No abstract

Bacterial resistance to biocides used as antiseptics, dyes, and disinfectants is a growing concern in food preparation, agricultural, consumer manufacturing, and health care industries, particularly among Gram-negative Enterobacteriaceae, some of the most common community and healthcare-acquired bacterial pathogens. Biocide resistance is frequently associated with antimicrobial cross-resistance leading to reduced activity and efficacy of both antimicrobials and antiseptics. Multidrug resistant efflux pumps represent an important biocide resistance mechanism in Enterobacteriaceae. An assortment of structurally diverse efflux pumps frequently co-exist in these species and confer both unique and overlapping biocide and antimicrobial selectivity. TolC-dependent multicomponent systems that span both the plasma and outer membranes have been shown to confer clinically significant resistance to most antimicrobials including many biocides, however, a growing number of single component TolC-independent multidrug resistant efflux pumps are specifically associated with biocide resistance: small multidrug resistance (SMR), major facilitator superfamily (MFS), multidrug and toxin extruder (MATE), cation diffusion facilitator (CDF), and proteobacterial antimicrobial compound efflux (PACE) families. These efflux systems are a growing concern as they are rapidly spread between members of Enterobacteriaceae on conjugative plasmids and mobile genetic elements, emphasizing their importance to antimicrobial resistance. In this review, we will summarize the known biocide substrates of these efflux pumps, compare their structural relatedness, Enterobacteriaceae distribution, and significance. Knowledge gaps will be highlighted in an effort to unravel the role that these apparent “lone wolves” of the efflux-mediated resistome may offer.

Members of the small multidrug resistant (SMR) efflux pump family known as SugE (more recently renamed to Gdx) are known for their narrow substrate selectivity to small guanidinium (Gdm+) compounds and disinfectant quaternary ammonium compounds (QACs). Gdx members have been identified on multidrug resistant plasmids in Gram-negative bacilli, but their functional role remains unclear as few have been characterized. Here, we conducted a survey of sequenced proteobacterial plasmids that encoded one or more SugE/Gdx sequences in an effort to 1) identify the most frequently represented Gdx member(s) on these plasmids and their sequence diversity, 2) verify if Gdx sequences possess a Gdm+ riboswitch that regulates their translation similar to chromosomally encoded Gdx members, and 3) determine the antimicrobial susceptibility profile of the most predominate Gdx member to various QACs and antibiotics in E. coli strains, BW25113 and KAM32. The results of this study determined 14 unique SugE sequences, but only one Gdx sequence annotated as ‘SugE(p)’ predominated among the >140 plasmids we surveyed. Enterobacterales plasmids encoding sugE(p) possessed a Guanidine-II riboswitch similar to the upstream region of E. coli gdx. Cloning and expression of sugE(p), gdx, and emrE sequences into a low copy expression vector (pMS119EH) revealed significant increases in QAC resistance to a limited range of detergent-like QACs only when gdx and sugE(p) transformants were grown as biofilms. These findings suggest that sugE(p) presence on proteobacterial plasmids may be driven by species that frequently encounter Gdm+ and QAC exposure. Importance: This study characterized the function of antimicrobial-resistant phenotypes attributed to plasmid encoded guanidinium selective small multidrug resistant (gdm/sugE) efflux pumps. These sequences are frequently monitored as biocide-resistance markers in antimicrobial resistance surveillance studies. Our findings reveal that Enterobacterial gdm sequences transmitted on plasmids possess a Guanidine-II riboswitch, which restricts transcript translation in the presence of guanidinium. Cloning and overexpression of this gdm sequence revealed that it confers higher resistance to quaternary ammonium compound (QAC) disinfectants (that possess guanidium moieties) when grown as biofilms. Since biofilms are commonly eradicated with QAC containing compounds the presence of this gene on plasmids and its biofilm-specific resistance are a growing concern to clinical and food safety prevention measures.

Qac efflux pumps from proteobacterial multidrug-resistant plasmids are integron-encoded and confer resistance to quaternary ammonium compound (QAC) antiseptics, however, many are uncharacterized and misannotated. A survey of >2000 plasmid-encoded qac identified 37 unique qac sequences that correspond to one of five representative motifs: QacE, QacEΔ1, QacF/L, QacH/I, and QacG. Antimicrobial susceptibility testing of each cloned qac member in Escherichia coli , highlighted distinctive antiseptic susceptibility patterns that were most prominent when cells grew as biofilms.

34Small multidrug resistance (SMR) efflux pump genes are commonly identified from integrons carried by 35 multidrug-resistant (MDR) plasmids. SMR pumps are annotated as 'qac' for their ability to confer resistance to 36 quaternary ammonium compounds (QACs) but few qac are characterized to date. Hence, we have examined SMR 37 sequence diversity, antimicrobial susceptibility, and gene expression from >500 sequenced proteobacterial 38 plasmids. SMR sequence diversity from plasmid database surveys identified 20 unique SMR sequences annotated 39 as qacE/EΔ1/F/G/H/I/L, or sugE. Phylogenetic analysis shows 'Qac' sequences are homologous to archetypical 40 SMR member EmrE, and share a single sequence origin. In contrast, SugE sequences are homologous to 41 archetypical member Gdx/SugE and likely originate from different species. SMR genes, qacE, qacEΔ1, qacF, 42 qacG, qacH, and sugE(p), were over-expressed in Escherichia coli to determine their QAC antimicrobial 43 susceptibility as planktonic, colony, and biofilms. SMRs (except qacEΔ1/sugE) expressed in biofilms 44 significantly increased its QAC tolerance as compared to planktonic and colony growth. Analysis of upstream 45 SMR nucleotide regions indicate sugE(p) genes are regulated by type II guanidinium riboswitches, whereas qacE 46 and qacEΔ1 have a conserved class I integron Pq promoter, and qacF/G/H are regulated by integron Pc promoter 47 in variable cassettes region. Beta-galactosidase assays were used to characterize growth conditions regulating Pq 48and Pc promoters and revealed that Pq and Pc have different expression profiles during heat, peroxide, and QAC 49 exposure. Altogether, this study reveals that biofilm growth methods are optimal for SMR-mediated QAC 50 susceptibility testing and suggests SMR gene regulation on plasmids is similar to chromosomally inherited SMR 51 members. 52 53 Introduction 54Small multidrug resistance (SMR) proteins belong to a family of proton motive force driven, multidrug 55 selective efflux pumps found in bacteria (and archaea) that are small (100-150 amino acids) and composed of 4 56 transmembrane (TM) α-helices as compared to most efflux pump family members with 12-14 TM α-helices (1). 57Depending on their phylogeny, SMR members confer low to moderate levels (2-8 fold increases in in minimum 58 inhibitory concentration (MIC) values from controls) of tolerance (reduced susceptibility or resistance) to a 59 variety of guanidinium (Gdm + ) and lipophilic cation containing chemical compounds when they are over-60 expressed in bacteria (2-5). The most notable Gdm + compounds associated with SMRs are quaternary ammonium 61 compounds (QACs). QACs include a wide range of disinfectants and antiseptics (e.g. benzalkonium and 62 cetrimide) as well as DNA/RNA inter-chelating lipophilic dyes (e.g. ethidium, acriflavine) (6-8). SMR family 63 members that confer antimicrobial tolerance by the expression of a single gene copy can be divided into two 64 major groups, 1) small multidrug proteins (SMPs) and 2) suppressor of groEL mutations (...