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Adaptation to biocides cetrimide and chlorhexidine in bacteria from organic foods: association with tolerance to other antimicrobials and physical stresses Rebeca Gadea, Nicolas Glibota, Ruben Pérez Pulido, Antonio Galvez, and Elena Ortega J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b04650 • Publication Date (Web): 08 Feb 2017 Downloaded from http://pubs.acs.org on February 9, 2017
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Journal of Agricultural and Food Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.
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Journal of Agricultural and Food Chemistry
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Adaptation to biocides cetrimide and chlorhexidine in bacteria from organic foods:
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association with tolerance to other antimicrobials and physical stresses
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Rebeca Gadea, Nicolás Glibota, Rubén Pérez Pulido, Antonio Gálvez*, Elena Ortega
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Área de Microbiología. Departamento de Ciencias de la Salud. Facultad de Ciencias
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Experimentales. Universidad de Jaén. 23071-Jaén, Spain.
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*Corresponding author. Present address: Área de Microbiología. Departamento de
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Ciencias de la Salud. Facultad de Ciencias Experimentales. Edif. B3. Universidad de
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Jaén. Campus Las Lagunillas s/n. 23071-Jaén, Spain. Tel.: 34-953-212160; fax: 34-
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953-212943.
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E-mail address:
[email protected] 14
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ABSTRACT
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Chlorhexidine (CH) and quaternary ammonium compounds (QAC), like cetrimide (CE)
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are widely used as disinfectants because of their broad antimicrobial spectrum.
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However, their frequent use for disinfection in different settings may promote bacterial
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drug resistance against both biocides and clinically relevant antibiotics. This study
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analyzes the effects of step-wise exposure to cetrimide (CE) and chlorhexidine (CH) of
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bacteria from organic foods and previously classified as biocide-sensitive. Gradual
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exposure of these strains to biocides resulted in mainly transient decreased antimicrobial
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susceptibility to other antibiotics and to biocides. Biocide-adapted bacteria also exhibit
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alterations in physiological characteristics, mainly decreased heat tolerance, or gastric
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acid tolerance in CE-adapted strains, while bile resistance does not seem to be
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influenced by biocide adaptation. Results from this study suggest that changes in
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membrane fluidity may be the main mechanism responsible for the acquisition of stable
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tolerance to biocides.
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Keywords: biocides; chlorhexidine; cetrimide; adaptation; antibiotics; resistance genes
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Journal of Agricultural and Food Chemistry
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INTRODUCTION
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Biocides have been extensively used as disinfectants by the pharmaceutical, chemical,
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and food industries. Non-negligible amounts are also used for domestic and
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environment purposes. Chlorhexidine (CH) and quaternary ammonium compounds
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(QAC), as cetrimide (CE) are biocides widely used because of their broad antimicrobial
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spectrum.
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Chlorhexidine is likely the most widely used biocide in antiseptic products,
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particularly in handwashing and oral products1 but also as a disinfectant and
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preservative. The uptake of chlorhexidine by bacteria occurs within 20 seconds2,
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causing damage of the outer cell layers.3 At the cytoplasmic membrane level,
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chlorhexidine has a biphasic effect on membrane permeability, inducing an initial high
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rate of leakage that is followed by a reduction of leakage as the concentration of
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chlorhexidine increases, due to coagulation of the cytosol.4
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QACs have also been used for a variety of clinical purposes, and for hard-
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surface cleaning and deodorization. QACs cause a loss of integrity and structural
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organization of the bacterial cytoplasmic membrane,5,6 together with other cell
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damaging effects.7 The QAC cetrimide has also been found to discharge the pH
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component of the Proton Motive Force at bacteriostatic concentrations.8 Unlike
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chlorhexidine, however, no biphasic effect on protoplast lysis has been observed.
