Non-antibiotic pharmaceuticals are toxic against <i>Escherichia coli</i> with no evolution of cross-resistance to antibiotics

Antimicrobial resistance can arise in the natural environment via prolonged exposure to the effluent released by manufacturing facilities. In addition to antibiotics, pharmaceutical plants also produce non-antibiotic pharmaceuticals, both the active ingredients and other components of the formulations. The effect of these on the surrounding microbial communities is less clear. We aimed to assess whether non-antibiotic pharmaceuticals and other compounds produced by pharmaceutical plants have inherent toxicity, and whether long-term exposure might result in significant genetic changes or select for cross-resistance to antibiotics. To this end, we screened four non-antibiotic pharmaceuticals (acetaminophen, ibuprofen, propranolol, metformin) and titanium dioxide for toxicity against Escherichia coli K-12 MG1655 and conducted a 30 day selection experiment to assess the effect of long-term exposure. All compounds reduced the maximum optical density reached by E. coli at a range of concentrations including one of environmental relevance, with transcriptome analysis identifying upregulated genes related to stress response and multidrug efflux in response ibuprofen treatment. The compounds did not select for significant genetic changes following a 30 day exposure, and no evidence of selection for cross-resistance to antibiotics was observed for population evolved in the presence of ibuprofen in spite of the differential gene expression after exposure to this compound. This work suggests that these compounds, at environmental concentrations, do not select for cross-resistance to antibiotics in E. coli

Prospecting for efficient enantioselective epoxide hydrolases

161-177Epoxide hydrolases (EHs) from microbial sources have recently been recognized as a versatile biocatalytic tool for the synthesis of enantiomerically pure epoxides and vicinal diols. Keeping in mind the potential of these compounds in pharmaceutical, agrochemical and flavour industries, a range of epoxide substrates have been analyzed using epoxide hydrolase as the catalyst. Enzymatic catalysis is often characterized by exquisite selectivity coupled with limited substrate scope. Hence, research efforts have been on to engineer known EHs for better enantioselectivity and to find novel enantioselective EHs with a wide substrate scope from culturables, non-culturables or from the genome database. Some of the results obtained are promising in terms of the practical utility of these enzymes in the asymmetric hydrolysis of epoxides

Novel LanT Associated Lantibiotic Clusters Identified by Genome Database Mining

<div><p>Background</p><p>Frequent use of antibiotics has led to the emergence of antibiotic resistance in bacteria. Lantibiotic compounds are ribosomally synthesized antimicrobial peptides against which bacteria are not able to produce resistance, hence making them a good alternative to antibiotics. Nisin is the oldest and the most widely used lantibiotic, in food preservation, without having developed any significant resistance against it. Having their antimicrobial potential and a limited number, there is a need to identify novel lantibiotics.</p><p>Methodology/Findings</p><p>Identification of novel lantibiotic biosynthetic clusters from an ever increasing database of bacterial genomes, can provide a major lead in this direction. In order to achieve this, a strategy was adopted to identify novel lantibiotic biosynthetic clusters by screening the sequenced genomes for LanT homolog, which is a conserved lantibiotic transporter specific to type IB clusters. This strategy resulted in identification of 54 bacterial strains containing the LanT homologs, which are not the known lantibiotic producers. Of these, 24 strains were subjected to a detailed bioinformatic analysis to identify genes encoding for precursor peptides, modification enzyme, immunity and quorum sensing proteins. Eight clusters having two LanM determinants, similar to haloduracin and lichenicidin were identified, along with 13 clusters having a single LanM determinant as in mersacidin biosynthetic cluster. Besides these, orphan LanT homologs were also identified which might be associated with novel bacteriocins, encoded somewhere else in the genome. Three identified gene clusters had a C39 domain containing LanT transporter, associated with the LanBC proteins and double glycine type precursor peptides, the only known example of such a cluster is that of salivaricin.</p><p>Conclusion</p><p>This study led to the identification of 8 novel putative two-component lantibiotic clusters along with 13 having a single LanM and 3 with LanBC genes. Putative lantibiotic clusters identified here hold the potential for the discovery of novel lantibiotic(s).</p></div

Approximate size of the lantibiotic biosynthetic proteins.

<p>*SBI_06987 identified in <i>Streptomyces bingchenggensis</i> BCW-1.</p><p>Approximate size of the proteins encoded in a type I lantibiotic biosynthetic cluster along with that of the identified ORF SBI_06987 (Protein ID: YP_004965239.1), in <i>Streptomyces bingchenggensis</i> BCW-1.</p

Cluster organization of the putative lantibiotic biosynthetic genes identified in actinobacteria, <i>Streptomyces</i> and <i>Mycobacterium</i>.

<p><i>S. hygroscopicus</i> ATCC 53653, <i>S. bingchenggensis</i> BCW-1, <i>S. viridochromogenes</i> DSM 40736 are shown here, encoding two LanM determinants and the phylogenetic analysis (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0091352#pone-0091352-g003" target="_blank"><b>Fig. 3</b></a>) of the precursor peptides suggested that these clusters might encode a putative two-component lantibiotic. LanA1 and LanA2 represents alpha and beta precursors. In <i>Mycobacterium tusciae</i>, the double glycine motif containing precursor peptide was found to be encoded with the LanB determinant, enzyme for dehydration of type IA precursor peptides. HP - Hypothetical Protein; TR - Transcriptional Regulator; immunity genes which cannot be assigned as LanF/E/G are shown in red color.</p

Phylogenetic analysis of the putative precursor peptides identified in <i>Ktedonobacter racemifer</i> DSM 44963.

<p>The ten identified precursor peptides in <i>K. racemifer</i> DSM 44963 formed a separate clade from the alpha and beta precursor peptides of well-known lantibiotics.</p

Sequence identity among the precursor peptides vs. the number of LanM.

<p>A comparison of the sequence identity of the precursor peptides identified in this study, including those of the well-known lantibiotics, bhtA, smb, lacticin 3147, haloduracin, staphylococcin C55, lichenicidin, procAs and cytolysin with the number of LanM processing enzymes required. Upto ∼20% sequence identity among the precursor peptides, two LanMs are required and 37% and above, a single LanM is sufficient for the processing.</p

Cluster organization of the putative lantibiotic biosynthetic genes identified in firmicutes other than <i>B. cereus</i>.

<p>Lantibiotic clusters identified in other <i>Firmicutes</i> besides the strains of <i>Bacillus cereus</i>. The clusters identified in <i>R. flavefaciens</i> FD-1 and <i>Bacillus</i> sp. 7_6_55CFAA_CT2 encode two LanM determinants and phylogenetic analysis of the identified precursor peptides suggested that these clusters might encode for a two-component lantibiotic (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0091352#pone-0091352-g003" target="_blank"><b>Fig. 3</b></a>).</p

Cluster organization of the putative lantibiotic biosynthetic genes identified in the strains of <i>Bacillus cereus</i>.

<p>Cluster organization representing a diversity among subspecies of <i>B. cereus</i>, which belongs to <i>Firmicutes</i>. Only the strain, <i>B.cereus</i> FRI-35 was found to encode a putative two-component lantibiotic cluster (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0091352#pone-0091352-g003" target="_blank"><b>Fig. 3</b></a>). Annotation with connected lines represent identical precursor peptides.</p