anaerobic conditions: Applicability to real postmortem cases and to postmortem blood derived microbial cultures Vassiliki A. Boumba a, *, Nikolaos Kourkoumelis b , Panagiota Gousia c , Vangelis Economou c , Chrissanthy Papadopoulou c , Theodore Vougiouklakis a a Department of Forensic Medicine & Toxicology, Medical School, University of Ioannina, 45110 Ioaninna, Greece b Department of Medical Physics, Medical School, University of Ioannina, 45110 Ioaninna, Greece c Department of Microbiology, Medical School, University of Ioannina, 45110 Ioaninna, Greece 1. Introduction Determination of blood alcohol concentration (BAC) in post- mortem cases is part of the death investigation process since ethanol might be a causal or contributory factor to the manner of death. The accurate scientic interpretation of the ethanol analysis results presents a critical task in cases where microbial ethanol neo-formation is suspected, or, alternatively, the origin of postmortem ethanol (ante mortem ingestion or microbial produc- tion) is questioned. In order to achieve a feasible accuracy in interpreting the results of postmortem ethanol analysis different approaches have been suggested. The analysis of multiple specimens and from multiple sampling sites could reveal atypical distribution of ethanol throughout the different compartments of the dead body and thus could be indicative of postmortem ethanol neo-formation [1 5]. The determination of various low molecular weight volatiles in postmortem specimens is suggested as a criterion to specify the origin of the measured postmortem ethanol and, if certain volatiles are detected then postmortem ethanol production should be suspected [3,4,610]. Finally, the determination of various non- oxidative metabolites of ethanol is used to distinguish the ante mortem ingestion from the postmortem synthesis of ethanol [4,1115]. Common feature in the aforementioned approaches is that the documentation, or not, of the microbially produced ethanol arises in qualitative terms. Recently we reported the formulation of mathematical models for calculating the microbial neo-formed ethanol in strict anaerobic cultures of the bacterial strains Escherichia coli, Clostridium perfringens, and Clostridium sporogenes [16]. This approach was the rst approximation to the quantication of the microbial ethanol production in cases where other low molecular weight alcohols were produced simultaneously with ethanol. Forensic Science International 232 (2013) 191198 A R T I C L E I N F O Article history: Received 26 April 2013 Received in revised form 19 July 2013 Accepted 26 July 2013 Available online 7 August 2013 Keywords: Postmortem ethanol Postmortem blood Multiple linear regression Autopsy E. coli Microbial ethanol A B S T R A C T The mathematical modeling of the microbial ethanol production under strict anaerobic experimental conditions for some bacterial species has been proposed by our research group as the rst approximation to the quantication of the microbial ethanol production in cases where other alcohols were produced simultaneously with ethanol. The present study aims to: (i) study the microbial ethanol production by Escherichia coli under controlled aerobic/anaerobic conditions; (ii) model the correlation between the microbial produced ethanol and the other higher alcohols; and (iii) test their applicability in: (a) real postmortemcases that had positive BACs (>0.10 g/L) and co-detection of higher alcohols and 1-butanol during the original ethanol analysis and (b) postmortem blood derived microbial cultures under aerobic/anaerobic controlled experimental conditions. The statistical evaluation of the results revealed that the formulated models were presumably correlated to 1-propanol and 1-butanol which were recognized as the most signicant descriptors of the modeling process. The signicance of 1-propanol and 1-butanol as descriptors was so powerful that they could be used as the only independent variables to create a simple and satisfactory model. The current models showed a potential for application to estimate microbial ethanol within an acceptable standard error in various tested cases where ethanol and other alcohols have been produced from different microbes. 