A Ternary Copper (II) Complex with 4-Fluorophenoxyacetic Acid Hydrazide in Combination with Antibiotics Exhibits Positive Synergistic Effect against Salmonella Typhimurium

Abstract

Salmonella spp. continues to figure prominently in world epidemiological registries as one of the leading causes of bacterial foodborne disease. We characterised 43 Brazilian lineages of Salmonella Typhimurium (ST) strains, characterized drug resistance patterns, tested copper (II) complex as control options, and proposed effective antimicrobial measures. The minimum inhibitory concentration was evaluated for seven antimicrobials, isolated and combined with the copper (II) complex [Cu(4-FH)(phen)(ClO₄)₂] (4-FH = 4-fluorophenoxyacetic acid hydrazide and phen = 1,10-phenanthroline), known as DRI-12, in planktonic and sessile ST. In parallel, 42 resistance genes were screened (PCR/microarray). All strains were multidrug resistant (MDR). Resistance to carbapenems and polymyxins (86 and 88%, respectively) have drawn attention to the emergence of the problem in Brazil, and resistance is observed also to CIP and CFT (42 and 67%, respectively), the drugs of choice in treatment. Resistance to beta-lactams was associated with the genes bla_TEM/bla_CTX-M in 39% of the strains. Lower concentrations of DRI-12 (62.7 mg/L, or 100 μM) controlled planktonic and sessile ST in relation to AMP/SUL/TET and AMP/SUL/TET/COL, respectively. The synergistic effect provided by DRI-12 was significant for COL/CFT and COL/AMP in planktonic and sessile ST, respectively, and represents promising alternatives for the control of MDR ST.

Keywords: food safety; multi drug resistance (MDR); salmonellosis.

Figure 1

The effect of antimicrobial treatment, with and without addition of the copper complex, on the free and biofilm forms of five ST strains. The results represent means with the standard deviation (error bars) of three independent repetitions with three replicates. +DRI-12: supplementation with [Cu(4-FH)(phen)(ClO₄)₂] at concentrations of 10 and 45 μM, respectively, for planktonic (PL) and biofilm (BF) forms of ST. ATM concentration: antimicrobial concentration. 1–14: doubling and increasing variations in concentrations for each antimicrobial. AMP: ampicillin (1: <0.5 μg/mL, 14: >1024 μg/mL); TET: tetracycline (1: <0.5 μg/mL, 14: >1024 μg/mL); MER: meropenem (1: <0.25 μg/mL, 14: >512 μg/mL); COL: colistin (1: <0.25 μg/mL, 14: >512 μg/mL); CFT: ceftriaxone (1: <0.125 μg/mL, 14: >256 μg/mL); CIP: ciprofloxacin (1: <0.0078 μg/mL, 14: >16 μg/mL); and SUL: sulfisoxazole (1: <16 μg/mL, 12: >8192 μg/mL). Red line: cutoff point according to CLSI (2021). ns: no statistical difference in the analysis between strains for each treatment. * p < 0.05; using Mann–Whitney test.

Figure 2

SEM images of biofilms with and without 100 μM of the copper complex treatment in two strains of ST. (A,B) control group with normal biofilm structure; (C) group treated with DRI-12, demonstrating the presence of a thick layer of extracellular matrix, and razing of the matrix structure and bacterial exposure; and (D) treatment with DRI-12, with biomass fragmentation.

Figure 3

The chemical structure of [Cu(4-fh)(phen)(ClO₄)₂].