RamAp Is an Efflux Pump Regulator Carried by an IncHI2 Plasmid

Abstract

In investigating the epidemiological trends of Salmonella enterica serovar Goldcoast, we previously identified several closely related strains with different MICs to azithromycin and quinolones. Genome sequencing and comparison of two very similar multidrug-resistant (MDR) strains, R18.0877 and R18.1656, has led to the identification of an extra plasmid-borne ramA gene, ramAp, on the large IncHI2 plasmid carried by R18.0877. The ramAp gene is located in a 953-bp region on the plasmid, which is identical to that of the Klebsiella quasipneumoniae chromosomal ramA loci. A truncated ISEcp1 located at the adjacent upstream area of the putative regulatory region of ramAp may likely contribute to its mobilization and expression. Introducing the ramAp gene and the truncated ISEcp1 into Escherichia coli has resulted in elevated expression of efflux pump genes and elevated MICs to chloramphenicol, azithromycin, nalidixic acid, ciprofloxacin, sulfamethoxazole, trimethoprim, tetracycline, and tigecycline. The ramAp is an extra efflux pump activator gene that potentially could be transmitted with the IncHI2 plasmid among bacteria. It is plausible that, with high interspecific conservation, the plasmid-encoded regulator reduces drug susceptibility by activating existing efflux pump systems of the host and thus can be regarded as a new type of auxiliary antimicrobial resistance determinant. Sequences of similar plasmids were found worldwide. Its impact on the emergence of antimicrobial resistance among bacterial pathogens is worrisome.

Keywords: ISEcp1; Salmonella enterica serovar Goldcoast; antimicrobial drug resistance; efflux pump regulator; plasmid-borne; ramA.

FIG 1

Schematic diagram of plasmid pR18.0877_278k compared to plasmid pR18.1656_270k. Open reading frames (black stripes) carried by plasmid pR18.0877_278k (GenBank accession number CP037959) are depicted at the outer circle. Pseudogenes are shown as gray stripes. At the inner circle, the conserved region shared by both of the plasmids is shown. The 6.6-kb extra ramAp region that is present only in pR18.0877_278k (98,026 to ∼104,647 bp) is shown. Significant genes on the plasmid are labeled as follows: aph(3′)-Ia, aminoglycoside O-phosphotransferase; floR, chloramphenicol/florfenicol efflux MFS; aph(6)-Id, aminoglycoside O-phosphotransferase; tetR, TetR family transcriptional regulator; tetA, tetracycline efflux MFS transporter; sul3, sulfonamide-resistant dihydropteroate synthase Sul3; lnu(F), lincosamide nucleotidyltransferase Lnu(F); aadA22, ANT(3′)-Ia family aminoglycoside nucleotidyltransferase; aac(3)-IId, aminoglycoside N-acetyltransferase AAC(3)-IId; bla_TEM-1, beta-lactamase TEM-1; bla_CTX-M-55, beta-lactamase CTX-M-55; qnrS13, quinolone resistance protein QnrS13; arr-2, NAD(+)-rifampin ADP-ribosyltransferase Arr-2; dfrA14, trimethoprim-resistant dihydrofolate reductase DfrA14.

FIG 2

The 6.6-kb extra ramAp-region in plasmid pR18.0877_278k. The significant gene features and the adjacent regions on the plasmid are depicted. The ramAp gene is depicted in black. IS or Tn are depicted as gray rectangles. Putative genes of other or unknown functions are shown in white arrows. The incomplete open reading frames or truncated IS/Tn elements were marked with an asterisk (*). PII, putative promoter region for ramAp. IRL/IRR indicates the inverted repeats of the IS or transposon (Tn) elements. The 953-bp region spanning ramAp is identical to that of Klebsiella quasipneumoniae strain ATCC 700603 (CP014696.2). The significant sequence features at the putative promoter region upstream of ramAp are shown below. The 1,147-bp region containing ramAp that was cloned into pMiniT is also indicated.

FIG 3

Expression levels of efflux pump genes. (A) Expression levels of acrA, acrB, and tolC genes in E. coli constructs (E. coli pMiniT-control and E. coli pMiniT-ramAp). Values on the y axis are relative expression levels (fold increase) normalized against levels of the endogenous reference rpsL. (B) Expression levels of acrA, acrB, and tolC genes in Salmonella Goldcoast (R18.1656 and R18.0877). Values on the y axis are relative expression levels (fold increase) normalized against levels of the endogenous reference rpoB. The data correspond to the mean values of three biological replications. Error bars correspond to the standard deviation. Asterisks indicate statistically significant differences (P < 0.05) in t tests.