Increased growth temperature and vitamin B12 supplementation reduces the lag time for rapid pathogen identification in BHI agar and blood cultures

Background: Rapid diagnostics of pathogens is essential to prescribe appropriate antibiotic therapy. The current methods for pathogen detection require the bacteria to grow in a culture medium, which is time-consuming. This increases the mortality rate and global burden of antimicrobial resistance. Culture-free detection methods are still under development and are not common in the clinical routine. Therefore, decreasing the culture time for accurately detecting infection and resistance is vital for diagnosis. Methods: This study investigated easy-to-implement factors (in a minimal laboratory set-up), including inoculum size, incubation temperature, and additional supplementation ( e.g., vitamin B12 and trace metals), that can significantly reduce the bacterial lag time (t lag). These factors were arranged in simple two-level factorial designs using Gram-positive cocci ( Staphylococcus aureus), Gram-positive bacilli ( Bacillus subtilis), and Gram-negative bacilli ( Escherichia coli and Pseudomonas aeruginosa) bacteria, including clinical isolates with known antimicrobial resistance profiles. Blood samples spiked with a clinical isolate of E. coli CCUG 17620 (Culture Collection University of Gothenburg) were also tested to see the effect of elevated incubation temperature on bacterial growth in blood cultures. Results: We observed that increased incubation temperature (42°C) along with vitamin B12 supplementation significantly reduced the t lag (10 – 115 minutes or 4% - 49%) in pure clinical isolates and blood samples spiked with E. coli CCUG17620. In the case of the blood sample, PCR results also detected bacterial DNA after only 3h of incubation and at three times the CFU/mL. Conclusion: Enrichment of bacterial culture media with growth supplements such as vitamin B12 and increased incubation temperature can be a cheap and rapid method for the early detection of pathogens. This proof-of-concept study is restricted to a few bacterial strains and growth conditions. In the future, the effect of other growth conditions and difficult-to-culture bacteria should be explored to shorten the lag phase.


Introduction
The discovery of antibiotics was a breakthrough in therapeutic medicines, which has enabled us to treat critical bacterial infections (Ali et al., 2020). It has been more than 35 years since a new class of antibiotics was discovered, and bacteria are resistant to almost all the presently used antibiotics (Hoffman, 2020). The unavailability of new antibiotics further increases the need for rapid diagnostics of pathogens and their AMR. Early and appropriate antibiotic therapy is crucial in cases of severe bacterial infections and can significantly reduce mortality. One such example is sepsis, a major health problem that accounts for an estimated 31.5 million cases with a mortality rate of 5.3 million deaths globally per year (Fleischmann et al., 2016;Taxt et al., 2020). In the case of sepsis, early treatment with appropriate antibiotics is vital for the patient's survival (Bloos et al., 2017;Ferrer et al., 2014). In septic patients, delaying each hour in prescribing appropriate antibiotics can have lethal consequences (Avershina et al., 2022). Due to the lack of adequate and early diagnostics, clinicians are pushed to prescribe empirical antibiotics to the patients, which in most cases are not very effective and further disseminate antibiotic resistance (Khaledi et al., 2020).
Optimizing the existing diagnostic methods and making them faster and more cost-effective is crucial to decrease the use of empirical and broad-spectrum antibiotics. The current diagnostic procedures rely on culture-based techniques where bacteria are cultured and then identified using different biochemical tests. After that, antibiotic susceptibility testing (AST) is performed, which gives an idea about the resistance or sensitivity of bacteria to different antibiotics. The culturebased methods have a turnaround time of two to four days in most samples. However, in the case of bloodstream infections where the bacterial load is lower, the identification and AST can take up to five days (Briggs et al., 2021;Metzgar et al., 2016). There are many emerging micro-and nanotechnologies for pathogen identification and AST, including both phenotypic (microfluidic-based bacterial culture) and molecular methods (PCR, hybridization probes, nanoparticles, synthetic biology, and mass spectrometry) (Li et al., 2017). Whole genome sequencing (WGS) using the Oxford Nanopore Technology's (ONT) MinION has the potential to detect pathogens and their associated antibiotic resistance genes (ARGs) rapidly, but this technique is still in development, is costly, and not used in clinical settings (Avershina et al., 2022;Taxt et al., 2020). All these techniques are expensive, need dedicated infrastructure, or are timeconsuming. Therefore, there is an urgent need to develop cost-and resource-effective methods that can speed up pathogen diagnostics and reduce the mortality from these infections.
We need to understand the bacterial growth requirements to evaluate the possible solutions for the rapid diagnosis of pathogens. Mesophilic bacteria are typically grown at 30 to 37°C in lab procedures. In rich growth media (i.e., Luria-Bertani, Terrific Broth), dense cultures are raised in a reasonably short time of two to six hours using inoculums 1-2% of overnight cultures (ca. 12-14 h). When growing bacteria from clinical samples in blood cultures, however, a far lower inoculum is experienced, i.e., <100 CFU/mL and growth must be pursued for a minimum of 24 h up to several days to detect potential pathogens (Doualeh et al., 2022;Hardy et al., 1992;Waldeisen et al., 2011). The lengthy growth of fastidious bacteria in blood cultures to reach detectable titers is a significant bottleneck in the workflow of testing pathogens for AMR (Menchinelli et al., 2019;Somily et al., 2018;Verroken et al., 2015). Brain heart infusion (BHI) is a nutrient growth medium suitable for blood culture and to culture various fastidious organisms. BHI broth is often used in several application areas, including clinical microbiology testing, life science, biopharmaceuticals, food safety, water safety, and antibiotic sensitivity tests. The bacterial growth curve shows four phases: lag phase, log (exponential) phase, stationary, and death phase. Clearly, the time for a culture to reach detectable density, i.e., the length of lag phase growth, is a bottleneck in the diagnostic workflow. In this study, we wanted to investigate easy-to-implement factors (in a minimal laboratory set-up), including inoculum size, incubation temperature, and additional supplementation (e.g., vitamin B12 and trace metals), that can significantly reduce the lag time (t lag ). These factors were arranged in simple two-level factorial designs using different Gram-positive (Staphylococcus aureus and Bacillus subtilis) and Gram-negative (Escherichia coli and Pseudomonas aeruginosa) bacteria, including clinical isolates with known AMR profiles (encoding methicillin resistance or extendedspectrum β-lactamases). BHI was used as the growth medium as it is suitable for use as a blood culture medium. Based on the results from experiments with pure bacterial isolates, blood samples spiked with a clinical isolate of E. coli CCUG17620 were also tested to see the effect of elevated incubation temperature on bacterial growth in blood cultures.  Table 1.

