Public health concern of antimicrobial resistance and virulence determinants in E. coli isolates from oysters in Egypt

Abstract

The emergence of critical-priority E. coli, carrying a wide array of resistance and virulence factors through food sources, poses a significant challenge to public health. This study aimed to investigate the potential role of oysters sold in Egypt as a source for E. coli, identify their resistance and virulence-associated gene profiles, and assess associated zoonotic risks. A total of 33 pooled fresh oyster samples were obtained from various retail fish markets in Egypt and examined bacteriologically for the presence of E. coli. Antimicrobial resistance was performed by the disk-diffusion method, and the multiple antibiotic resistance index (MAR) was calculated. All isolates were screened for extended-spectrum beta-lactamase (ESBL) (bla_TEM, bla_SHV, bla_CTX-M, and bla_OXA-1), plasmid-mediated AmpC bla_CMY-2, and carbapenemases (bla_KPC, bla_NDM, bla_VIM, and bla_OXA-48) genes by Polymerase chain reaction. Moreover, the presence of virulence-encoding genes was investigated. The virulent MDR strains were clustered using R with the pheatmap package. The prevalence of E. coli was 72.7% (24 out of 33), with 66.7% of the isolates classified as multi-drug resistant, and 75% exhibited MAR values exceeding the 0.2 threshold. Different antimicrobial sensitivity phenotypes and genotype profiles were identified in E. coli isolates. The most prevalent gene detected among all isolates was bla_TEM (22/24, 91.7%). Notably, all non-ESBL producers were positive for bla_CMY2. Carbapenem-resistant and carbapenem-intermediate strains were carbapenemase producers, with the predominance of the bla_KPC gene (11/24, 45.8%). Remarkably, twelve out of sixteen virulence genes were identified, with papC (21/24, 87.5%) and sfa (16/24, 66.7%) genes being the most prevalent. Most isolates carry virulence genes primarily associated with extra-intestinal pathogenic E. coli (ExPEC) (87.5%) and enteropathogenic (EPEC) (70.8%) pathotypes. Four E. coli isolates exhibit cluster patterns. This study provides the first insight into the emergence of virulent MDR E. coli among oysters in Egypt. It underscores the potential role of oysters as a source for disseminating these strains within aquatic ecosystems, presenting a possible threat to public health.

Keywords: E. Coli pathotypes; Carbapenems; Extended-spectrum β-lactamases; Multi-drug resistance; Plasmid AmpC; Virulence factors.

Fig. 1

Percentage of antimicrobial resistance and intermediate resistance determined by disc diffusion method in E. coli isolates from retail oysters in Egypt. Antibiotic discs: Ampicillin (AMP), Cefoxitin (FOX), Cefotaxime (CTX), Ceftriaxone (CRO), Ceftazidime (CAZ), Meropenem (MRP), Ertapenem (ETP), Amikacin (AK), Azithromycin (AT), Erythromycin (Eo), Doxycycline (Do), Ciprofloxacin (CIP), Levofloxacin (LE), Trimethoprim-Sulfamethoxazole (COT), and Chloramphenicol (C).

Fig. 2

Heatmap of E. coli strains clustered according to both phenotypes and genotypes. G1, G2 and G3 represent the clustering of the isolates according to their antimicrobial resistance phenotype and genotype profiles, while the top of the heatmap (C1, C2, C3 and C4) represents the pattern of antibiotic resistance and resistance genes tested. The varied colors represent positive resistance genes (light yellow) or negative (blue) results, regarding the antimicrobial resistance; resistant phenotype (red), intermediate resistant (light yellow) and sensitive (blue). The phenotypic resistance patterns were screened against fifteen antibiotic discs: Ampicillin (AMP), Cefoxitin (FOX), Cefotaxime (CTX), Ceftriaxone (CRO), Ceftazidime (CAZ), Meropenem (MRP), Ertapenem (ETP), Amikacin (AK), Azithromycin (AT), Erythromycin (Eo), Doxycycline (Do), Ciprofloxacin (CIP), Levofloxacin (LE), Trimethoprim-Sulfamethoxazole (COT), and Chloramphenicol(C).The resistance genotypes are also provided; β-lactamases (bla_TEM, TEM beta-lactamase; bla_CTX−M, CTX-M beta-lactamase; bla_SHV, SHV beta-lactamase; bla_OXA−1, oxacillin-hydrolyzing β-lactamases ; bla_CMY−2, plasmid-mediated AmpC β-lactamase gene), and carbapenemases (bla_KPC,K. pneumoniae carbapenemases; bla_NDM, New Delhi metallo-beta-lactamase; bla_OXA−48, oxacillin-hydrolyzing metallo-β-lactamases; bla_VIM ,Verona vntegron-encoded metallo-beta-lactamase).

Fig. 3

(a) Heatmap of E. coli strains clustered according to both virulence genes and their association with pathotypes. G1, G2 represent the clustering of the isolates according to their virulence genes, the top of the heatmap (C1, C2 and C3) illustrate the different pathotypes present in each isolate; (b) Percentage of occurrence of different pathotypes among the recovered E. coli isolates.

Fig. 4

Heatmap of MDR E. coli isolates clustered according to both antimicrobial resistance phenotype and virulence genes carriage. The varied colors represent positive virulence genes (red) or negative (blue) results; regarding the antimicrobial resistance; resistant phenotype (yellow), intermediate resistant (light blue), and sensitive (blue). Antibiotic discs: Ampicillin (AMP), Cefoxitin (FOX), Cefotaxime (CTX), Ceftriaxone (CRO), Ceftazidime (CAZ), Meropenem (MRP), Ertapenem (ETP), Amikacin (AK), Azithromycin (AT), Erythromycin (Eo), Doxycycline (Do), Ciprofloxacin (CIP), Levofloxacin (LE), Trimethoprim-Sulfamethoxazole (COT), and Chloramphenicol(C).