We recently showed that high g c content

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We recently showed that high G + C content actinomycetes use a hydrolytic ring-opening reaction as a common strategy of daptomycin inactivation [37], and this work further exposes the susceptibility of daptomycin’s structure to hydrolytic cleavage. We also provide the first evidence that daptomycin inactivation can occur within the low G + C content bacteria Firmicutes , for which daptomycin’s use is approved; a mechanism that clinical microbiologists should be on alert for emergence in pathogens. The inducible activity in P. lautus is very likely catalyzed by an EDTA-sensitive ring-opening esterase/protease [Figure S3]. Our efforts to purify the associated protein were unsuccessful due to instability of the activity (possibly by autocatalytic digestion); however, a similar inducible activity was recapitulated in a surface strain of P. lautus . This is intriguing as it suggests either involvement of a specific receptor for daptomycin or a non-specific response to the physiological impact of daptomycin bioactivity. The observation of two distinct macrolide inactivation mech- anisms in the Lechuguilla bacterial isolates was also intriguing. In the resistant Streptomyces strains we determined that antibiotic modification by glycosylation was the primary mechanism of inactivation, a mechanism that is known in surface actinomycetes [11]. On the other hand, we established that the mechanism of macrolide inactivation in B. paraconglomeratum is through phos- phorylation at position 2 9 catalyzed by a member of the MPH class of antibiotic kinases. Previously identified mph genes are encoded on plasmids found in clinically resistant isolates of the pathogens Escherichia coli , Staphylococcus aureus , Pasteurella multocida and Pseudo- monas aeruginosa [36]. This is the first report of mph genes from environmental bacteria as a potential source of the genes currently circulating in pathogens. The presence of a transposase-like gene upstream of mph from a surface strain of B. faecium points to a potential history of horizontal gene transfer (Figure 6). It is possible that mph genes have been circulating among bacterial populations before the cave was sealed off millions of years ago, resulting in an mph gene that is a shared trait between both terrestrial and cave bacteria. There are two likely explanations for retention of the mph genes with same biochemical properties despite the long isolation of the Brachybacterium strains: (i) these genes could serve a physiologic or metabolic function unrelated to antibiotic resistance (although the genetic context (Figure 6) does not suggest an obvious role); or (ii) these genes are resistance elements for conferring antibiotic resistance. We could not detect any macrolide biosynthetic gene clusters in bacteria collected in the same region as B. paraconglomer- atum (as evidenced by a absence of the signature macrolide D- desosamine biosynthesis gene, eryCVI , not shown); however the actinomycete small sample size does not rule out the possibility of the presence of hitherto undetected macrolide producers within the cave.
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