical analysis was components. Whole blood was diluted in 1:5 v/v using 1X PBS and performed only when each group size was at least n = 3 independent was added to 7.5 mL of Ficoll solution. The mixture was centrifuged variables. The threshold for statistical significance was p < 0.05 throughout. ■ for 30 min at 400g without break. Pure lymphocytes were carefully removed and centrifuged for 10 min at 350g. The supernatant containing PBS was discarded and the pellet was resuspended in PBS. ASSOCIATED CONTENT Isolated lymphocytes were stained with calcein green AM dye for * sı Supporting Information visualization and incubated with the lysis buffer at room temperature The Supporting Information is available free of charge at for 15 min in the dark to avoid photobleaching of the fluorescent dye. https://pubs.acs.org/doi/10.1021/acssensors.1c01219. Eukaryotic Cell Staining. WBC staining was performed using CellTrace calcein green AM dye (Thermo Fischer Scientific). Ficoll- Effect of concentration of lysis buffer on whole blood separated pure WBCs were stained by adding 1 μL of calcein green (Figure S1); effect of lysis buffer and its components on AM dye to 1 mL of the sample and incubated at room temperature for blood cells over time (Figure S2 with GIF); effect of 15 min in the dark and viewed using a fluorescence microscope before components of lysis buffer on E. coli viability in the exposing to the lysis buffer. To visualize platelets, Ficoll-separated blood (Figure S3); detection of E. coli in the LB medium plates were centrifuged again at 1200 rpm for 5 min and the pellet was by PB color formation (Figure S4); detection of the resuspended in 100 μL of 1X PBS. Platelets were stained with a 10 Gram-negative and Gram-positive mixture using PB μg/mL final concentration of Alexa Fluor 647 anti-human CD61 antibody. This was incubated in the dark for 25 min at 4 °C. This formation (Figure S5); antibiotic susceptibility testing mixture was centrifuged at 1200 rpm for 5 min, and the supernatant using ampicillin at 720 nm (Figure S6); antibiotic was discarded. The pellet was resuspended in 100 μL of 1X PBS and susceptibility testing using ampicillin for low concen- then visualized using a fluorescence microscope before exposure to tration of E.coli (Figure S7); antibiotic susceptibility the lysis buffer. testing using gentamicin at 720 nm (Figure S8) (PDF) ■ Colorimetric Bacterial Detection. An LED spotlight (15 W, 6400 K, 85−256 V Vanessa-15) was used as a light source (a range of AUTHOR INFORMATION visible light wavelength, mostly in blue color) to induce the photocatalytic reaction. In addition to qualitative detection based Corresponding Authors on color change, we also measured the absorbance by spectrometry to Xavier Muñoz-Berbel − Instituto de Microelectrónica de semiquantitatively measure bacterial concentration based on the PB Barcelona (IMB-CNM, CSIC), Universitat Autónoma de 3364 https://doi.org/10.1021/acssensors.1c01219 ACS Sens. 2021, 6, 3357−3366 ACS Sensors pubs.acs.org/acssensors Article Barcelona, Barcelona 08193, Spain; orcid.org/0000- of effective antimicrobial therapy is the critical determinant of survival 0002-6447-5756; Email: Xavier.Munoz@imb-cnm.csic.es in human septic shock. Crit. Care Med. 2006, 34, 1589−1596. Aman Russom − Division of Nanobiotechnology, Department (5) Frank, U.; Malkotsis, D.; Mlangeni, D.; Daschner, F. D. of Protein Science, Science for Life Laboratory, KTH Royal Controlled clinical comparison of three commercial blood culture Institute of Technology, Stockholm 17165, Sweden; AIMES - systems. Eur. J. Clin. 