2013 Elsevier Ireland Ltd. All rights reserved. * Corresponding author. Tel.: +30 26510 07724; fax: +30 26510 07857. E-mail addresses: vboumba@cc.uoi.gr, metsbou@yahoo.gr (V.A. Boumba). Contents lists available at ScienceDirect Forensic Science International j our nal h o mepage: w ww. el sevi er . co m/ l oc at e/ f o r sc i i nt 0379-0738/$ see front matter 2013 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.forsciint.2013.07.021 The present contribution describes the development of simple, albeit accurate, mathematical models which could quantify the microbial produced ethanol in correlation with other alcohols in E. coli cultures under initially aerobic and then anaerobic, controlled laboratory conditions. The applicability of the constructed models was tested in real postmortem cases that had positive BACs (BAC > 0.10 g/L) and co-detection of higher alcohols and 1-butanol during the original ethanol analysis. The applicability of the models was also tested to microbial cultures of blood culture media inoculated with postmortem blood from autopsy cases under aerobic/anaerobic controlled experimental conditions. 2. Materials and methods 2.1. Microbial cultures 2.1.1. Bacterial strain The bacterial strain used in this study was E. coli ATCC 35218. The E. coli strain was stored at 80 8C (Mikrobank beads Prolab Diagnostics, Canada) and prior to use it was revived by transferring 35 beads into 10 mL of Brain Heart Infusion (BHI) (CM0225, Oxoid) and was incubated at 37 8C for 1824 h. The bacterial inoculum was adjusted to a working concentration of 10 6 cells/mL using spectrophotometry. In brief, a standard solution of 10 8 cells/mL was prepared from an initial culture after overnight incubation at 37 8C aerobically and the concentration was determined using the spectrophotometer (Model 6305, Jenway, England) at 600 nm. In order to obtain a nal concentration of 10 6 cells/mL, 100 mL of the standard inoculum were added in 9.9 mL of BHI in sterile tubes. The tubes were incubated aerobically for the rst 5 days. After this period 1 mL of sterile parane oil was added in each tube as an overlay and the tubes were placed into GENbag anaer incubation systems (45534, bioMe rieux, France) so as to provide anaerobic conditions from day 6 to day 30. The incubation, both anaerobic and aerobic, was performed at 25 8C for 30 days. 2.1.2. Bacterial enumeration The bacterial concentration was determined by the pour plate count technique [16]. Decimal dilutions were performed in tubes with 9 mL of Maximal Recovery Diluent (MRD, 02-510, Scharlau Chemie, Spain). From each tube an inoculum of 1 mL was transferred into 2 petri dishes and 15 mL of Plate Count Agar (1.05463, Merck KGaA, Germany) melted at approximately 45 8C were added. After solidication all plates were incubated aerobically at 37 8C for 48 h. Colonies were counted under a colony counter (Stuart SC6, Barloworld Scientic Ltd., UK). 2.2. Postmortem blood derived microbial cultures 2.2.1. Identication of microbes Seven postmortem blood samples were examined for the presence of E. coli, Staphylococcus aureus, Enterococcus spp., and Clostridium spp. Ten mL of aseptically collected blood was added into 1 mL of Buffered Peptone Water BPW (CM1049, Oxoid, UK). E. coli was isolated by plating a loopful of BPW on Violet Red Bile agar (4021852, Biolife, Italy) and Chromocult Coliform Agar (1.10426.0500, Merck KGaA, Germany). The inoculated media were incubated aerobically at 37 8C for 24 h. Characteristic colonies were selected, pure cultured and identied to the genus level using the API20E system (bioMe rieux, France). For the isolation of Gram positive strains (S. aureus and Enterococcus spp.), Manitol Salt agar (CM0085, Oxoid, UK) and D-Coccosel Bile Esculin Agar (51025, bioMe rieux, France) were used, respectively. The plates were incubated at 37 8C aerobically. Characteristic colonies were selected, pure cultured and identied to the genus level by biochemical tests. For Staphylococci the catalase test, the coagulase test (Staphytect Plus, Oxoid, UK) and the API STAPH system (bioMe rieux, France) were used. Biochemical identication of the Enterococci was performed using the catalase test and the API 20 STREP system (bioMe rieux, France). For the isolation of Clostridium species a loopful was streaked onto blood agar plates, the inoculated plates were incubated anaerobically using the GENbag anaer incubation systems (bioMe rieux, France) for 48 h at 37 8C and were examined for b-haemolysis. Identication was performed microscopically after Gram stain and with the use of the API 20A system (bioMe rieux, France). 