Chemicals
All reagents were obtained from standard lab suppliers. Bacteriological agar and Brain Heart Infusion (BHI) broth for the media preparation were the products of HiMedia Laboratories (Mumbai, India; lot number: BCBH4004V), with BHI consisting of (g/L) calf brain infusion from 200 g (12.5), beef heart infusion from 250 g (5), peptone (10), sodium chloride (5), disodium chloride D (+) glucose (2) Pre-culture and growth procedures BHI agar and liquid culture media were used to maintain cultures and to examine growth curves. Bacterial samples stored at -80°C were thawed gently at room temperature, inoculated to BHI broth, and incubated overnight at 37°C. BHI agar plates were prepared by adding 1.5% bacteriological agar powder to BHI broth. An overnight inoculum was streaked out, and plates were incubated at 37°C for 24 h. Single colonies were selected and inoculated into fresh BHI broth. The inoculated BHI broth was incubated at 37°C overnight and served as inoculum for culture experiments (See Supplementary figure S1 for an overview of the complete process, Extended data). Experimental liquid cultures were cultivated in shake flasks (500 mL broth in 2L Erlenmeyer bottles). Cultures within the same experimental design were inoculated from the same bacterial preculture suspension to ensure identical starting conditions.
All cultures in BHI broth were incubated at 37°C or 42°C under shaking conditions for up to 6 h, whereas blood cultures were cultivated for 12 h. During incubation, the optical density (OD) was measured at 600 nm every 20-30 minutes, starting from the time of inoculation (0 min) until the bacterial growth reached the stationary phase (approximately 4-5 h). Culture aliquots were immediately cooled and kept on ice until measurement. The OD 600 was plotted versus time (min) to obtain the growth curves.
As a practical response value to estimate the effectiveness of treatments, an endpoint of the lag time (t lag ) was defined, the t lag being the time (min) from inoculation until the OD 600 value reached a net increase of 0.5. The net OD increase was obtained by subtracting the OD at t=0 from all OD values. The t lag was obtained by extrapolating at OD 0.5 from the zeroadjusted growth curve. Although this provisional end point is beyond the actual end of lag (Bertrand, 2019), an extended OD cut-off value was selected to reduce the estimation error when growth curves go from an undefined shallow rise to a steady exponential growth ( Figure 1).
The doubling time (t d ) for bacteria was calculated based on the steepest rise in the exponential phase of the growth curve.
The OD (log) and time interval in the exponential phase were selected at two points, which showed a specific growth rate (μ). The following formula was used to calculate the bacterial doubling time.
In the formula above, 0.693 is constant, and "μ" is the specific growth rate, which is calculated by using the following equation: where; Ln is the natural log OD 2 is the OD at the endpoint of the exponential phase OD 1 is the OD at the starting point of the exponential phase t 2 is the time corresponding to OD 2 t 1 is the time corresponding to OD 1 Design of experiments for screening of growth variables A set of growth screening experiments examined the ability of selected variables to cause a reduced lag time. A structured two-level factorial design (Design of Experiments, DOE) was used to observe the effect of incubation temperatures (37°C and 42°C), low (0.05%), and high (0.5%) inoculum, vitamin B12 (AE50 μM), and trace metal (present y/n) supplements. The design was tested on the wild-type strains (3) and the clinical isolates (4) but with a selection of two or three variables or factors. Table 2 shows the coded and decoded values of the factors tested (X 1 , X 2 , X 3 ) at a low (-1) and high (+1) level. Thus, either four (2 2 ) or eight (2 3 ) growth experiments were performed for each strain. For a detailed overview of conditions for the individual strains, Table 1 is provided as well as in Supplementary Tables 2-4 (Extended data).
The estimated t lag values from DOE were presented in interaction effect plots, illustrating the impact of a variable (e.g., temperature) with connected experiments on another variable (e.g., vitamin B12 supplement). The main effects of tested variables are shown numerically in descending order in bar charts. The bars are the absolute effect values computed as twice the regression coefficients from fitted DOE models. They show the change in t lag when a variable goes from a low to a high level when other factors are kept at their average.

