# Microbial Activity in Subterranean Ecosystems: Recent Advances
## 3 The Search of Antibiotics Produced by Subsurface Bacteria
### Antibiotics from Subsurface Bacteria
The screening of only antimicrobial activity without a structural elucidation of the involved metabolites may not be useful for the discovery of new antibiotics. A few examples of studies where the bioactive compounds were fully identified are those of [PERSON] et al. [82], who provided the chemical structure of cervimycin A-D, a polyketide glycoside complex obtained from _Streptomyces tendae_, isolated from Grotta dei Cervi, in Italy, and the huanglongmycin A-C complex, synthesized by a strain of _Streptomyces_ found in a Chinese cave [83]. [PERSON] et al. [84] isolated a _Streptomyces_ sp. from a cave moon milk which produced cyclodysidin D and chaxalactin B. Another 120 metabolites were observed in the liquid extract, from which a total of 102 compounds could not be identified and appeared to be novel. These three cases exemplify how caves are a research field with high potential for novel drug discovery. In addition to conventional isolation techniques, several authors explored the possibilities of increasing the recovery of novel or interesting bacteria by improving isolation and cultivation techniques in order to extend the number of bacteria producing bioactive compounds under laboratory conditions [85]. Some of these methodologies include pretreating samples under different conditions--air drying, dry heating, moist incubation, desiccation, setting different pH, design of new culture media mimicking nature conditions, etc.--for an effective isolation of rare _Actinobacteria_[71; 86; 87]. A list of non-specific and specific methods that enhance the production of bioactive compounds can be found in Manteca and Yague [88]. Another approach is represented by NGS techniques in combination with genome mining which revolutionized the field of antibiotic research. According to [PERSON] et al. [89] genome mining analyses suggest that less than 10% of the genetic potential of antibiotic producers is currently being used, which indicates that there is a huge untapped genetic reservoir waiting to be exploited for drug discovery. Currently, more than 1555 completed genome sequences of _Streptomyces_ are available in EzBiocloud [90] and, for instance, _Streptomyces coelicolor_ harbors 22 secondary metabolite gene clusters but really produces only four of the encoded metabolites under standard laboratory conditions [58]. Screening for biosynthetic genes is an effective strategy to characterize bioactivity. For [PERSON] et al. [91] information on the expression of biosynthetic genes encoding for various bioactive compounds in cave bacteria is either limited or missing, and genome mining for PKS and NRPS genes in parallel with transcriptional analysis of the identified genes would be the more effective strategy to analyze and exploit the bioactivity of cave bacterial strains. Along with genomic mining, the combination of culturing techniques and transcriptomics would complement the systematic investigation for bioactive compounds in bacteria. These techniques have been recently used to investigate the production of the antibiotic andrimid in the marine bacterium _Vibrio comalliilyticus_[92]. In this study, the authors reported the differential expression of five biosynthetic gene clusters as well as an overproduction of andrimid in the presence of chitin rather than glucose in the culture medium. Alteration of cultivation parameters, such as, solid/liquid culturing, the presence/absence of nutrients, variations of pH and temperature, or changes in aeration supplying, would lead to the activation/inactivation of metabolic pathways involved in the biosynthesis of antimicrobials. These conditions or phenotypic variations are used in transcriptomic analyses to identify the clusters of genes differentially expressed in every condition, and afterwards, expressed in heterologous hosts by means of vectors such as plasmids, cosmids, fosmids, and artificial bacterial chromosomes or bioactive compounds.