2.2.2. Normal human blood culture media inoculated with postmortem blood (postmortem blood derived microbial cultures) The postmortem blood derived cultures were performed by a procedure based to that described by Appenzeller and colleagues [15] modied to t the conditions of microbial cultures used herein. Specically, ethanol free whole human blood from healthy individuals was inoculated with postmortem blood (100 mL/mL of normal human blood). Postmortem blood samples were collected into sterilized collection tubes containing anticoagulant, during autopsy from cases with putrefaction and also were subjected to the procedure of microbial identication (described in Section 2.2.1). The blood samples were kept at 4 8C until further use. One mL portions of the inoculated blood were transferred into 40 sterilized blood tubes (Vacuette EDTAK3, 3 mL). Twenty tubes were spiked with the appropriate volume of a concentrated glucose solution (sterile dextrose 5% (w/v), pyrogen free solution for intravenous infusion, DEMO Ltd., Greece) so as to obtain 200 mg/dL nal glucose concentration. The other tubes were incubated without additional glucose. All the tubes were capped and incubated at 25 8C. On the seventh day a sterile paran oil overlay was added into each tube and the tubes were placed into the GENbag anaer incubation system. At days 0, 1, 2, 3, 5, 7, 9, 11, 15 and 20 two tubes were removed from the incubation system and alcohols concentrations were determined by HS- GCFID. 2.3. Determination of volatiles The alcohols concentration in the culture medium and the blood samples were determined by the previously described procedure [16]. 2.4. Statistical analysis Regression analysis was employed to model the correlation between the microbial produced ethanol and the other higher alcohols. The experimental data were consistent with a Gaussian distribution (normality test) and were analyzed with multiple linear regression (MLR) methods to model the relationship between ethanol concentrations as the dependent variable being in linear correlation with the concentrations of the other alcohols (independent variables). Each descriptors relevance to the current model was assessed by partial least squares (PLS) regression utilizing the PLS1 algorithm. During the analysis, one outlier value was removed from the dataset. 3. Results and discussion 3.1. Experimental determination of alcohols produced in pure E. coli cultures E. coli is a Gram negative, aerobic and facultative anaerobic, rod- shaped bacterium that is commonly found in the lower intestine of warm-blooded organisms. It is considered one of the main colonizers in corpses and of the main ethanol producers [17]. The growth curve of E. coli during the 30 days incubation at 25 8C is presented in Fig. 1. During the rst 5 days of incubation the conditions were aerobic. On the fth day up to the end of the incubation the conditions of microbial growth changed to anaerobic. By the fth day of incubation the bacterial population reached the stationary phase of growth. The change from aerobic to anaerobic conditions was selected in order to approximate the usual conditions where a postmortem blood evolves from death to autopsy (initially air is available, which gradually decreases and nally depletes) [1,19] and the duration of changing was selected in order to obtain the optimal microbial growth. At 4 8C no growth was observed as expected (not shown) [15,16,18]. The amounts of ethanol, and other alcohols produced under the applied experimental conditions were variable (Table 1). Ethanol 1,0E+06 1,0E+07 1,0E+08 1,0E+09 1,0E+10 30 25 20 15 10 5 0 Days B a c t e r i a l
c o u n t
( L o g
1 0
C F U
/
m L Fig. 1. Growth curve of E. coli showing the number of bacteria during the incubation period of 30 days at 25 8C. The time of change between aerobic and anaerobic incubation is shown by the dotted line at 5 days. V.A. Boumba et al. / Forensic Science International 232 (2013) 191198 192 was produced in higher concentrations compared to the other alcohols. Considering the ethanol concentration at days 0 and 5 (0 g/L and 0.55 g/L, respectively), it is concluded that the mean rate of ethanol increase is 0.11 g/L/day (or 11 mg/dL/day) during the rst ve-days period of incubation. After switching to anaerobic conditions, from day six to day nine, the rate of ethanol production decreased to a mean rate of 0.017 g/L/day (or 1.7 mg/dL/day). Afterwards, from day 10 to the end of the experiment, ethanol kept a plateau concentration of about 0.62 g/L. The most abundantly produced alcohol (except ethanol) was 1- propanol which was continuously increasing during the rst 17 days. Interestingly, the mean rate of 1-propanols increase was 0.42 mg/dL/day during the rst ve days, while for the next twelve days the increase rate declined, and afterwards, from day 18 to the end, 1-propanol showed no considerable change. The third in abundance alcohol was 1-butanol which increased the rst ve days with a rate approximately 10 times slower (0.042 mg/dL) than the respective increase rate of 1-propanol, while afterwards it remained practically stable. Finally, isobutanol and methyl- butanol increased slowly for twelve days and then remained stable. Our