Blood culturing
Clinical strain E. coli CCUG17620 was spiked with blood to mimic the sepsis infection. The blood was spiked with 40 CFU/mL from an overnight plate culture of the clinical strain. The lower CFU was used as an inoculum because, in the case of sepsis, the CFU/mL ranges typically from 10-100 (Doualeh et al., 2022). E. coli was grown overnight on a BHI agar plate and then suspended in saline to measure the OD. After measuring OD, the suspensions were plated on agar plates to calculate the CFU at different OD values. After optimizing the relation of OD with CFU counts, the desired concentration in the range of 10-100 CFU/mL was used for spiking with human blood. Fresh human blood obtained from healthy donors at Inland Norway University was used for this experiment. Human blood spiked with E. coli was mixed with BHI broth medium and incubated at the two study temperatures, i.e., 37 and 42°C. CFU/mL was calculated every 3 h for 12 h by plating out the samples on BHI agar plates. The CFU/mL was calculated using the formula: number of colonies Â dilution factor/volume of culture plated.

DNA extraction and PCR from blood cultures
Bacterial DNA from blood culture samples was extracted using QIAamp BiOstic Bacteremia DNA Kit from Qiagen (Germany). The concentration and purity of isolated DNA were determined using the Qubit system with a dsDNA HS assay kit (Thermo Fisher Scientific, USA) and the NanoDrop spectrophotometer. PCR was performed to confirm the presence of E. coli CCUG17620 DNA and human DNA in samples taken at 3 h, 6 h, 9 h, and 12 h of incubation at 37°C and 42°C. The primers used for identifying E. coli were uspA-F CCGATACGCTGCCAATCAGT and uspA-R ACGCAGACCGTAGGCCAGAT, and human DNA was β-actin (F-CGGCCTTGGAGTGTGTATTAAGTA and R-TGCAAAGAACACGGCTAAGTGT) (Hasan et al., 2016). The PCR reaction was set up in a total volume of 20 μL per sample containing 4 μL HOT FIREPol MultiPlex Mix Ready to Load master mix from Solis Biodyne (Estonia), 0.5 μL of 10 μM forward and reverse primers, 8 ng/μL of template DNA and nuclease-free water. The thermal cycling conditions for PCR were initial denaturation at 95°C for 12 min followed by 35 cycles of 95°C for 25 s, 61°C for 50 s and 72°C for 1 min, and a final extension of 72°C for 7 min. The PCR product was run on 1% agarose gel for 45 min at 100V and then visualized on the Gel doc system to determine the presence or absence of our desired bands.

Statistical analysis
The MODDE 13.0 Pro (Umetrics/Sartorius Stedim Data Analytics AB, Sweden) was used to generate the design matrix, regression coefficients, and statistical analysis. The significance testing (p < 0.05) of the DOE screening was performed with ANOVA. All the growth curve graphs are generated using GraphPad Prism 9.4.1.

Results
Higher incubation temperature, along with vitamin B12 supplementation, reduces the lag time for bacterial growth Escherichia coli E. coli HB101 K2 In the absence of vitamin B12 supplementation, the t lag for bacterial growth was 265 minutes and 255 minutes at 37°C and 42°C, respectively, when an inoculum size of 0.05% was used for culturing ( Figure 2A). When using a higher inoculum of 0.5% in the absence of vitamin B12 supplement, the t lag recorded was 220 minutes and 145 minutes at 37°C and 42°C, respectively ( Figure 2A). In the presence of vitamin B12 supplement and using an inoculum size of 0.05%, the bacterial t lag was 315 minutes (37°C) and 160 minutes (42°C). Similarly, when the inoculum size was increased to 0.5%, with vitamin B12 supplementation, the t lag was 200 minutes (37°C) and 190 minutes (42°C). The shortest t lag was recorded to be 145 minutes, which was observed using an inoculum size of 0.5% in the absence of vitamin B12 supplement and incubation at 42°C (Figure 2 and Supplementary Table 2 [Extended data]).
Using an inoculum size of 0.05% and a vitamin B12 supplement, we observed 95 minutes of reduction in the lag time at 42°C compared to 37°C ( Figure 2B). In contrast, when the inoculum size was increased to 0.5% along with vitamin B12 supplementation, the t lag was reduced by 10 minutes at 42°C. Based on the effect plot, it is reported that the sample with no vitamin B12 supplement and 0.5% inoculum size showed the shortest t lag . But when the inoculum size was decreased to 0.05%, we observed that the sample incubated at 42°C in the presence of vitamin B12 supplement showed the shortest t lag ( Figure 2B). The reduction in t lag was between 4% and 49% for the above mentioned eight tested conditions. Overall, the effect plot of E. coli CCUG17620 and E. coli NCTC13441 at inoculum size 0.5% indicated that when the incubation temperature increased (42°C instead of 37°C), the lag time decreased by 15-25% in the tested strains ( Figure 3B).

