Tn6188 - a novel transposon in Listeria monocytogenes responsible for tolerance to benzalkonium chloride

Abstract

Controlling the food-borne pathogen Listeria (L.) monocytogenes is of great importance from a food safety perspective, and thus for human health. The consequences of failures in this regard have been exemplified by recent large listeriosis outbreaks in the USA and Europe. It is thus particularly notable that tolerance to quaternary ammonium compounds such as benzalkonium chloride (BC) has been observed in many L. monocytogenes strains. However, the molecular determinants and mechanisms of BC tolerance of L. monocytogenes are still largely unknown. Here we describe Tn6188, a novel transposon in L. monocytogenes conferring tolerance to BC. Tn6188 is related to Tn554 from Staphylococcus (S.) aureus and other Tn554-like transposons such as Tn558, Tn559 and Tn5406 found in various Firmicutes. Tn6188 comprises 5117 bp, is integrated chromosomally within the radC gene and consists of three transposase genes (tnpABC) as well as genes encoding a putative transcriptional regulator and QacH, a small multidrug resistance protein family (SMR) transporter putatively associated with export of BC that shows high amino acid identity to Smr/QacC from S. aureus and to EmrE from Escherichia coli. We screened 91 L. monocytogenes strains for the presence of Tn6188 by PCR and found Tn6188 in 10 of the analyzed strains. These isolates were from food and food processing environments and predominantly from serovar 1/2a. L. monocytogenes strains harboring Tn6188 had significantly higher BC minimum inhibitory concentrations (MICs) (28.5 ± 4.7 mg/l) than strains without Tn6188 (14 ± 3.2 mg/l). Using quantitative reverse transcriptase PCR we could show a significant increase in qacH expression in the presence of BC. QacH deletion mutants were generated in two L. monocytogenes strains and growth analysis revealed that ΔqacH strains had lower BC MICs than wildtype strains. In conclusion, our results provide evidence that Tn6188 is responsible for BC tolerance in various L. monocytogenes strains.

Figure 1. Organization of the L. monocytogenes transposon Tn6188 and comparison with other related transposons Tn554 (X03216), Tn5406 (AF186237), Tn558 (AJ715531), Tn559 (FN677369).

The transposase genes tnpABC are shown in dark blue, blue and light blue, respectively. Effector proteins conferring antimicrobial resistance are shown in red; proteins with other or unknown function are shown in orange. The 6 bp nucleotide sequences at the transposon junctions are shown in boxes. The disrupted radC genes are indicated by an asterisk.

Figure 2. Amino acid sequence alignment of QacH from Tn6188 and related Qac/smr/EmrE-like proteins.

The alignment was done with MAFFT [23], shading of conserved amino acid residues was performed with Boxshade. Amino acid residues important for function in EmrE and QacC are highlighted in red. Data for important amino acid residues are taken from [31-33]. The consensus is displayed at the bottom of each alignment block, asterisks indicate identical positions, dots indicate similar positions. Abbreviations and accession numbers: QacH_LM4423 (L. monocytogenes 4423, CCP37730), QacH_LM6179 (L. monocytogenes 6179, CCP37735), QacH_LMK15 (L. monocytogenes K15, HG329628), QacH_LMHPB2262 (L. monocytogenes HPB2262, EFF95069), QACC_STAAU (Staphylococcus aureus, P14319), QACH_STASA (Staphylococcus saprophyticus, O87868), QACJ_STAAU (Staphylococcus aureus, NP_783299), EMRE_ECOLI (E. coli, P23895).

Figure 3. Benzalkonium chloride MICs of L. monocytogenes strains.

BC MICs of ten L. monocytogenes strains with (+Tn6188, A) and without (-Tn6188, B) Tn6188 are shown. MICs represent mean values of three biological independent replicates, SD=0.

Figure 4. Gene expression of qacH and benzalkonium chloride MICs of ΔqacH strains.

Gene expression of Tn6188 qacH in the presence of different BC concentrations was determined by quantitative RT-PCR (A). Values, represented as x-fold of control (0 mg/l BC), were normalized according 16S rRNA expression levels. *p<0.02. Differences in MICs of BC using L. monocytogenes wildtype and ΔqacH strains (4423 and 6179) are shown in (B). MICs represent mean values of three biological independent replicates, SD=0.