An in vitro method for discriminating between live and dead microbes in a sample, comprising discriminating between transcriptionally-active and inert microbial nucleic acid sequences in the sample, wherein the method comprises the steps of: (a) sequencing a set of RNAs extracted from the sample, wherein the set of RNAs is obtained by culturing the sample in presence of an RNA-labelling agent and further by submitting the extracted RNAs to conditions promoting nucleotide substitution; thereby obtaining a set of sequence reads; (b) identifying a microbial nucleic acid sequence hit through: (i) optionally, filtering the set of sequence reads, (ii) optionally, assembling the sequence reads into contigs, (iii) aligning the sequence reads or contigs onto a database comprising microbial nucleic acid sequences, (iv) identifying at least one microbial nucleic acid sequence hit mapped against at least one sequence read or contig, and (v) determining a consensus microbial nucleic acid sequence built from these sequence reads that have mapped against the at least one microbial nucleic acid sequence hit at step (iv); (c) determining the number and/or rate of substituted nucleotides in the set of sequence reads or contigs that have mapped against the at least one microbial nucleic acid sequence hit at step (b)(iv) in comparison with the consensus microbial nucleic acid sequence determined at step (b)(v); and (d) concluding that the consensus microbial nucleic acid sequence belongs to a live microbe if, in the set of sequence reads or contigs that have mapped against the at least one microbial nucleic acid sequence hit at step (b)(iv), the number and/or rate of thymidine -to-cytidine substitution is greater than the average number and/or rate of substitution of all other nucleotides in the same sequence reads or contigs, preferably of T-to-adenine and T- to- guanosine substitutions in the same sequence reads or contigs. (a) sequencing a set of RNAs extracted from the sample, wherein the set of RNAs is obtained by culturing the sample in presence of an RNA-labelling agent and further by submitting the extracted RNAs to conditions promoting nucleotide substitution; thereby obtaining a set of sequence reads; (b) identifying a microbial nucleic acid sequence hit through: (i) optionally, filtering the set of sequence reads, (ii) optionally, assembling the sequence reads into contigs, (iii) aligning the sequence reads or contigs onto a database comprising microbial nucleic acid sequences, (iv) identifying at least one microbial nucleic acid sequence hit mapped against at least one sequence read or contig, and (v) determining a consensus microbial nucleic acid sequence built from these sequence reads that have mapped against the at least one microbial nucleic acid sequence hit at step (iv); (i) optionally, filtering the set of sequence reads, (ii) optionally, assembling the sequence reads into contigs, (iii) aligning the sequence reads or contigs onto a database comprising microbial nucleic acid sequences, (iv) identifying at least one microbial nucleic acid sequence hit mapped against at least one sequence read or contig, and (v) determining a consensus microbial nucleic acid sequence built from these sequence reads that have mapped against the at least one microbial nucleic acid sequence hit at step (iv); (c) determining the number and/or rate of substituted nucleotides in the set of sequence reads or contigs that have mapped against the at least one microbial nucleic acid sequence hit at step (b)(iv) in comparison with the consensus microbial nucleic acid sequence determined at step (b)(v); and (d) concluding that the consensus microbial nucleic acid sequence belongs to a live microbe if, in the set of sequence reads or contigs that have mapped against the at least one microbial nucleic acid sequence hit at step (b)(iv), the number and/or rate of thymidine -to-cytidine substitution is greater than the average number and/or rate of substitution of all other nucleotides in the same sequence reads or contigs, preferably of T-to-adenine and T- to- guanosine substitutions in the same sequence reads or contigs.