Staphylococcus aureus S. aureus NCTC8325
In the case of the clinical isolate S. aureus NCTC8325 with no vitamin B12 supplements and 0.05% inoculum size, the time to reach OD 0.5 was 285 minutes and 210 minutes at 37°C and 42°C, respectively ( Figure 4A and Supplementary Table 2). But in the presence of vitamin B12 supplementation and lower inoculum size of 0.05%, the t lag was 190 minutes (37°C) and 165 minutes (42°C). When the inoculum size was increased to 0.05%, the t lag decreased significantly at 42°C (135 minutes) in comparison to 37°C (215 minutes) in the presence of vitamin B12 supplementation ( Figure 4A and Supplementary Table 2). We can conclude that incubation at 42°C, along with vitamin B12 supplement, reduces the t lag in S. aureus NCTC8325.
The effect plot of S. aureus NCTC8325 at 0.5% inoculum shows a reduction of 121 minutes in t lag at 42°C incubation in the presence of vitamin B12 supplement. At the same time, a reduction of 45 minutes in t lag was observed at 0.05% inoculum and 42°C incubation in the presence of vitamin B12 supplement ( Figure 4B). The reduction in t lag was between 13% and 37% for the eight tested conditions.

S. aureus CCUG35600
Based on the above results, we tested the growth of another clinical strain of S. aureus. The effect plot of the clinical isolate S. aureus CCUG35600 at inoculum size 0.5% at 42°C co-cultured with vitamin B12 supplement showed a decline in lag time compared to without supplementation. Overall, the shortest t lag time was observed following the co-culture of bacteria with vitamin B12 supplement ( Figure 3A and B). So, the reduction in t lag was 20%.
Pseudomonas aeruginosa P. aeruginosa showed a significant reduction in the t lag when vitamin B12 supplements were added along with 42°C incubation. It reached OD 0.5 in 185 minutes when vitamin B12 supplement was added along with 42°C incubation. On the other hand, 235 minutes was required with no vitamin B12 supplement and 37°C incubation ( Figure 5A).
The effect plot showed a reduction of 30 minutes in t lag when bacteria were cultured at 42°C in the presence of vitamin B12 supplementation ( Figure 5B). The decline in t lag was between 9% and 20% for the four tested conditions.

Bacillus subtilis
B. subtilis also showed the same pattern as other bacteria, with a t lag of 155 minutes with vitamin B12 supplement and 42°C incubation. On the other hand, the t lag was 210 minutes and 215 minutes to reach OD 0.5 when incubated at 37°C with and without vitamin B12 supplement, respectively ( Figure 5A and Supplementary Table 3 [Extended data]). The effect plot showed a reduction of 45 minutes in t lag when bacteria were co-cultured at 42°C in the presence of vitamin B12 ( Figure 5B). The reduction in t lag was between 5% and 28% for the four tested conditions.
The main effects on t lag from the variables tested in three-(E. coli and S. aureus) and two-factor (P. aeruginosa and B. subtilis) experimental designs are shown in Figure 6. We tested the time to reach OD 0.5 for S. aureus, E. coli, P. aeruginosa, and B. subtilis and noticed that incubating the bacteria at 42°C reduces the lag time. However, the clinical isolates (E. coli CCUG17620, E. coli NCTC13441, and S. aureus CCUG35600), which were tested for trace metal solution complement, adversely affected bacterial growth by prolonging the lag time (details in Extended data).

Bacterial doubling time
The doubling time of all the tested isolates falls between 12 -55 minutes. The median of the calculated t d is 30.6 minutes (n=36), meaning the value closest to the midpoint of the population, i.e., the most 'normal' value. Although the difference in t d was insignificant, we still noticed that at 42°C, t d is lower (bacteria grow faster) than at 37°C. The difference in t d at both temperatures was more pronounced in E. coli isolates (E. coli HB101 K2, E. coli NCTC13441, and E. coli CCUG17620) compared to S. aureus isolates. We did not see any effect of temperature on t d in P. aeruginosa and B. subtilis isolates (Table 1