results show that 1-propanol and 1-butanol are the main alcohols that are produced simultaneously with ethanol by microbes under the applied experimental conditions. The amounts of ethanol and other alcohols produced by E. coli in BHI cultures under combined aerobical/anaerobical conditions were higher compared to those produced by E. coli in BHI cultures under strict anaerobic conditions [16]. The observed discrepancies are attributed to the presence/absence of air. Therefore it is observed that higher amounts of alcohols are produced by certain microbes (here E. coli) under aerobic conditions. It is of importance to underline that 1-butanol was produced during the rst ve days of incubation, in the presence of air, while during this period both ethanol and 1-propanol had the higher production rates; ethanol and 1-propanol continued to accumu- late, with a decreased production rate, for further 4 and 12 days, respectively, under anaerobic conditions. To explain these results the following aspects should be considered: (i) ethanol can be produced from all the available postmortem substrates but with higher rates and yields from carbohydrates; (ii) 1-propanol and 1- butanol (and the other higher alcohols) could be produced from all the available postmortem substrates through branched biochemi- cal pathways that depend on glucose metabolism; and (iii) 1- propanol, isobutanol and methyl-butanols are produced using amino acids as substrates [20]. Therefore, it should be concluded that carbohydrates (as the preferable substrate) were consumed by the microbes during the aerobic phase of growth, generating mainly ethanol, 1-propanol and 1-butanol. As carbohydrates were being depleted from the medium, other available substrates were consumed continuing to generate higher alcohols (1-propanol, isobutanol and methyl-alcohols) and ethanol in line with previous suggestion [20]. 3.2. Formulation of models for ethanol production by E. coli 3.2.1. Statistical evaluation of volatiles All volatiles during the rst 5 days follow a linear approxima- tion (r 2 > 0.9, not shown here) with the exception of ethanol. For the next days (630), MLR is not able to successfully t a similar linear model (smaller r 2 value) regarding all variables with the notable exception of 1-propanol. Probably, 1-propanol plays a signicant role in the successful model prediction while all data is better to be treated simultaneously since for the rst 5 days, data is ambiguous regarding the variance. By performing a principal component analysis (PCA) for the whole data to determine how original variables are projected to a smaller set of underlying variables, it becomes evident that ethanol explains 99% of the total variance while the remaining 1% is explained by 1-propanol. Table 1 Alcohols concentration produced during the 30 days incubation period of E. coli in BHI culture medium at 25 8C under aerobic (days 15) and anaerobic (days 630) conditions. Day Ethanol (g/L) 1-Propanol (mg/dL) Isobutanol (mg/dL) 1-Butanol (mg/dL) Methyl-butanol (mg/dL) 0 0 0 0 0 0 1 0.49 1.12 0.01 0.1 0.03 2 0.48 1.33 0.01 0.15 0.03 3 0.50 1.50 0.01 0.18 0.04 4 0.52 1.81 0.01 0.19 0.04 5 0.55 2.08 0.02 0.21 0.05 6 0.53 2.05 0.02 0.21 0.05 7 0.56 2.18 0.02 0.22 0.06 8 0.59 2.30 0.02 0.23 0.04 9 0.61 2.33 0.02 0.23 0.05 10 0.62 2.85 0.02 0.24 0.06 11 0.58 2.67 0.02 0.23 0.06 12 0.60 2.87 0.03 0.24 0.08 13 0.63 3.18 0.03 0.24 0.08 14 0.60 2.63 0.03 0.23 0.08 15 0.58 2.78 0.02 0.23 0.07 16 0.61 3.04 0.03 0.23 0.11 17 0.61 3.22 0.03 0.23 0.07 18 0.62 3.01 0.03 0.23 0.07 19 0.62 3.15 0.03 0.23 0.07 20 0.63 3.47 0.03 0.24 0.08 21 0.62 3.23 0.03 0.24 0.08 22 0.62 3.30 0.03 0.22 0.06 23 0.61 3.18 0.03 0.24 0.07 24 0.61 3.07 0.03 0.24 0.08 25 0.61 3.08 0.03 0.23 0.07 26 0.61 3.16 0.03 0.23 0.08 27 0.61 3.56 0.03 0.23 0.09 28 0.60 3.13 0.03 0.23 0.07 29 0.61 3.20 0.03 0.24 0.08 30 0.62 3.63 0.03 0.24 0.09 V.A. Boumba et al. / Forensic Science International 232 (2013) 191198 193 Therefore, it is shown again that the most important variations arise from ethanol and 1-propanol. Finally, by subtracting one by one variable, it is concluded that isobutanol and methyl-butanol are the variables that do not signicantly contribute to the total variation. 