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M.; Sweden; AIMES - Center for the Advancement of Integrated Fraley, S. I. Emerging Technologies for Molecular Diagnosis of Sepsis. Medical and Engineering Sciences at Karolinska Institutet Clin. Microbiol. Rev. 2018, 31, No. e00089. and KTH Royal Institute of Technology, Stockholm 17165, (9) Hosokawa, M.; Asami, M.; Nakamura, S.; Yoshino, T.; Sweden; orcid.org/0000-0001-5348-3526 Tsujimura, N.; Takahashi, M.; Nakasono, S.; Tanaka, T.; Jiri Dietvorst − Instituto de Microelectrónica de Barcelona Matsunaga, T. Leukocyte counting from a small amount of whole (IMB-CNM, CSIC), Universitat Autónoma de Barcelona, blood using a size-controlled microcavity array. Biotechnol. Bioeng. Barcelona 08193, Spain 2012, 109, 2017−2024. Amparo Ferrer-Vilanova − Instituto de Microelectrónica de (10) JiriDietvorst, L. NaroaUria, Maria-PilarMarco, XavierMuñoz- Barcelona (IMB-CNM, CSIC), Universitat Autónoma de Berbel, Current and near-future technologies for antibiotic suscept- ibility testing and resistant bacteria detection. TrAC, Trends Anal. Barcelona, Barcelona 08193, Spain; orcid.org/0000- Chem. 2020, 127. 0002-4223-8442 (11) Chu, Y. W.; Wang, B. Y.; Engebretson, D. A.; Carey, J. R. Single Gonzalo Guirado − Department de Química, Universitat step, rapid identification of pathogenic microorganisms in a culture Autònoma de Barcelona, Barcelona 08193, Spain bottle. Analyst 2013, 138, 5879−5885. Complete contact information is available at: (12) Rios-Toro, J. J.; Marquez-Coello, M.; Garcia-Alvarez, J. M.; https://pubs.acs.org/10.1021/acssensors.1c01219 Martin-Aspas, A.; Rivera-Fernandez, R.; Saez de Benito, A.; Giron- Gonzalez, J. A. Soluble membrane receptors, interleukin 6, Author Contributions procalcitonin and C reactive protein as prognostic markers in patients ⊥ with severe sepsis and septic shock. PLoS One 2017, 12, S.N.I. and J.D. contributed equally. S.N.I. and J.D. together No. e0175254. performed all the experiments and S.N.I. wrote the manuscript. (13) Buchegger, P.; Preininger, C. Point-of-Care Chip for Diagnosis A.F.-V. and G.G. helped in the determination of bacterial of Sepsis: Joining Biomarker Quantification and Bacterial Class sensing mechanisms. A.R. and X.M.-B. are responsible for Identification. Biomed. Tech. 2013, 58, 000010151520134146. reviewing and editing the manuscript. A.R. and X.M.-B. were (14) Kemmler, M.; Sauer, U.; Schleicher, E.; Preininger, C.; the principal supervisors for this work. A.R. and X.M.-B. are Brandenburg, A. Biochip point-of-care device for sepsis diagnostics. responsible for funding and resource acquisition. All authors Sens. Actuators, B 2014, 192, 205−215. have approved the final version of the manuscript. (15) Min, J.; Nothing, M.; Coble, B.; Zheng, H.; Park, J.; Im, H.; Notes Weber, G. F.; Castro, C. M.; Swirski, F. K.; Weissleder, R.; Lee, H. The authors declare no competing financial interest. Integrated Biosensor for Rapid and Point-of-Care Sepsis Diagnosis. ■ ACS Nano 2018, 12, 3378−3384. ACKNOWLEDGMENTS (16) Ghonge, T.; Koydemir, H. C.; Valera, E.; Berger, J.; Garcia, C.; Nawar, N.; Tiao, J.; Damhorst, G. L.; Ganguli, A.; Hassan, U.; Ozcan, This project has received funding from the European Union A.; Bashir, R. Smartphone-imaged microfluidic biochip for measuring Horizon 2020 research and innovation programme under the CD64 expression from whole blood. Analyst 2019, 144, 3925−3935. 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