Higher incubation temperature increases bacterial growth in blood cultures
We also observed that higher incubation temperature could increase bacterial growth in blood cultures as in pure bacterial isolates. The CFU of the blood samples, which were spiked with E. coli CCUG17620 and incubated at the two study temperatures, 37°C and 42°C, showed that bacteria could grow much faster in the early stages of incubation at 42°C compared to 37°C. When we calculated the CFU after 3 h and 6 h of incubation, we could see that the CFU/mL of bacteria were three times higher at 42°C than at 37°C. This higher growth of bacteria in the initial hours of incubation is critical for  . Main effects on t lag from the variables tested in three-(E. coli and S. aureus) and two-factor (P. aeruginosa and B. subtilis) experimental designs. Bars represent the change in t lag (min) when a factor varies from its low to a high level (cfr. identifying the causative pathogen. This information is valuable for infection diagnostic purposes. Further incubation beyond 6 h, narrowed the gap between CFU/mL at 37°C and 42°C. At 12 h of incubation, we got the same CFU at both temperatures (Figure 7).
PCR results show higher purity of DNA at an increased growth temperature We performed PCR to confirm the presence of bacterial DNA versus human DNA at different incubation time points, as described in Methods. The PCR product run on 1% agarose gel was visualized for the presence of DNA and compared with a 100 bp DNA ladder, which was also run on the gel. We could see the DNA bands for the E. coli CCUG 17620 sample even after 3 h incubation. Although the CFU/mL after 3 h of incubation was low, i.e., 40 at 37°C and 120 at 42°C, it was still enough bacterial DNA to be amplified through PCR (Figure 8). For the samples which were incubated for a longer time and had greater CFU, the band signals for E. coli DNA appeared stronger in comparison to the human DNA (Figures 8 and 9). We can see from the PCR results that we obtained amplification at both the incubation temperatures (37°C and 42°C), but the DNA band strength and purity were higher at 42°C. The DNA concentration was also higher at increased incubation temperature (42°C) compared to 37°C. The DNA concentration of the samples after 3 h of incubation was 52.4 and 34.3 μg/mL at 42°C and 37°C, respectively. Similarly, we also observed an increased DNA concentration at 6 h (110 μg/mL at 42°C and 82 μg/mL at 37°C) and 9 h (134.4 μg/mL at 42°C and 119.2 μg/mL at 37°C) of incubation in samples which were incubated at a higher temperature. This higher concentration of DNA is beneficial for nanopore sequencing, which can be used to identify the bacteria in real time.