3.2.2. Modeling whole data By performing a PLS model the descriptors relevance becomes also evident as it is shown in Fig. 2A. The signicance of each alcohol in building the mathematical models is in relevance with its produced amount. The corresponding model (r 2 = 0.90) is described by Eq. (1A): Ethanol 0:05 1-propanol 1:13 isobutanol 0:95 1-butanol 1:60 methyl-butanol 0:31 (1A) By modeling the data with only the most signicant variables (1-propanol and 1-butanol) the prediction shows a considerably satisfactory linear correlation (r 2 = 0.75), indicating the power of these two alcohols as descriptors of the process, as it is shown in Fig. 2B. Therefore we obtain the following model: Ethanol 0:05 1-propanol 0:53 1-butanol 0:32 (1B) 3.2.3. Modeling data from day 1 to 5 If we analyze the data separately for the rst 5 days of aerobical incubation, we see that the ethanol concentration is heavily dependent on 1-propanol, followed by isobutanol, and then by 1- butanol concentration (Fig. 3). The variance of methyl-butanol is of least signicance. The relevant PLS model is described by the Eq. (2): Ethanol 0:07 1-propanol 3:77 isobutanol 0:26 1-butanol 0:07 methyl-butanol 0:39 (2) 3.2.4. Modeling data from day 6 to 30 Using similar methodology and the data for the anaerobic cultivation period from day 6 to day 30 the PLS model is as described by Eq. (3): Ethanol 0:05 1-propanol 1:06 isobutanol 1:76 1-butanol 1:62 methyl-butanol 0:14 (3) The descriptors relevance shows again the signicance of 1- propanol and 1-butanol in describing the process which is very similar with the whole dataset (Fig. 4). 3.2.5. Validation modeling with PLS Validation modeling was performed by creating a small dataset of 13 randomly selected values of measurements made every 3 days (validation dataset). The rest of the data values were used as the training dataset. The resulted PLS model is described by the following equation: Ethanol 0:06 1-propanol 1:34 isobutanol 1:00 1-butanol 1:74 methyl-butanol 0:29 This equation was applied to the validation dataset with r 2 = 0.93 showing that the model for the whole dataset can indeed successfully describe the ethanol production from E. coli under the applied experimental conditions conrming our results. 3.2.6. Aspects of microbial ethanol production modeling The models for estimating ethanol production by E. coli under a combination of aerobic/anaerobic conditions were presumably correlated to 1-propanol and 1-butanol, as the relevant descrip- tors signicance revealed. This result is in discordance with the models constructed previously for E. coli under strict anaerobic conditions, where the main descriptors of ethanol modeling were 1-butanol followed by methyl-butanol. On the other hand, the result is in concordance with the models constructed previously for C. perfringens and C. sporogenes [16]. It is worth mentioning that 1-propanol [6,810] and 1-butanol [7] have been recognized, in previous studies by other authors to be correlated with the postmortem ethanol production basically in qualitative terms. Our results conrm and enhance the signicance of the presence of 1-propanol and 1-butanol as main indicators of microbial ethanol production and further, recognize them as the main descriptors of the quantication process. The relevance of both these alcohols as descriptors of ethanol modeling is so powerful that the model constructed by using only these two variants is quite satisfactory and in agreement with the general consideration that the simplest the model the easier its applica- tion. 3.3. Applicability of the models to death-related forensic cases 3.3.1. Real postmortem cases The applicability of the models presented in this report was tested in 60 postmortem cases. The cases were selected by reviewing retrospectively our chromatogram archives and select- Fig. 2. (A) Four descriptors (4D) model and (B) two descriptors (2D) model for ethanol production by E. coli by analyzing the whole dataset for the 30 days of incubation. V.A. Boumba et al. / Forensic Science International 232 (2013) 191198 194 ing those that had positive blood alcohol concentrations (BAC 0.10 g/L) and co-detection of higher alcohols and 1-butanol during the original ethanol analysis. The concentrations of 1- butanol and higher alcohols were calculated for the selected cases. Cases were classied according to the manner of death as resulted from the retrospective review of the forensic pathologists report as natural (11 cases, 18.3%), violent (14 cases, 23.3%) or undetermined (35 cases, 58.3%) cases. Also, it was revealed that 58 out of the 60 samples were from corpses with putrefaction, one from a corpse with extended trauma and one from a totally carbonized body. In Table 2 are presented the measured BACs, the estimated ethanol concentrations after applying each model (Eqs. (1A), (1B), (2) and (3)) and the relative standard error for each one of the selected cases. The higher acceptable standard error (in order to consider successful the application of the models) in the estimation of the produced ethanol when compared to the measured original BACs, was selected to be 40%, the same as in