Global Association between Thermophilicity and Vancomycin Susceptibility in Bacteria

Chayan Roy, Masrure Alam, Subhrangshu Mandal, Prabir K. Haldar, Sabyasachi Bhattacharya, Trinetra Mukherjee, Rimi Roy, Moidu J. Rameez, Anup K. Misra, Ranadhir Chakraborty, Ashish K. Nanda, Subhra K. Mukhopadhyay, Wriddhiman Ghosh

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.