title: Global Association between Thermophilicity and Vancomycin Susceptibility in Bacteria.
abstract: Exploration of the aquatic microbiota of several circum-neutral (6.0-8.5 pH) mid-temperature (55-85°C) springs revealed rich diversities of phylogenetic relatives of mesophilic bacteria, which surpassed the diversity of the truly-thermophilic taxa. To gain insight into the potentially-thermophilic adaptations of the phylogenetic relatives of Gram-negative mesophilic bacteria detected in culture-independent investigations we attempted pure-culture isolation by supplementing the enrichment media with 50 μg ml(-1) vancomycin. Surprisingly, this Gram-positive-specific antibiotic eliminated the entire culturable-diversity of chemoorganotrophic and sulfur-chemolithotrophic bacteria present in the tested hot water inocula. Moreover, it also killed all the Gram-negative hot-spring isolates that were obtained in vancomycin-free media. Concurrent literature search for the description of Gram-negative thermophilic bacteria revealed that at least 16 of them were reportedly vancomycin-susceptible. While these data suggested that vancomycin-susceptibility could be a global trait of thermophilic bacteria (irrespective of their taxonomy, biogeography and Gram-character), MALDI Mass Spectroscopy of the peptidoglycans of a few Gram-negative thermophilic bacteria revealed that tandem alanines were present in the fourth and fifth positions of their muropeptide precursors (MPPs). Subsequent phylogenetic analyses revealed a close affinity between the D-alanine-D-alanine ligases (Ddl) of taxonomically-diverse Gram-negative thermophiles and the thermostable Ddl protein of Thermotoga maritima, which is well-known for its high specificity for alanine over other amino acids. The Ddl tree further illustrated a divergence between the homologs of Gram-negative thermophiles and mesophiles, which broadly coincided with vancomycin-susceptibility and vancomycin-resistance respectively. It was thus hypothesized that thermophilic Ddls have been evolutionarily selected to favor a D-ala-D-ala bonding. However, preference for D-ala-D-ala-terminated MPPs does not singlehandedly guarantee vancomycin susceptibility of thermophilic bacteria as the large and relatively-hydrophilic vancomycin molecule has to cross the outer membrane before it can inhibit peptidoglycan biosynthesis. Literature shows that many mesophilic Gram-negative bacteria also have D-ala-D-ala-terminated MPPs, but they still remain resistant to vancomycin due to the relative impermeability of their membranes. But the global vancomycin-susceptibility phenotype of thermophilic bacteria itself testifies that the drug crosses the membrane in all these cases. As a corollary, it seems quite likely that the outer membranes of thermophilic bacteria have some yet-unknown characteristic feature(s) that invariably ensures the entry of vancomycin.
authors.affiliation: Department of Microbiology, Bose Institute Kolkata, India.
authors.forename: Chayan
authors.initials: C
authors.lastname: Roy
authors.affiliation: Department of Microbiology, Bose Institute Kolkata, India.
authors.forename: Masrure
authors.initials: M
authors.lastname: Alam
authors.affiliation: Department of Microbiology, Bose Institute Kolkata, India.
authors.forename: Subhrangshu
authors.initials: S
authors.lastname: Mandal
authors.affiliation: Department of Microbiology, Bose Institute Kolkata, India.
authors.forename: Prabir K
authors.initials: PK
authors.lastname: Haldar
authors.affiliation: Department of Microbiology, Bose Institute Kolkata, India.
authors.forename: Sabyasachi
authors.initials: S
authors.lastname: Bhattacharya
authors.affiliation: Department of Microbiology, The University of Burdwan Burdwan, India.
authors.forename: Trinetra
authors.initials: T
authors.lastname: Mukherjee
authors.affiliation: Department of Microbiology, Bose Institute Kolkata, India.
authors.forename: Rimi
authors.initials: R
authors.lastname: Roy
authors.affiliation: Department of Microbiology, Bose Institute Kolkata, India.
authors.forename: Moidu J
authors.initials: MJ
authors.lastname: Rameez
authors.affiliation: Division of Molecular Medicine, Bose Institute Kolkata, India.
authors.forename: Anup K
authors.initials: AK
authors.lastname: Misra
authors.affiliation: Department of Biotechnology, University of North Bengal Siliguri, India.
authors.forename: Ranadhir
authors.initials: R
authors.lastname: Chakraborty
authors.affiliation: Department of Chemistry, University of North Bengal Siliguri, India.
authors.forename: Ashish K
authors.initials: AK
authors.lastname: Nanda
authors.affiliation: Department of Microbiology, The University of Burdwan Burdwan, India.
authors.forename: Subhra K
authors.initials: SK
authors.lastname: Mukhopadhyay
authors.affiliation: Department of Microbiology, Bose Institute Kolkata, India.
authors.forename: Wriddhiman
authors.initials: W
authors.lastname: Ghosh
keywords: D-alanine-d-alanine ligase; MALDI-TOF mass spectrometry; NGS; peptidoglycan; thermophilic bacteria; vancomycin susceptibility