# Microbial Activity in Subterranean Ecosystems: Recent Advances
## 3 The Search of Antibiotics Produced by Subsurface Bacteria
### Antibiotics from Subsurface Bacteria
This acidity and metal pollution has caused the loss of most forms of aquatic life, with the exception of acidophilic microorganisms which inhabit these extreme environments [96]. In this scenario, microorganisms are subjected to stress and the need to develop a metabolic system capable of coping with oligotrophy (lack of organic nutrients) and the expression of genes that produce bioactive compounds to compete with other organisms for the scarce nutrients available in the acidic environment. The inclusion of mines in this project represents a further step and an innovative research area, since as far as we know there are no previous reports considering mine microbiomes as a source of bioactive compounds. Preliminary results (Table 1) showed that about 14% of the bacteria isolated from caves and mines produced bioactive compounds against the tested pathogens. It was noteworthy that bacteria isolated from sediments of two submarine cave and marine organisms inhabiting the caves, located in the Algarve coast, only yielded two positive strains, in contrast with the higher number of terrestrial isolates. In addition, it is remarkable that 29% of bacteria isolated from the Iberian Pyrite Belt mines showed inhibitory activity against pathogens, which doubled the percentage of cave bacteria. Most of the bacteria showed high activity against Gram-positive bacteria and lower against Gram-negative bacteria. This is a reason to focus the search on having antibiotics active against Gram-negative bacteria, as demanded by the WHO. A complete screening of bacteria from air, water, and sediments from mines provided a bacterium collected with an air sampler [97] inside the mine of Lousal (Portugal). Based on the study of the 16S rRNA gene and subsequent sequencing of the genome, the strain was identified as a species within the genus _Pseudomonas_ and showed a high bioactivity against all the tested pathogenic \begin{table}
\begin{tabular}{c c c c c c c c c} \hline \hline \multirow{2}{*}{**Origin**} & **Tested** & **Positive** & \multirow{2}{*}{**1**} & \multirow{2}{*}{**2**} & \multirow{2}{*}{**3**} & \multirow{2}{*}{**4**} & \multirow{2}{*}{**5**} & \multirow{2}{*}{**6**} \\  & **Strains** & & & & & & & \\ \hline Terrestrial caves & 863 & 126 & 77 & 48 & 116 & 18 & 31 & 24 \\ Marine caves & 144 & 2 & 0 & 0 & 2 & 0 & 1 & 0 \\ Mines & 79 & 23 & 13 & 7 & 14 & 3 & 8 & 3 \\ Total (bacteria) & 1086 & 151 & 90 & 55 & 132 & 21 & 40 & 27 \\ Total (\%) & 100\% & 13.9\% & 8.29\% & 5.06\% & 12.15\% & 1.93\% & 3.68\% & 2.49\% \\ \hline \hline \end{tabular}  
* 1: _Bacillus cereus_ CET 148; 2: _Staphylococcus aureus_ CET 4630; 3: _Arthobacter_ sp. LR584284; 4: _Pseudomonas aeruginosa_ CET 110; 5: _Escherichia coli_ DSM 105182; 6: _Acinetobacter humammii_ DSM 300007.  
\end{table} 
Table 1: Screening of bacteria from mines and marine caves in Alentejo, Algarve, and Andalucia, as well as from the IRNAS-CSIC cave bacterial collection. bacteria. Genome mining analysis focused on the secondary metabolism with antiSMASH [98] which resulted in the prediction of 19 biosynthetic gene clusters from different domains such as polyketide synthases, non-ribosomal peptide synthases, tRNA-dependent cyclodopeptide synthases, aryl polyenes, phenazines, bacteriocins, N-acetylglutaminylglutamine amides, betalactones, flavin-dependent tryptophan halogenases, homoserine lactones, and siderophores (Figure 3). An \"One Strain Many Compounds\" (OSMAC) approach [99] was used to check the bioactivity of _Pseudomonas_ sp. with the aim of performing a comparative analysis of gene expression by means of transcriptomics techniques. A variation of pattern in the inhibition of pathogens was observed between solid and liquid cultures. The bioactivity of _Pseudomonas_ sp. was limited to Gram-positive bacteria in a solid culture with nutrient agar and 2% of glycerol, whereas for the liquid version of the same culture, the inhibition of pathogens was total, for both Gram-positive and Gram-negative bacteria.