our previous relevant study [16]. The model described by Eq. (1A) estimated successfully the microbial produced ethanol in 24 out of the 60 cases (38%), the model described by Eq. (2) estimated successfully the microbial produced ethanol in 16 out of the 60 cases (25%), and the model described by Eq. (3) estimated successfully the microbial produced ethanol in 22 out of the 60 cases (35%). However, the simplest model, described by Eq. (1B) that was constructed by only two descriptors 1-propanol and 1-butanol was applied successfully in the majority of cases (26 out of 60 cases, 42%). The overall picture was that at least one of the current models (Eqs. (1A), (1B), (2) and (3)) were applied successfully in: 41 out of the 60 cases (68%), 39 of them with putrefaction (39/41, 95%); in 28/43 cases (65%) with BAC lower than 0.7 g/L; and, in 13 out of 17 cases (76%) with BAC higher than 0.7 g/L. These results are consistent with the general expressed consideration that decom- position is an aggravating factor for postmortem ethanol produc- tion and, in line with the statement that concentrations of ethanol lower than 0.7 g/L are most probably due to microbial ethanol neo- formation [2,21,22]. 3.3.2. Postmortem blood-derived microbial cultures Post-mortem blood samples from seven autopsy cases were used to inoculate normal human blood, with and without additional glucose, as described in Section 2.2. The initial glucose Fig. 3. Four descriptors (4D) model for ethanol production by E. coli by analyzing the data for the rst ve days of incubation under aerobic conditions. Fig. 4. Four descriptors (4D) model for ethanol production by E. coli by analyzing the data from day 6 to day 30 of incubation under anaerobic conditions. V.A. Boumba et al. / Forensic Science International 232 (2013) 191198 195 concentration of the spiked blood samples was chosen to be 200 mg/dL in order to be the same with the glucose content of the BHI culture medium. The selection of cases was randomized amongst routinely autopsied corpses with obvious signs of putrefaction at autopsy. The characteristics of the selected cases (microbes detected, original BACs, estimated BACs and relative standard error ranges after applying the models to the autopsy samples) and, the results after applying the models described by Eqs. (1B) and (2) to the inoculated samples for each case (mean estimated BACs and standard deviations, relative standard error ranges) are presented in Table 3. Five out of the seven blood samples carried microbial strains (E. coli, Enterococcus faecalis, Klebsiella species) commonly found in human corpses [1,23]. Clostridium species were not identied in blood samples from cases 1, 5 and 6, while for the cases 2, 3, 4 and 7 the results were inconclusive. Four out of the seven blood samples were positive for ethanol and other alcohols during the original ethanol analysis (these were presented as cases 15, 37, 50 and 59 in Table 2). When Table 2 Application of each model constructed for E. coli (Eqs. (1A), (1B), (2) and (3)) in postmortem cases to calculate the microbially produced ethanol. The original ethanol concentration measured for each case along with the manner of death are also provided. Case no. Manner of death Measured ethanol (g/L) Estimated ethanol (g/L) Relative standard error (E%) Eq. (1A) Eq. (1B) Eq. (2) Eq. (3) Eq. (1A) Eq. (1B) Eq. (2) Eq. (3) 1 Undetermined 0.10 0.35 0.37 0.45 0.17 244 259 337 68 2 Undetermined 0.10 0.61 0.63 0.77 0.41 489 504 637 294 3 Natural 0.16 0.59 0.45 1.01 0.25 268 184 530 58 4 Natural 0.16 0.35 0.36 0.44 0.17 113 123 171 3 5 Violent 0.19 0.44 0.45 0.58 0.26 135 138 208 36 6 Natural 0.20 0.33 0.34 0.42 0.15 64 70 111 25 7 Violent 0.20 0.60 0.60 0.77 0.39 198 201 283 97 8 Natural 0.21 0.51 0.50 0.71 0.32 145 138 236 52 9 Undetermined 0.21 0.54 0.41 1.00 0.35 158 94 376 66 10 Natural 0.22 0.47 0.48 0.60 0.28 116 121 178 29 11 Undetermined 0.25 0.38 0.40 0.48 0.20 52 57 92 21 12 Undetermined 0.27 0.31 0.32 0.39 0.13 13 19 44 52 13 Violent 0.28 0.68 0.67 0.79 0.53 148 142 187 92 14 Undetermined 0.29 0.49 0.49 0.63 0.30 70 72 121 3 15 Violent 0.33 1.00 0.81 0.55 1.20 200 141 63 261 16 Undetermined 0.33 1.34 0.97 0.72 1.76 304 193 117 433 17 Violent 0.34 0.66 0.62 0.64 0.57 92 81 86 66 18 Undetermined 0.35 0.62 0.52 1.03 0.42 78 49 194 20 19 Natural 0.38 0.31 0.32 0.42 0.14 17 15 10 63 20 Natural 0.39 0.77 0.56 1.34 0.23 98 43 245 42 21 Undetermined 0.39 0.40 0.42 0.51 0.22 2 6 29 45 22 Undetermined 0.42 0.84 0.85 1.05 0.61 101 104 151 47 23 Violent 0.42 0.67 0.66 0.88 0.46 59 57 110 9 24 Natural 0.42 0.50 0.52 0.63 0.31 20 23 51 26 25 Natural 0.42 0.38 0.58 0.70 0.52 9 38 67 23 26 Undetermined 0.47 1.11 0.81 2.03 