CmeABC Multidrug Efflux Pump Contributes to Antibiotic Resistance and Promotes Campylobacter jejuni Survival and Multiplication in Acanthamoeba polyphaga

Abstract

Campylobacter jejuni is a foodborne pathogen that is recognized as the leading cause of human bacterial gastroenteritis. The widespread use of antibiotics in medicine and in animal husbandry has led to an increased incidence of antibiotic resistance in Campylobacter In addition to a role in multidrug resistance (MDR), the Campylobacter CmeABC resistance-nodulation-division (RND)-type efflux pump may be involved in virulence. As a vehicle for pathogenic microorganisms, the protozoan Acanthamoeba is a good model for investigations of bacterial survival in the environment and the molecular mechanisms of pathogenicity. The interaction between C. jejuni 81-176 and Acanthamoeba polyphaga was investigated in this study by using a modified gentamicin protection assay. In addition, a possible role for the CmeABC MDR pump in this interaction was explored. Here we report that this MDR pump is beneficial for the intracellular survival and multiplication of C. jejuni in A. polyphaga but is dispensable for biofilm formation and motility.IMPORTANCE The endosymbiotic relationship between amoebae and microbial pathogens may contribute to persistence and spreading of the latter in the environment, which has significant implications for human health. In this study, we found that Campylobacter jejuni was able to survive and to multiply inside Acanthamoeba polyphaga; since these microorganisms can coexist in the same environment (e.g., on poultry farms), the latter may increase the risk of infection with Campylobacter Our data suggest that, in addition to its role in antibiotic resistance, the CmeABC MDR efflux pump plays a role in bacterial survival within amoebae. Furthermore, we demonstrated synergistic effects of the CmeABC MDR efflux pump and TetO on bacterial resistance to tetracycline. Due to its role in both the antibiotic resistance and the virulence of C. jejuni, the CmeABC MDR efflux pump could be considered a good target for the development of antibacterial drugs against this pathogen.

Keywords: Acanthamoeba polyphaga; Campylobacter jejuni; CmeB; antibiotic resistance; biofilm formation; host cell invasion; motility; multidrug efflux pumps; survival and multiplication.

FIG 1

C. jejuni 81-176 is able to survive and to multiply inside A. polyphaga. (A) Intracellular survival was determined by CFU counting at 0, 5, and 24 h after gentamicin treatment at 25°C, under aerobic conditions. (B) Intracellular multiplication was determined at 0, 24, 48, and 72 h after gentamicin treatment at 37°C, under aerobic conditions. Black bars represent bacterial counts obtained with the standard gentamicin protection assay, and gray bars represent bacterial counts obtained with a modified version developed in this study. P values, referring to comparisons between the samples at each time point, were as follows: panel A: 0 h, P = 0.583; 5 h, P = 0.031; 24 h, P = 0.00003; panel B: 0 h, P = 0.166; 24 h, P = 0.03; 48 h, P = 0.001; 72 h, P = 0.00028. *, 0.01 < P ≤ 0.05; **, 0.001 < P ≤ 0.01; ***, P ≤ 0.001.

FIG 2

CmeB is required for survival and multiplication of C. jejuni 81-176 in A. polyphaga. (A) Intracellular survival was determined by CFU counting at 0, 5, and 24 h after gentamicin treatment at 25°C, under aerobic conditions. (B) Intracellular multiplication was determined at 0, 24, 48, and 72 h after gentamicin treatment at 37°C, under aerobic conditions. Black bars, 81-176; white bars, 81-176/cmeB::kan^r mutant; gray bars, 81-176/cmeB::kan^r/cmeB complementation derivative. P values, referring to comparisons between the values for WT and mutant strains at each time point, were as follows: panel A: 0 h, P = 0.018; 5 h, P = 0.015; panel B: 0 h, P = 0.021; 24 h, P = 0.031; 48 h, P = 0.028; 72 h, P = 0.000004. *, 0.01 < P ≤ 0.05; ***, P ≤ 0.001. ND, not detected.

FIG 3

The cmeB mutation does not affect biofilm formation and motility. (A) Quantification of biofilms at the air-liquid interface of the glass tubes. The absorbance values measured for the WT, cmeB mutant, and complemented strains were 0.179 ± 0.06, 0.151 ± 0.01, and 0.145 ± 0.02, respectively. No statistically significant difference (P = 0.443) in biofilm quantities between the WT and cmeB mutant strains was observed. (B) Quantification of growth zones in BHI soft-agar motility plates inoculated with different C. jejuni strains. The average diameters of bacterial growth for the WT, cmeB mutant, and complemented strains were 34.2 ± 6.37 mm, 26.7 ± 3.33 mm, and 32.2 ± 4.54 mm, respectively. No statistically significant difference in growth zones (P = 0.15) between the WT and cmeB mutant strains was observed. Black bars, 81-176; white bars, 81-176/cmeB::kan^r mutant; gray bars, 81-176/cmeB::kan^r/cmeB complementation derivative.

FIG 4

Hypothetical model of the interaction between C. jejuni and A. polyphaga. The following possible stages of bacterial entry are depicted: 1, adhesion to and invasion of amoebic cells via phagocytosis; 2, gathering within amoebic vacuoles (38, 40); 3, escape to the extracellular (EC) medium (39); 4a, bacterial cell lysis; 4b, intracellular survival (ICS) and escape without lysis; 4c, intracellular multiplication (ICM); 4d, escape after lysis followed by release into the extracellular medium (39); 5, presence in the extracellular medium. In the extracellular medium, C. jejuni is able to multiply and to reinfect other amoebic cells. Stages 1, 3, 4a to 4c, and 5 are based on the observations reported in this study.