Adaptation to Biocides Cetrimide and Chlorhexidine in Bacteria from Organic Foods: Association with Tolerance to Other Antimicrobials and Physical Stresses.

Chlorhexidine (CH) and quaternary ammonium compounds (QAC), such as cetrimide (CE), are widely used as disinfectants because of their broad antimicrobial spectrum. However, their frequent use for disinfection in different settings may promote bacterial drug resistance against both biocides and clinically relevant antibiotics. This study analyzes the effects of stepwise exposure to cetrimide (CE) and chlorhexidine (CH) of bacteria from organic foods and previously classified as biocide-sensitive. Gradual exposure of these strains to biocides resulted in mainly transient decreased antimicrobial susceptibility to other antibiotics and to biocides. Biocide-adapted bacteria also exhibit alterations in physiological characteristics, mainly decreased heat tolerance, or gastric acid tolerance in CE-adapted strains, while bile resistance does not seem to be influenced by biocide adaptation. Results from this study suggest that changes in membrane fluidity may be the main mechanism responsible for the acquisition of stable tolerance to biocides.

Effects of exposure to quaternary-ammonium-based biocides on antimicrobial susceptibility and tolerance to physical stresses in bacteria from organic foods.

In the present study, a collection of 76 biocide-sensitive bacterial strains isolated from organically produced food were adapted by repeated exposure to increasing concentrations of the quaternary ammonium compounds (QACs) benzalkonium chloride (BC) and hexadecylpyridinium chloride (HDP). The sensitivity of both wildtype strains and their corresponding QAC-adapted strains to other biocides and to antibiotics was studied. QAC tolerance increased in 88.2% of strains for BC and in 30.3% of strains for HDP, with increases in minimum inhibitory concentrations between 2 and over 100 fold. Adaptive resistance was stable after 20 subcultures in biocide-free medium for 7 and 5 of the BC- and HDP-adapted strains, respectively. Adaptation to BC and HDP also reduced the susceptibility to other biocides, mainly hexachlorophene (CF), didecyldimethylammonium bromide (AB), triclosan (TC) and chlorhexidine (CH). BC-adapted strains showed increased antibiotic resistance to ampicillin (AM) followed by sulfamethoxazol (SXT) and cefotaxime (CTX), and some showed increased sensitivity to ceftazidime (CAZ), CTX, AM and STX. Changes in antibiotic resistance in HDP-adapted strains were more heterogeneous and strain-dependent. Main efflux pump genes detected in QAC-adapted strains were acrB, sugE, norC, qacE and qacH, as well as antibiotic resistance genes aac(6_)-Ie-aph(2_)-Ia, aph(2_)-Ic, ant(4_)-Ia, lsa, mrsA/B, ereA, ermB and cat. Membrane anisotropy experiments revealed that QAC adaptation induced an increase in membrane rigidity in the case of BC, while response to HDP was more heterogeneous and strain-dependent. Growth capacity was significantly higher in some QAC-adapted strains and strain-dependent changes in heat tolerance were also detected in QAC-adapted strains. Gastric acid or bile resistances do not seem to be influenced by QAC adaptation.

Adaptive tolerance to phenolic biocides in bacteria from organic foods: Effects on antimicrobial susceptibility and tolerance to physical stresses.

The aim of the present study was to analyze the effects of step-wise exposure of biocide-sensitive bacteria from organic foods to phenolic biocides triclosan (TC) and hexachlorophene [2,2'-methylenebis(3,4,6-trichlorophenol)] (CF). The analysis included changes in the tolerance to the biocide itself, the tolerance to other biocides, and cross-resistance to clinically important antibiotics. The involvement of efflux mechanisms was also studied as well as the possible implication of modifications in cytoplasmic membrane fluidity in the resistance mechanisms. The influence of biocide tolerance on growth capacity of the adapted strains and on subsequent resistance to other physical stresses has also been analyzed. Repeated exposure of bacteria from organic foods to phenolic biocides resulted in most cases in partially increased tolerance to the same biocide, to dissimilar biocides and other antimicrobial compounds. Nine TC-adapted strains and six CF-adapted strains were able to develop high levels of biocide tolerance, and these were stable in the absence of biocide selective pressure. Most strains adapted to TC and one CF-adapted strain showed significantly higher anisotropy values than their corresponding wildtype strains, suggesting that changes in membrane fluidity could be involved in biocide adaptation. Exposure to gradually increasing concentrations of CF induced a decrease in heat tolerance. Biocide adaptation had no significant effects of gastric acid or bile resistance, suggesting that biocide adaptation should not influence survival in the gastrointestinal tract.

Synergistic activity of biocides and antibiotics on resistant bacteria from organically produced foods.

Synergism between biocides and antibiotics was investigated in 20 biocide and antibiotic-resistant bacterial strains that were previously isolated from organically produced foods, according to their antimicrobial resistance profiles. Most of the antibiotic/biocide combinations yielded synergistic interactions, reducing the inhibitory concentrations of biocides and antibiotics by 4- to 16-fold. Among enterococci, synergism with biocides was detected for amoxicillin (AM), cefuroxime (CX), erythromycin (EM), ciprofloxacin (CP), and trimethoprim/sulphametoxazol (T/S). Among staphylococci, interactions were synergistic (AM) and either synergistic or indifferent (CX and EM, depending on biocide). Among the three methicillin-resistant Staphylococcus aureus clinical strains included in the study, the combinations of methicillin and triclosan or hexachlorophene acted synergistically in all strains, but interactions were either synergistic or indifferent for the other biocides, depending on the strain. All combinations tested were synergistic for Lactobacillus (AM, CX, EM, and CP) and Micrococcus (AM, EM). In Salmonella, interactions were indifferent (AM, CX, EM, and CP) or synergistic (T/S). Synergism with biocides was also detected in Klebsiella isolates (AM, CX, and T/S), Enterobacter sp. (AM, CX, EM, and T/S), Pantoea (AM, CX, EM, CP, and T/S), and Chryseobacterium sp. (EM). These results suggest that combinations of biocides and antibiotics may open new possibilities to combat antimicrobial resistance.