Novel N-Acyl Homoserine Lactone-Degrading Bacteria Isolated From Penicillin-Contaminated Environments and Their Quorum-Quenching Activities

Abstract

N-Acyl homoserine lactones (AHLs) are signaling molecules used in the quorum sensing (QS) of Gram-negative bacteria. Some bacteria interfere with the QS system using AHL-inactivating enzymes, commonly known as quorum-quenching (QQ) enzymes. We have recently isolated a new QQ bacterium showing high resistance to multiple β-lactam antibiotics, and its QQ enzyme (MacQ) confers β-lactam antibiotic resistance and exhibits QQ activities. This observation suggests the possibility of isolating novel QQ bacteria from β-lactam antibiotic-resistant bacteria. In this direction, we attempted to isolate penicillin G (PENG)-resistant bacteria from penicillin-contaminated river sediments and activated sludge treating penicillin-containing wastewater and characterize their QQ activities. Of 19 PENG-resistant isolates, six isolates showed high QQ activity toward a broad range of AHLs, including AHLs with 3-oxo substituents. Five of the six AHL-degraders showed AHL-acylase activity and hydrolyzed the amide bond of AHLs, whereas the remaining one strain did not show AHL-acylase activity, suggesting that this isolate may likely possess alternative degradation mechanism such as AHL-lactonase activity hydrolyzing the lactone ring of AHLs. The 16S rRNA gene sequence analysis results categorized these six AHL-degrading isolates into at least five genera, namely, Sphingomonas (Alphaproteobacteria), Diaphorobacter (Betaproteobacteria), Acidovorax (Betaproteobacteria), Stenotrophomonas (Gammaproteobacteria), and Mycobacterium (Actinobacteria); of these, Mycobacterium sp. M1 has never been known as QQ bacteria. Moreover, multiple β-lactam antibiotics showed high minimum inhibitory concentrations (MICs) when tested against all of isolates. These results strongly demonstrate that a wide variety of β-lactam antibiotic-resistant bacteria possess QQ activities. Although the genetic and enzymatic elements are yet unclear, this study may infer the functional and evolutionary correlation between β-lactam antibiotic resistance and QQ activities.

Keywords: AHL-acylase; AHL-lactonase; quorum quenching; quorum sensing; β-lactam antibiotic resistance.

FIGURE 1

The general structure of AHL signals and the corresponding degradation mechanisms of AHL-lactonase (A) and AHL-acylase (B). Cleavage of the lactone ring by an AHL-lactonase enzyme (dashed arrow) yields the corresponding acyl homoserine. Cleavage of the amide bond by an AHL-acylase enzyme (filled arrow) yields the corresponding fatty acid and homoserine lactone (HSL) ring.

FIGURE 2

Phylogenetic affiliations of the AHL-degrading isolates obtained in this study and the previously known AHL degraders based on their almost full length 16S rRNA gene sequences. The phylogenetic tree was constructed by neighbor-joining (NJ) method with Kimura’s correction. The 16S rRNA gene sequence of Aquifex pyrophilus (M83548) was used as an outgroup. Bootstrap values of >50% and >80% estimated using neighbor-joining and maximum-likelihood methods (1,000 replications) are shown by circle and square at branching points, respectively. The six new AHL-degrading strains isolated in this study are shown in name with boldface.

FIGURE 3

Metabolite analysis of C₁₀-HSL degradation products by cell extract of the AHL degrading isolates. HPLC profiles of unreacted DANSYL chloride solution (A), C₁₀-HSL (B), dansylated HSL standard (C), reaction product of C₁₀-HSL after incubation with cell extract of Acidovorax sp. MR-S7 (D), strain S1 (E), strain S2 (F), strain S17 (G), strain M2 (H), and strain M6 (I). Note that a dansylated digestion product and a dansylated HSL standard both eluted with retention time at 5.8–6.0 min.