Marine Bivalve Mollusks As Possible Indicators of Multidrug-Resistant Escherichia coli and Other Species of the Enterobacteriaceae Family

Abstract

The mechanisms for the development and spread of antibacterial resistance (ABR) in bacteria residing in environmental compartments, including the marine environment, are far from understood. The objective of this study was to examine the ABR rates in Escherichia coli and other Enterobacteriaceae isolates obtained from marine bivalve mollusks collected along the Norwegian coast during a period from October 2014 to November 2015. A total of 549 bivalve samples were examined by a five times three tube most probable number method for enumeration of E. coli in bivalves resulting in 199 isolates from the positive samples. These isolates were identified by biochemical reactions and matrix Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry, showing that 90% were E. coli, while the remaining were species within the genera Klebsiella, Citrobacter, and Enterobacter. All 199 isolates recovered were susceptibility tested following the European Committee on Antimicrobial Susceptibility Testing disk diffusion method. In total, 75 of 199 (38%) isolates showed resistance to at least one antibacterial agent, while multidrug-resistance were seen in 9 (5%) isolates. One isolate conferred resistance toward 15 antibacterial agents. Among the 75 resistant isolates, resistance toward extended-spectrum penicillins (83%), aminoglycosides (16%), trimethoprim (13%), sulfonamides (11%), tetracyclines (8%), third-generation cephalosporins (7%), amphenicols (5%), nitrofurans (5%), and quinolones (5%), were observed. Whole-genome sequencing on a selection of 10 E. coli isolates identified the genes responsible for resistance, including bla_CTX-M genes. To indicate the potential for horizontal gene transfer, conjugation experiments were performed on the same selected isolates. Conjugative transfer of resistance was observed for six of the 10 E. coli isolates. In order to compare E. coli isolates from bivalves with clinical strains, multiple-locus variable number tandem repeats analysis (MLVA) was applied on a selection of 30 resistant E. coli isolates. The MLVA-profiles were associated with community-acquired E. coli strains causing bacteremia. Our study indicates that bivalves represent an important tool for monitoring antibacterial resistant E. coli and other members of the Enterobacteriaceae family in the coastal environment.

Keywords: Enterobacteriaceae; Escherichia coli; antibacterial resistance; bivalve mollusks; horizontal gene transfer.

FIGURE 1

Number of E. coli and other bacteria in the Enterobacteriaceae family showing phenotypic resistance to antibacterial agents applied in accordance with the EUCAST, CLSI, and ICMR clinical breakpoint tables. The two E. coli isolates B177 and B184 (marked by arrows) displayed resistance against nine or more antibacterial agents.

FIGURE 2

Minimal spanning tree showing the phylogenetic relationships between 212 E. coli isolated from blood stream infection (BSI) according to patient outcome (non-severe, 3failure = organ failure ≥3 organs within one day of admission to hospital, death14d = death within 14 days of admittance to hospital), 38 ECOR strains of human origin with main phylogroup, 30 E. coli isolates from bivalves, and four HUS-associated EHEC strains. The distance between circles are indicated by the thickness and dotting of lines, hence a thicker line indicate a closer relation than a thin line, and a thin line indicate a closer relation than a dotted line. Shared MLVA-profiles are shown as shared circles.