Prevalence, Virulence Genes, Phylogenetic Analysis, and Antimicrobial Resistance Profile of Helicobacter Species in Chicken Meat and Their Associated Environment at Retail Shops in Egypt

Abstract

Helicobacter pylori (H. pylori) and Helicobacter pullorum (H. pullorum) are frequently reported pathogens in humans and poultry, respectively. Nevertheless, the source of H. pylori is still unclear. This study aimed to detect Helicobacter spp. in chicken carcasses and to assess the antibiogram and the virulence genes of Helicobacter isolates. Three hundred chicken meat samples (100 each of chicken breast, liver, and gizzard), besides 60 swab samples from chicken processing surfaces, were collected from retail shops in Qalyubia Governorate, Egypt, and examined for the prevalence of H. pylori and H. pullorum. The 16S rRNA of three H. pylori and two H. pullorum isolates were sequenced to determine the genetic relationship between these two Helicobacter spp. Of the 300 chicken samples tested, 16 (5.33%) and 14 (4.67%) were positive for H. pylori and H. pullorum, respectively. Multiplex PCR revealed that the virulence genes vacuolating cytotoxin A (vacA)s1, cytotoxin-associated gene A (cagA), and restriction endonuclease-replacing gene A (hrgA) were detected in 66.7%, 77.8%, and 100% of H. pylori strains tested, respectively. H. pylori showed the highest resistance for clarithromycin, while H. pullorum exhibited the highest resistance towards erythromycin and ciprofloxacin. The study concluded that the chicken meat and giblets are potential sources of the virulent and antimicrobial-resistant strains of H. pylori of human origin.

Keywords: Helicobacter pullorum; Helicobacter pylori; antibiogram; chickens; virulence.

Figure 1

(A) H. pylori (lanes 1–8). (B) H. pullorum (lanes 1–7). Agarose gel electrophoresis showing 16S rRNA PCR amplicon (398 bp) for Helicobacter genus. Five microliters from the PCR product were separated by electrophoresis on a 1.5% agarose gel and visualized under UV light. M: DNA marker (Gene Ladder 100) used as a reference for a fragment size; Lane C+: positive control from H. pylori ATCC 43,504 strain. C−: negative control of Escherichia coli (E. coli) K12 DH5α as a negative control.

Figure 2

(A) Agarose gel electrophoresis of PCR amplicon (447 bp) of the 16S rRNA specific for H. pullorum strains (lanes 1–7). (B) Agarose gel electrophoresis of PCR product of glmM gene (294 bp) specific for the characterization of H. pylori strains (lanes 1–9). Lane M: 100 bp ladder as a molecular-size DNA marker. Lane 1: H. pylori positive control for the glmM gene. Lane 2: negative control from E. coli K12DH5α. Lanes from 1 to 9: positive H. pylori strains.

Figure 3

Identity percentages detected through the homology search (BLAST-N) of the 16S rRNA sequences of the isolated H. pylori and H. pullorum.

Figure 4

Phylogeny analysis of the 16S rRNA sequences of the isolated three H. pylori (a red circular shape) and the two H. pullorum (a blue rhombus shape) isolates.

Figure 5

Agarose gel electrophoresis of the multiplex PCR of vacA (259 bp), cagA (499 bp), and hrgA (594 bp) as virulence genes of H. pylori strains. Lane M: 100 bp ladder as a molecular-size DNA marker. Lane C+: H. pylori ATCC 43504 strain positive control. Lane C−: E. coli K12 DH5α negative control. Lanes 2, 3, 6, 7, and 8: H. pylori positive control for vacA, cagA, and hrgA genes. Lanes 4 and 5: positive control of H. pylori for cagA and hrgA genes. Lane 1: positive control of H. pylori for vacA and hrgA genes. Lane 9: positive control of H. pylori for the hrgA gene. Lanes 1 and 2 represent chicken meat; lanes 3 and 4 represent chicken gizzard; lanes 5 to 9 represent chicken liver.