High Throughput Screening of Antimicrobial Resistance Genes in Gram-Negative Seafood Bacteria

Abstract

From a global view of antimicrobial resistance over different sectors, seafood and the marine environment are often considered as potential reservoirs of antimicrobial resistance genes (ARGs) and mobile genetic elements (MGEs); however, there are few studies and sparse results on this sector. This study aims to provide new data and insights regarding the content of resistance markers in various seafood samples and sources, and therefore the potential exposure to humans in a global One Health approach. An innovative high throughput qPCR screening was developed and validated in order to simultaneously investigate the presence of 41 ARGs and 33 MGEs including plasmid replicons, integrons, and insertion sequences in Gram-negative bacteria. Analysis of 268 seafood isolates from the bacterial microflora of cod (n = 24), shellfish (n = 66), flat fishes (n = 53), shrimp (n = 10), and horse mackerel (n = 115) show the occurrence of sul-1, ant(3″)-Ia, aph(3′)-Ia, strA, strB, dfrA1, qnrA, and blaCTX-M-9 genes in Pseudomonas spp., Providencia spp., Klebsiella spp., Proteus spp., and Shewanella spp. isolates and the presence of MGEs in all bacterial species investigated. We found that the occurrence of MGE may be associated with the seafood type and the environmental, farming, and harvest conditions. Moreover, even if MGE were detected in half of the seafood isolates investigated, association with ARG was only identified for twelve isolates. The results corroborate the hypothesis that the incidence of antimicrobial-resistant bacteria (ARB) and ARG decreases with increasing distance from potential sources of fecal contamination. This unique and original high throughput micro-array designed for the screening of ARG and MGE in Gram-negative bacteria could be easily implementable for monitoring antimicrobial resistance gene markers in diverse contexts.

Molluscs—A ticking microbial bomb

“…They found isolates of S. algae resistant to β-lactams ( blaOXA-55-LIKE, ampC ), tetracyclines ( tetR, tet34 , and tet35 ), fluoroquinolones ( qnrA3, qnrA7, mepAB —multi-drug efflux pump, mfpA—qnr homolog, emrD - multi-drug efflux pump, and mtdN —multi-drug resistance protein), polymyxin ( eptA ), chloramphenicol ( cat ). Also, Delannoy et al (2022) found Shewanella spp. ( S. indica, S, haliotis, S. algae , and unidentified Shewanella spp.)…”

“…Although global seafood production is growing yearly, systematic surveillance of AMR in this sector is mainly lacking or has only been at a pilot study level. One main reason for this gap is the difficulties of investigating AMR phenotypes of marine bacteria, which often have specific growth requirements, and often lack standardized antimicrobial susceptibility testing methods and interpretation criteria ( Delannoy et al, 2022 ). Among the most frequently found bacteria in bivalve molluscs are those belonging to the Proteobacteria phylum ( Pierce and Ward, 2019 ).…”

“…A work published in 2022 by Delannoy et al (2022) concerned the presence of ARGs and mobile genetic elements (MGE) in the marine environment, including seafood. The team tested 268 seafood isolates of the bacterial microflora for the presence of 41 ARGs and 33 MGEs, including plasmid replicons, integrons, and insertion sequences in Gram-negative bacteria.…”

Antibiotic Resistance Profiles and ARG Detection from Isolated Bacteria in a Culture-Dependent Study at the Codfish Industry Level

“…Many authors, based on the biomolecular ARG trades between food matrices and human microbiota, performed PCR assays, amplifying numerous genetic determinants from many bacterial species isolated in fresh fillets (fish and mammalian species). Molecular tests were designed to discover nucleotide sequences, which codify resistance against the most frequently administrated antibiotic molecules in veterinary and human medicine (i.e., tetracyclines, beta-lactams, and quinolones) [ 10 , 11 , 12 , 13 ]. They discovered the phenotypic expression of many antibiotic resistance patterns against the veterinary legally permitted molecules and the so-called Critical Important Antimicrobials (CIA), whose usage is strongly indicated for humans only [ 2 ].…”

Antibiotic Resistance Properties among Pseudomonas spp. Associated with Salmon Processing Environments

“…isolated from retail seafood revealed that 98% of the tested isolates were resistant to three or more antibiotics [ 18 ]. However, in general, the occurrence of antimicrobial-resistant bacteria in seafood isolates decreases with increasing distance from possible sources of fecal contamination [ 19 ].…”