0.87 137 73 333 85 27 Violent 0.52 0.74 0.74 0.95 0.52 43 43 84 1 28 Undetermined 0.54 0.31 0.32 0.39 0.13 44 41 28 76 29 Undetermined 0.58 1.36 0.76 2.97 1.10 135 32 414 90 30 Undetermined 0.59 0.49 0.49 0.64 0.29 18 17 8 50 31 Undetermined 0.60 0.76 0.71 1.07 0.54 27 20 79 9 32 Undetermined 0.60 0.36 0.38 0.52 0.18 40 37 14 70 33 Undetermined 0.61 0.79 0.81 0.98 0.57 29 32 61 7 34 Violent 0.62 1.09 0.96 1.65 0.84 76 55 166 36 35 Undetermined 0.62 0.66 0.68 0.83 0.46 7 9 34 27 36 Undetermined 0.63 1.19 0.93 2.06 0.94 89 48 226 49 37 Undetermined 0.64 0.63 0.61 0.72 0.48 2 5 13 26 38 Violent 0.65 1.02 0.96 1.03 0.95 57 48 58 45 39 Violent 0.66 0.51 0.40 0.91 0.32 22 39 38 51 40 Undetermined 0.66 0.28 0.57 2.15 1.35 57 14 224 104 41 Undetermined 0.68 0.93 0.78 1.20 0.27 36 14 76 139 42 Undetermined 0.69 0.48 0.46 0.68 0.29 30 33 1 58 43 Violent 0.69 0.79 0.54 1.55 0.57 12 24 119 19 44 Undetermined 0.71 0.66 0.57 1.04 0.45 7 19 46 36 45 Undetermined 0.71 0.48 0.49 0.62 0.29 31 32 12 59 46 Natural 0.72 0.75 0.75 1.11 0.09 4 4 54 88 47 Undetermined 0.73 0.79 0.74 1.10 0.57 7 2 50 22 48 Undetermined 0.75 0.96 0.98 1.19 0.73 28 30 59 3 49 Undetermined 0.81 0.63 0.59 1.02 0.12 23 28 25 85 50 Violent 0.84 0.88 0.87 1.15 0.65 5 4 37 21 51 Undetermined 0.87 1.23 1.11 1.76 0.25 41 28 102 72 52 Undetermined 0.91 0.91 0.92 1.14 0.68 1 2 26 25 53 Undetermined 0.94 2.13 0.82 5.42 1.81 127 13 477 93 54 Undetermined 0.98 0.73 0.56 1.30 0.52 26 43 32 47 55 Violent 1.06 0.42 0.44 0.55 0.25 60 59 48 77 56 Undetermined 1.08 0.49 0.49 0.65 0.30 55 55 40 72 57 Violent 2.15 0.98 0.95 1.05 0.85 55 56 51 61 58 Undetermined 2.58 1.10 1.00 1.59 0.85 57 61 38 67 59 Undetermined 2.94 0.83 0.80 0.97 0.70 72 73 67 76 60 Natural 3.56 0.78 0.78 1.01 0.56 78 78 72 84 V.A. Boumba et al. / Forensic Science International 232 (2013) 191198 196 ethanol and other alcohols were produced during the incubation of the inoculated samples the procedure was considered positive and the applicability of the models was tested. Therefore the procedure was positive only for two out of the seven tested postmortem bloods (cases 1 and 2 from Table 3). The application of the current models for the original autopsy blood of the rst case, where E. coli was identied, gave acceptable standard errors (selected to be <40%). In the inoculated blood samples in the presence of additional glucose higher amounts of ethanol were produced and, the models gave better scores when applied to these samples compared to the inoculated blood samples without additional glucose (Table 3). These results show that the current models could be used to estimate neo-formed ethanol by E. coli either under unspecied environmental conditions or in laboratory conditions. The observed discrepancies should be apparently attributed to the differences in the glucose content of the medium and possibly to differences in the succession of microbes in the inoculated blood samples compared to the original blood. In the second case the models estimated that only a portion of the detected ethanol could be of microbial origin. Since E. faecalis is not an ethanol producing microorganism [16] we should assume that another microbe (not identied) was activated in the original blood, from death to autopsy, and then during incubation of the inoculated samples. In the inoculated blood samples with additional glucose, more ethanol was produced compared to the samples without additional glucose. Interestingly, in this latter case, the application of the models succeeded better scores than those obtained for the inoculated samples with glucose. These results show that the current models could be used successfully to estimate the neo-formed ethanol in cases where ethanol producing microbe(s) (different than E. coli) had grown either under unspecied environmental conditions or under laboratory conditions. In the autopsy blood samples from cases 3 and 4, Klebsiella species had produced the alcohols to each autopsy blood sample. Previously, it was reported that Klebsiella pneumoniae was capable of producing ethanol in urine spiked with glucose, by fermentation at 35 8C [24]. However, the application of the current models was within acceptable standard error range only in case 4 (Table 3). The results suggest that in cases where Klebsiella species were activated, occasionally, the models could be used effectively. Alternatively, we cannot exclude the possibility that more microbe(s) could have grown to each original blood, and had produced the alcohols. Finally, the inoculation procedure was negative for cases 3 and 4, probably indicating that the ethanol producing microbes that generated the alcohols in the original blood (Klebsiella