Effect of biofilm formation by clinical isolates of Helicobacter pylori on the efflux-mediated resistance to commonly used antibiotics

Abstract

Aim: To evaluate the role of biofilm formation on the resistance of Helicobacter pylori (H. pylori) to commonly prescribed antibiotics, the expression rates of resistance genes in biofilm-forming and planktonic cells were compared.

Methods: A collection of 33 H. pylori isolates from children and adult patients with chronic infection were taken for the present study. The isolates were screened for biofilm formation ability, as well as for polymerase chain reaction (PCR) reaction with HP1165 and hp1165 efflux pump genes. Susceptibilities of the selected strains to antibiotic and differences between susceptibilities of planktonic and biofilm-forming cell populations were determined. Quantitative real-time PCR (qPCR) analysis was performed using 16S rRNA gene as a H. pylori-specific primer, and two efflux pumps-specific primers, hp1165 and hefA.

Results: The strains were resistant to amoxicillin, metronidazole, and erythromycin, except for one strain, but they were all susceptible to tetracycline. Minimum bactericidal concentrations of antibiotics in the biofilm-forming cells were significantly higher than those of planktonic cells. qPCR demonstrated that the expression of efflux pump genes was significantly higher in the biofilm-forming cells as compared to the planktonic ones.

Conclusion: The present work demonstrated an association between H. pylori biofilm formation and decreased susceptibility to all the antibiotics tested. This decreased susceptibility to antibiotics was associated with enhanced functional activity of two efflux pumps: hp1165 and hefA.

Keywords: Antibiotic resistance; Biofilm; Efflux genes; Helicobacter pylori; hefA; hp1165.

Figure 1

Polymerase chain reaction products of hefA (162 bp), hp1165 (117 bp) and 16S rRNA genes on 1.5% agarose gel. A: Detection of hefA gene: 1: 100 bp size marker, 2: Control, 3: Hp932 strain, 4: Hp141 strain; B: Detection of hp1165 gene: 1: HP70 strain, 2: Control, 3: Size marker, 4: Hp932 strain, 5: Hp141 strain; C: Detection of 16S rRNA gene: 1: Hp932 strain, 2: Hp141 strain, 3: Hp70 strain, 4: Control, 5: Size marker.

Figure 2

Real-time reverse transcription-polymerase chain reaction products of hp1165 (117 bp), hefA (162 bp), and 16S rRNA (136 bp) genes in biofilm-forming (B) and planktonic (P) populations on 1.5% agarose gel. A: Detection of hp1165 gene: 1: Hp141 strain (B), 2: Hp141 strain (P), 3: Hp932 strain (B), 4: Hp932 strain (P), 5: 100 bp size marker; B: Detection of hp1165 gene: 1-2: Hp70 strain (B), 3: Hp70 strain (P), 4: Size marker; C: Detection of hefA gene: 1: Size marker, 2: Hp932 strain (B), 3: Hp932 strain (P), 4: Hp141 strain (B), Hp141 strain (P), 5: Hp70 strain (B), 6: Hp70 strain (P); D: Detection of 16S rRNA gene: 1: Hp932 strain (B), 2: Hp932 strain (P), 3: Hp141 strain (B), 4: Hp141 strain (P), 5: Hp70 strain (B), 6: Hp70 strain (P), 7: Size marker.

Figure 3

Expression rates of Helicobacter pylori efflux pump genes, hefA and hp1165. Data are expressed as the means of multiple experiments ± standard deviation. Significantly different (^aP ≤ 0.05) relative to the mRNA expression level (planktonic vs biofilm-forming).