Toward Rapid Detection of Viable Bacteria in Whole Blood for Early Sepsis Diagnostics and Susceptibility Testing

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.


Effect of concentration of lysis buffer
Incubation of blood samples with the lysis buffer implies the dilution of the already low bacterial concentration in blood, although a too low concentration of buffer may reduce the efficiency of cell lysis. Eight different dilutions from 1:2 to 1:10 (blood : buffer ratio in v/v) were evaluated after 5min of incubation and analyzed by microscopy. The ratio mixture is defined based on volume to volume ratio (v/v). For instance, 1:2 denotes 1mL of whole blood mixed with 2mL of lysis buffer. Volume of blood was always kept constant to 1mL while the volume of lysis buffer was varied. Only in the 1:2 sample, some intact bacteria were still observed in the microscopic preparation ( Fig.S1), suggesting that the cut off concentration ensuring complete blood cell lysis was in between 1:2 and 1:4. The incubation of blood samples in a 1:4 dilution for 5min was selected as the optimal lysis protocol. However, 1:10 dilution was used in further experiments to retain the highest concentration of lysis buffer.  S1. Effect of concentration of lysis buffer on whole blood. Different volume to volume ratio of whole blood to lysis buffer from 1:2 to 1:10 was tested. The results showed that there were few blood cells still intact in 1:2 case while cases above 1:4 had completely ruptured all blood cells. The cut off ratio was thus found to be between 1:2 to 1:4.

Effect of lysis buffer and its components on blood cells over time
The effect of lysis buffer and its components on the blood cells were investigated in separate experiments over time and bright field images were captured. After exposing to 2% saponin, few blood cells (RBCs) were still observed even after 1hr of exposure. In addition, few blood cell clumps were also observed in few cases after 1hr (highlighted in red box). On the other hand, all the blood we completely ruptured after exposing to 4% sodium cholate and lysis buffer after 110s and 100s respectively.

Effect of components of lysis buffer on E.coli viability in blood
Effect of lysis buffer and components of lysis buffer were compared to study their effect on bacterial viability over time in blood. Initial concentration of 10^3 CFU/mL E.coli was spiked into whole blood and exposed to 2% sodium cholate, 1% saponin and lysis buffer separately and incubated over time (0 to 4hr) at room temperature. No lysis buffer condition was used as negative control (E.coli in PBS). After every hour, agar plating was performed and colonies were counted after 17hr of incubation at 37 o C (n=3). Statistical analysis revealed no significant (ns) difference for all the cases up to 1hr of incubation. As the incubation time was increased no significant difference was obtained between E.coli in MH (No lysis ) and E.coli exposed to saponin. This showed that saponin did not affect the E.coli viability over time. On the other hand, we observed a decrease in E.coli count which reduced to zero after exposing to sodium cholate and a significant difference; p<0.001 (***) was observed for sodium cholate after 2hr of incubation signifying that sodium cholate when exposed alone, kills bacteria. However, mixing saponin and sodium cholate in the lysis buffer revived the E.coli viability over time, signifying the importance of lysis buffer on bacterial viability with a value of p<0.01 (**).

Fig.S3. Effect of lysis buffer and its components on viability of E.coli in blood over time. 1%
saponin, 2% sodium cholate and lysis buffer were exposed to E.coli spiked in blood. Agar plating was performed over time from 1hr to 4hr for all the cases (n=3). It was observed that after 2hr of exposure to sodium cholate, there was a decrease in E.coli growth and no growth was observed after 4hr of exposure. However, for both saponin and lysis buffer, the viability of E.coli was retained for all the time points. showed a high background OD values of 1 and 3 A.U respectively with a significant difference; p<0.001 (***). This is due to some reaction between components present in LB, PB and lysis 6 buffer solution which is still under evaluation. The sample having Lysis buffer, PB and E.coli also showed a very similar O.D value of 3.5 A.U which is not a significant change (ns) in value to differentiate from 'LB + Lysis buffer + PB'. As a result, LB media was replaced by MH (Mueller-Hinton) media for future experiments. To understand the change in viability in a sample containing mixture of bacteria, two different bacterial species were spiked in MH media. Equal concentration (1000 CFU/mL) of gram negative (E.coli) and gram positive (Staphylococcus Capitis) and absorbance was measured after incubating the filter paper for 17hr in PB solution as shown in graph of Fig.7A (n=3). It was observed that the absorbance was similar for the samples with and without lysis buffer exposure (''MH + E.coli + Staph'' and ''MH + E.coli + Staph+ Lysis'') signifying very low effect of lysis buffer on bacterial viability. In addition, the filter paper were taken out from the tubes and were compared to see the PB color formed on the filter paper as shown in Fig.7B It was observed that the sample containing bacteria formed blue coloration on the filter paper confirming the need to bacterial metabolism for PB formation. Antibiotic susceptibility testing using ampicillin at 720nm 8 As an extension to Fig.8, OD values were also measured at 720nm for the antibiotic susceptibility testing that was performed using ampicillin on E.coli (n=3). As expected similar trend of OD values were seen with increase in antibiotic concentration.

Antibiotic susceptibility testing using ampicillin for low concentration of E.coli
To determine the MIC of ampicillin for low concentration of E.coli (1000 CFU/mL), the antibiotic susceptibly testing was repeated and OD values were measures at 600nm and 720nm for 'Blood +E.coli + Lysis' as sample (n=3). 'MH + Lysis' was used as a negative control (NC). OD measurements and the PB color formation on the filter paper showed that the MIC was at 16mg/L, showing the applicability of this method to low concertation of bacteria as shown in Fig.S7.  Fig.S7. Antibiotic susceptibility testing using ampicillin at 600 and 720nm for low concertation of E.coli. Low concentration of E.coli (1000 CFU/mL) was spiked into whole blood and MIC was determined by performing antibiotic susceptibility testing by following the protocol in Fig.1 (n=3). Decrease in OD values were observed as the bacterial growth get suppressed with increasing antibiotic concentration and the MIC was determined to be 16mg/L..

Antibiotic susceptibility testing using gentamicin at 720nm
Antibiotic susceptibly testing was performed at three different concentration of gentamicin (0.5, 2 and 8mg/L) to determine the MIC of gentamicin for E.coli. A similar trend of decreasing OD values measured at 600nm were seen with increase in the concentration of gentamicin concentration as shown in Fig.S6 (n=3). Correspondingly decreased blue color intensity was observed in filter paper with increasing concentration of gentamicin.