Discussion
Early and appropriate detection of pathogens is crucial for treating bacterial infections. However, since the bacterial load is usually very low for infection detection, it is difficult to identify the pathogen and recommend appropriate antibiotic therapy. In the clinical routine, bacteria are grown overnight, requiring a longer time to grow in fresh culture media. Therefore, any method which increases bacterial growth in the initial hours of culturing can be extremely useful in treating patients with different bacterial infections. In the case of sepsis, early and appropriate antibiotic therapy in the first hour of infection can significantly reduce the mortality rate (Avershina et al., 2022;Bloos et al., 2017;Ferrer et al., 2014).
The most common method currently used for identifying bacterial pathogens in clinical samples is based on culture. Although these culturing methods, in most cases, provide information on the causative agent, they are slow and timeconsuming. The routine culture-based identification takes around 48-72 hours, and an additional 24-48 hours for antibiotic susceptibility testing. Still, this method is most widely used in clinical diagnosis because it is cost-effective and does not require advanced lab infrastructure. Many other methods, such as whole genome sequencing (WGS), fluorescent in-situ hybridization (FISH), PCR, and matrix-assisted laser desorption/ionization time-of-flight mass spectrometer (MALDI-TOF MS), have the potential for rapid identification of pathogens. However, most of these techniques still require bacterial culturing as the initial step and currently need to be well-developed to be used in clinical settings.   In this study, we have used different inoculation sizes (0.5% and 0.05%), incubation temperatures (37°C and 42°C), and bacterial growth supplements (vitamin B12 and trace metal solution) to study their effect on the growth of bacteria. We used BHI culture media for bacterial growth because it can support the growth of a wide variety of bacteria. BHI is also suitable for using as a blood culture medium and is similar to the BACTEC blood culture medium, which is extensively used in the clinical microbiology routine. Also, it has been previously used for short-term incubation of positive blood cultures to accelerate the identification of bacteria by MALDI-TOF MS (Torres et al., 2017). We used an incubation temperature of 37°C and 42°C because it is the most commonly used temperature in microbiology for incubating bacteria. Also, it can easily be implemented in a standard laboratory setting (Gutierrez et al., 2018). We have shown that incubating the bacteria at a higher temperature of 42°C instead of 37°C and adding vitamin B12 supplementation in the culture media can significantly reduce the t lag of bacterial growth. The effect of higher incubation temperature and vitamin B12 supplementation was insignificant on the bacterial doubling time. We did observe a shorter doubling time for E. coli isolates at 42°C (Table 1). We hypothesize that it is relatively more important for the bacteria to reduce the lag time than the doubling time. When the exponential growth phase is reached, the bacteria multiply at different rates as the growth machinery has been activated.
We observed reduced t lag in the pure bacterial isolates and the E. coli blood culture using 42°C incubation. The current methods require the blood cultures to be incubated for 24 h or more before identifying pathogens using biochemical tests and then AST. Using this higher incubation temperature (42°C), we can see that after 3 h of incubation, the bacterial CFU/mL was 120, which is three times higher than the standard incubation temperature (37°C). Although the PCR results show bacterial DNA's presence at both temperatures, we can see the better quality of DNA at 42°C by looking at the intensity of bands (Figure 8). This high-quality DNA can be beneficial for performing downstream analyses like PCR or real-time metagenomic sequencing for identifying pathogens and ARGs.
The duration of the lag phase is influenced by several factors such as pH, temperature, nutrients (carbon, nitrogen, phosphorus), oxygen availability, physiological history of the cells, and size of the inoculum (Bertrand, 2019; Mellefont et al., 2003;Rolfe et al., 2012). Many studies have investigated the effect of environmental factors such as pH, temperature, and nutrients on the duration of the bacterial lag phase (Francois et al., 2006(Francois et al., , 2007. However, so far, no studies have been done to investigate the effect of vitamin B12 supplements on the bacterial lag phase at different conditions, such as temperature (37°C and 42°C) in connection with inoculum size (0.05% and 0.5%). Other studies have also reported that bacterial lag time depends on the nutrient concentration, such as potassium bicarbonate (KHCO 3 ), amino acids, ferrous sulfate, sodium pyruvate, and mucin (Berthelet and MacLeod, 1989). Jiang et al. have shown that adding 0.3% mucin to brain heart infusion horse serum (BHI-HS) medium reduced the lag time of Helicobacter pylori and enhanced its growth (Jiang & Doyle, 2000). These results are similar to our study, where we have also shown that vitamin B12 supplement in the BHI medium reduces the lag time of bacteria. Vitamin B12 can affect the bacterial lag phase, possibly because it can contribute to different metabolic activities in the bacterial cell, such as the synthesis of nucleic acid and amino acids. Some bacteria can synthesize vitamin B12 on their own, while others require it to be preset in the growth medium (LeBlanc et al., 2011). If vitamin B12 is unavailable in the medium, bacteria need additional time to adapt to the environment and synthesize the necessary enzymes required for vitamin B12 production, which can increase their t lag (Sultana et al., 2023). Malani et al. reported that higher concentrations of sucrose, biotin and pantothenate and lower concentrations of cyanocobalamin (i.e., vitamin B12), CaSO 4 , and glutathione in a growth medium result in larger bacterial inclusion bodies and product expression (Malani et al., 2020). The microbial cell is composed mainly of carbon, hydrogen, nitrogen, and oxygen elements. Other elements such as phosphorus, sulfur, calcium, magnesium, potassium, iron, and sodium are equally crucial for the vital activities of bacterial metabolism (dos Santos et al., 2022). These micronutrients are essential for the enzymatic activities of the bacterial cell and act as prosthetic groups or cofactors for necessary metabolic enzymes (Hood & Skaar, 2012).
It has also been shown that an extended lag phase is advantageous for bacterial survival and helps in the regrowth of bacteria upon the removal of antibiotics (Bertrand, 2019). Li et al. evaluated the effect of the bacterial lag phase on the resistance and sensitivity to 12 different antibiotics. They observed that tetracycline and quinolone were the most common antibiotics to which the bacteria showed resistance due to their extended lag phase (Li et al., 2017). Therefore, reducing bacterial lag time is even more critical as it can contribute to drug resistance against the clinically relevant antibiotics used to treat bacterial infections. For all four tested bacteria (S. aureus, E. coli, P. aeruginosa, and B. subtilis at OD 0.5), we reported that incubating the bacteria at 42°C reduces the lag time (t lag ). Studies have reported that bacteria have evolved to regulate their virulence factors due to changes in temperature when they enter from the external environment to the host. Bacteria recognize the signal provided by the temperature and enhance the production of their virulence factors accordingly within the host (Konkel & Tilly, 2000;Lam et al., 2014). We hypothesize that faster bacterial growth could lead to the rapid spread of the pathogen and, therefore, a more severe infection that can be fatal.
With an increasing volume of cells in culture media, it takes less time for the bacteria to adapt to the new environment and therefore has a shorter lag time (Robinson et al., 2001;Aamir et al., 2015). In our study, we observed a short lag time for some of the bacteria by using a larger inoculum size of 0.5%, but in E. coli HB101 K2 and S. aureus NCTC8325, we did not observe the same effect. We did not see any effect of the inoculum concentration on the lag phase in some bacteria because of several factors, such as inherent bacterial characteristics, culture growth phase, and environmental adaptation. Also, some bacteria may have limited ability to utilize the available nutrients even when the inoculum size is larger. Therefore, the inoculum concentration in some of these bacteria might not significantly affect their lag phase (Bidlas et al., 2008).
In this study, we have shown that using vitamin B12 supplements in culture media and incubation at a higher temperature of 42°C can significantly reduce the t lag . This reduction in t lag is significant for rapidly detecting pathogens and early and appropriate antibiotic therapy. We have also shown that incubating E. coli blood culture at a higher temperature of 42°C reduces the t lag and enhances the growth of bacteria. It will be beneficial in the future to enrich the culture media with different bacterial growth supplements, which can reduce bacterial lag time and thus allow rapid identification of pathogens. However, to increase the method's reliability and for any potential future use in clinical microbiology, we need to increase the number of bacterial strains analyzed and focus on additional AMR profiles and clinically relevant pathogens. Moreover, the effect of other elements, such as phosphorus, magnesium, calcium, sodium, and iron, as a bacterial growth supplement will be interesting to explore in future research. These results demonstrate the potential that, unlike other molecular-based detection methods, these measures can be cost-effective and do not need any specialized instruments or expertise.