species or other) could not been regenerated to the inoculated samples under the applied experimental conditions. Postmortem blood samples that were negative for ethanol in the original volatile analysis (cases 57) did not produce alcohols during incubation of the inoculated samples, irrespectively whether microbes had been detected (case 5) or not (cases 6 and 7). For these last three cases it can be concluded that there were not present ethanol producing microbes in the autopsy bloods and, conse- quently, there was not alcohols production during the incubation period of the postmortem blood derived cultures. In general, these results indicate that whenever a blood sample is contaminated with microbe species that are ethanol produ- cers, a prole of ethanol and other alcohols would be generated if the conditions are favorable for microbial growth. In such a case the current models show a potential for application, irrespectively of the activated microbe(s). Inuencing factors appear to be the culture composition and more specically the glucose content, the conditions of microbial growth and probably, the succession of microbes. 3.3.3. Aspects, limitations and outcomes of the application of models The working hypothesis for our study is that although the postmortem or putrefactive conditions could be extremely different and variable among different cases, the patterns of other alcohols would follow more or less, in qualitative and quantitative terms, the ethanol production, since the biochemical pathways of their microbial production are interactive [20]. The patterns of produced ethanol and other alcohols, under controlled experi- mental conditions, could be described by mathematical equations (models); the patterns of ethanol and alcohols produced under different, real conditions, could be approximated by the models, and this is why ethanol estimation should be made within an acceptable standard error. Ideally, a model should exist for every postmortem case (predisposing that it was constructed under the same conditions) which is impossible, since the conditions could not be dened accurately. In our view, each model represents a tool which could be used as an alternate choice to estimate the microbially produced ethanol in real cases. In every given case, the effectiveness of each Table 3 Characteristics of the autopsy bloods used to inoculate normal human blood (original BACs, estimated BACs and relative standard error ranges (jE%j)) and results for the inoculated samples with and without additional glucose in each case (produced ethanol concentration, relative standard error ranges after applying the models described by Eqs. (1B) and (2), respectively). Case Microbes detected BAC (g/L) Estimated BAC range (g/L) Range of jE%j Fermentation experiment Blood without glucose Blood with glucose Produced ethanol (g/L), mean SD Range of jE%j (Eqs. (1B) and (2)) Produced ethanol (g/L), mean SD Range of jE%j (Eqs. (1B) and (2)) 1 E. coli, E. faecalis 0.64 0.480.72 226 0.17 0.06 26 a 0.69 0.10 2740 b 2 E. faecalis 2.94 0.710.97 6776 0.76 0.11 040, 127 1.05 0.12 3140, 335 3 K. pneumoniaea, E. faecalis 0.33 0.551.20 63261 0 0 4 Klebsiella spp., E. faecalis 0.84 0.651.15 437 0 0 5 E. faecalis 0 0 0 0 6 ND 0 0 0 0 7 ND 0 0 0 0 Abbreviation: ND, none (microbe) detected. a This value represents the best score achieved by applying the simplest model described by Eq. (1B). b Values resulted for the simplest model described by Eq. (1B). V.A. Boumba et al. / Forensic Science International 232 (2013) 191198 197 model in achieving the goal is different (due to the different postmortem conditions, resulting in different alcohol patterns); thus, one tool could be more effective and accurate than the others for each case. Incidentally, all the proposed so far models, these presented herein, as well as, those presented in our previous relevant study [16], were applied successfully for 57 out of 60 cases presented in Table 2. Nevertheless, lack of adequate evidence in respect to the origin of ethanol for these cases does not allow a denite conclusion, although putrefaction and extensive trauma of the body are recognized aggravating factors for ethanol production [3,4,21,25,26]. 4. Concluding remarks The present approach, approximates the problem of microbial ethanol production with a novel, simple methodology. The models are suggested as tools which could make feasible the quantication of the microbial neo-formed ethanol in postmortem cases. The alcohols 1-propanol and 1-butanol are proven consistent with microbial production of ethanol and the two most signicant alcohols in the modeling process. 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