Open Peer Review I confirm that I have read this submission and believe that I have an appropriate level of
The manuscript entitled as "Increased growth temperature and vitamin B12 supplementation reduces the lag time for rapid pathogen identification in BHI agar and blood cultures" published on F1000 Research by Ali et al. is an interesting work of applied microbiology. The ms is a proof-ofconcept study for future diagnostic improvements. Before its indexing, the following are some comments and observations which are recommended to authors for appropriate response during further revision of their manuscript: Please explain the rationale for incubation temperature setting (37 to 42°C) and also provide potential reasons for lag phase changes with/without vitamin B12 in different bacterial stains.

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The bacterial inoculum concentration showed different trends for the lag phase on different temperatures and vitamin B12 supplementation, please discuss different reasons to explain these shifts. Are there any other factors (except the ones studied here) to affect the bacterial lag phase? ○ Due to the unique ways in which various bacteria behave and function, the responsiveness and functional capabilities fluctuate as the environment changes. When various environmental factors are activated, not all bacteria or pathogens behave the same way. Some act in opposition to the change, while others react positively. So, how likely is it to expand the observed modification for tens of bacterial pathogens? More precisely, the environmental changes may affect the bacterial physiological functions; in this context, do the tested incubation temperature and vitamin supplementation affect the microbes' pathogenicity or efficiency as well?

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The nutrients are necessary for the bacteria to grow; some are present in significant amounts, while others are scarce. What is the rationale, for authors, to test B12 out of several microbial nutrients?
As per the study-observations, increased incubation temperature (42°C) reduced the T lag , may it do the same in real life body-temperature situations? Reduced lag time may enhance the pathogens' survival in compromised immunity scenarios with raised body temperature. Please discuss this practical aspect as well.

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As per the ms title and abstract, the 1 st impression of the study is more like diagnostic improvements while the introduction start with AMR does not fit in appropriately. 2 nd paragraph or even the 2 nd sentence of 2 nd paragraph might be something to start the introduction and whole AMR scenario may follow in later phase.

Are sufficient details of methods and analysis provided to allow replication by others? Yes
If applicable, is the statistical analysis and its interpretation appropriate? I cannot comment. A qualified statistician is required.
Are all the source data underlying the results available to ensure full reproducibility? Yes

Are the conclusions drawn adequately supported by the results? Yes
Competing Interests: No competing interests were disclosed.

Reviewer Expertise: Microbial interactions, Antimicrobial resistance
I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above.

Rafi Ahmad
We thank the esteemed reviewer for taking the time to provide us with feedback. The reviewer's comments have helped improve the manuscript. We hope that we have addressed the reviewer's comments sufficiently. Please explain the rationale for incubation temperature setting (37 to 42°C) and also provide potential reasons for lag phase changes with/without vitamin B12 in different bacterial stains.

Response:
The incubation temperature (37 & 42°C) was used in this study because these temperatures can easily be maintained in standard laboratory settings and the most commonly used temperature range (37 to 42°C) for the majority of bacterial strains in microbiology. We used the higher (42°C) and lower (37°C) temperatures in this range to compare their effect on the bacterial growth (Gutierrez et al., 2018).
The relevant text is added in the discussion section of the manuscript (Paragraph 3). The lag time for bacterial growth is dependent on many factors, such as temperature, bacterial strain, and growth medium (availability of nutrients). According to our study, vitamin B12 can affect the lag phase of bacterial growth, possibly because it can contribute to different metabolic activities in bacteria, such as the synthesis of nucleic acid and amino acids. Some bacteria can synthesize vitamin B12, while others require it to be present in the growth medium (LeBlanc et al., 2011). The text has been added in paragraph 4 of the discussion section). If the bacteria do not have vitamin B12 available in the growth medium, they require time to synthesize different enzymes, which is required for the production of vitamin B12 (Sultana et al., 2023). Therefore, the lag time of the bacteria, in that case, is longer.
The relevant text is added in the discussion section of the manuscript (Paragraph 4). The bacterial inoculum concentration showed different trends for the lag phase on different temperatures and vitamin B12 supplementation, please discuss different reasons to explain these shifts. Are there any other factors (except the ones studied here) to affect the bacterial lag phase? ○ Response: The reason we did not see any effect of the inoculum concentration on the lag phase in some of the bacteria may be due to several factors, such as inherent bacterial characteristics, culture growth phase, and adaptation to the environment. Some bacteria may have limited ability to utilize the available nutrients even when the inoculum size is larger. Therefore, the inoculum concentration in some of these bacteria may not have any significant effect on their lag phase (Bidlas et al., 2008).
The relevant text is added in the discussion section of the manuscript (Paragraph 6).
Many other factors besides the ones tested here can affect the bacterial lag phase. Some of them are the availability of different nutrients such as carbon, nitrogen, and phosphorus. The pH of the growth medium can also affect the bacterial growth rate and metabolism. Oxygen availability is also important for the optimal growth of bacteria as some bacteria require oxygen for their growth, and others are anaerobic (Bertrand, 2019;Mellefont et al., 2003;Rolfe et al., 2012).
The relevant text is added in the discussion section of the manuscript (Paragraph 4). Due to the unique ways in which various bacteria behave and function, the responsiveness and functional capabilities fluctuate as the environment changes. When various environmental factors are activated, not all bacteria or pathogens behave the same way. Some act in opposition to the change, while others react positively. So, how likely is it to expand the observed modification for tens of bacterial pathogens? More precisely, the environmental changes may affect the bacterial physiological functions; in this context, do the tested incubation temperature and vitamin supplementation affect the microbes' pathogenicity or efficiency as well?
○ Response: The effect of higher incubation temperature and vitamin B12 supplementation on tens of bacteria is difficult to predict unless we have specific experimental data for each species. But in this study, we have shown that tested bacterial strains show reduced lag time in the presence of vitamin B12 and higher incubation temperature. This method can potentially be tested for other clinically important bacteria as well. The effect of higher incubation temperature and vitamin B12 supplementation on bacterial pathogenicity and efficiency is difficult to generalize because microbial pathogenicity and efficiency are complex phenomena involving many other factors, such as bacterial virulence factors, hostpathogen interactions, and immune responses. In some cases, higher incubation temperature and/or vitamin B12 can enhance bacterial pathogenicity by increasing its growth rate or virulence factor expression, while in other cases, it may have the opposite or no effect at all (Konkel & Tilly, 2000).
We can hypothesize that faster growth will lead to the rapid spread of the pathogen and, therefore, a more serious infection. As the reviewer has stated, reduced lag time may enhance the pathogens' survival in compromised immunity scenarios with raised body temperature, which will be more pathogenic.
The text has been incorporated into the revised manuscript (Paragraph 5 -Discussion).
The nutrients are necessary for the bacteria to grow; some are present in significant amounts, while others are scarce. What is the rationale, for authors, to test B12 out of several microbial nutrients? ○ Response: Bacteria require several nutrients to grow, such as carbon, nitrogen, phosphorus, sulfur, etc. Out of all these nutrients, we selected vitamin B12 for testing because it is cheap and easily available (Fang et al., 2017). In our study, we have also used trace elements to see their effect on bacterial growth (Supplementary data). Also, in future studies, the effect of different nutrients on bacterial growth will be interesting to explore, as we have discussed in the manuscript.
The text has been incorporated into the revised manuscript (Concluding paragraph -Discussion).
As per the study-observations, increased incubation temperature (42°C) reduced the Tlag, may it do the same in real life body-temperature situations? Reduced lag time may enhance the pathogens' survival in compromised immunity scenarios with raised body temperature. Please discuss this practical aspect as well.
○ Response: An increase in the body temperature is usually called fever which is the body's response to fight any pathogen or infection. In this study, we have shown that higher incubation temperature can reduce the t lag, which will enhance bacterial growth. However, it is difficult to replicate the identical effect as observed in the human body in the lab. According to some studies, bacteria have evolved to regulate their virulence factors due to changes in temperature when they enter from the external environment to the host. Bacteria recognize the signal provided by the temperature and enhance the production of their virulence factors accordingly within the host (Lam et al., 2014). The growth of bacteria at higher temperatures in the human body can be fatal. We can hypothesize that faster growth will lead to the rapid spread of the pathogen and, therefore, a more serious infection.
The relevant text is added in the discussion (paragraph 5) to highlight this aspect. As per the ms title and abstract, the 1st impression of the study is more like diagnostic improvements while the introduction start with AMR does not fit in appropriately. 2nd paragraph or even the 2nd sentence of 2nd paragraph might be something to start the introduction and whole AMR scenario may follow in later phase ○ Response: The text in the manuscript has been updated as per the reviewer's suggestion Almost half of the 2nd paragraph is on 'Sepsis', I think it narrates the examples of different fatal diseases, so please say it so.
difference of the bacterial count has been noticed in the initial period of incubation however, most of the experiments showing similar growth pattern over a longer incubatory period. Hence, more number of isolates need to be tested.

Response:
In this study, we tested three E. coli, two S. aureus, and one isolate of P. aeruginosa and B. subtilis. We agree that bacterial counts fluctuate more during the initial incubation period. However, this difference in the bacterial count during the initial incubation time can be advantageous for quickly detecting pathogens. In our unpublished work, we have observed that in blood cultures, pathogens with higher bacterial counts during the early stages of incubation allow for faster detection using nanopore sequencing. Sequencing at a timely stage enables the identification of pathogens and their antimicrobial resistance, allowing for swift therapeutic intervention. As this was a proof-of-concept study, we could only test available bacterial strains which we already possess -most of them known pathogens. Future studies should focus on testing more pathogens and using different nutrients to see their effect on bacterial growth.
Pseudomonas aeruginosa is known pathogen and its growth at 42°C is taken as one of the identification characters for P. aeruginosa. It would be helpful if other Gramnegative bacilli, such as the ESKAPE group of pathogens, were included in the experiment.