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While the strong killing effects of biocides are clinically and industrially
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beneficial, their frequent use may lead to the development of bacterial drug resistance9
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not only against biocides but also against clinically relevant antibiotics.10 In fact,
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reduced sensitivity to chlorhexidine digluconate, benzalkonium chloride and Irgasan(®)
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has been associated with resistance to carbapenems, aminoglycosides, tetracycline and
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ciprofloxacin in Acinetobacter baumannii in previous studies.11 We have also
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previously described increased tolerance to biocides and antibiotics in bacteria from
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organic foods after exposure to phenolic biocides.12 Moreover, methods for the
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detection of antibiotic residues in foods continue to be described.13 Even so, residues of
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biocides can cause an increase in the dissemination and accumulation of antibiotic-
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resistant bacteria or antibiotic resistance genes along the food chain.
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This study aimed at analyzing the effects of step-wise exposure of biocide-
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sensitive bacteria from organic foods to chlorhexidine (CH) and cetrimide (CE), and to
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determine possible modifications in sensitivity to antibiotics, environmental stress and
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membrane fluidity following adaptation to these biocides.
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MATERIAL AND METHODS
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Antimicrobials. The biocides and antibiotics employed for the assays together
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with their commercial suppliers were described in a previous study:12 benzalkonium
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chloride
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didecyldimethylammonium bromide (AB), triclosan (TC), hexachlorophene [2,2′-
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methylenebis(3, 4,6-trichlorophenol)] (CF), chlorhexidine (CH), ampicillin (AM),
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ceftazidime (CAZ), cefotaxime (CTX), imipenem (IPM), ciprofloxacin (CIP),
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sulfamethoxazol (SXT), sulfamethoxazol/trimethoprim (TMP/SXT), tetracycline (TE),
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and nalidixic acid (NA) (for Salmonella).
(BC),
cetrimide
(CE),
hexadecylpyridinium
chloride
(HDP),
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Bacterial strains. A collection of 76 biocide-sensitive strains previously
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isolated from 39 foods certified as organically grown according to European regulations
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(http://www.wipo.int/edocs/lexdocs/laws/en/eu/eu122en.pdf)14 were used in this study
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(Table S1). Strains were grown in Brain Heart Infusion (BHI) broth (Scharlab,
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Barcelona, Spain) for 15 h at 37°C.
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Strain identification. Most strains were identified in previous studies.13 Only
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Chryseobacterium sp. UJA48q was identified in the present study, using the
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methodology previously reported.15,16
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Strain adaptation to biocides. Strains were adapted to the biocides cetrimide
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(CE) and chlorhexidine (CH) by serial inoculation on Trypticase Soy Broth (TSB;
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Scharlab) supplemented with a range of concentrations of the biocides as described
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previously.12,17 The suspensions from the last tubes with recorded bacterial growth were
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seeded on TSA plates and the bacterial colonies were collected, resuspended in 1 ml
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BHI broth (Scharlab) containing 20% glycerol and stored at -80 °C. The stability of the
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adaptive tolerance and minimum inhibitory concentrations (MICs) after 5, 10, 15 and 20
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passages were also determined as described previously.12
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Determination of adaptive tolerance, sensitivity to biocides and antibiotics.
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The MICs for other biocides and for antibiotics of wildtype, CE- and CH-adapted
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strains were determined in TSB (Scharlab) by the broth microdilution method using 96-
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well microtiter plates inoculated in triplicate with bacterial suspensions adjusted to
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5x105 colony-forming units (CFU)/ml as described previously.12 For biocide dilutions
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with high turbidity, the minimum bactericidal concentration (MBC) was determined.12
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The final concentrations used for antibiotics correspond to the MICs and twice
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the MICs interpretative standard of antibiotic-resistant strains defined by Clinical and
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Laboratory Standards Institute.18
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PCR detection of resistance genes. The following efflux pump genes were
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investigated by PCR amplification: acrB and mdfA,19; sugE,20 yhiUV and evgA,21
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emeA,22 qacA/B, qacC, qacG, qacH and qacJ,23 qacE∆1,24 efrA and efrB,
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mepA, norA, norB, norC, sdrM and sepA.26
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mdeA,
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Genes conferring resistance to β-lactam antibiotics (bla, mecA), chloramphenicol
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(cat),26,27 aminoglycosides (aac(6_)-Ie-aph(2_)-Ia, aph(2_)-Ib, aph(2_)-Ic, aph(2_)-Id,
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aph(3_)-IIIa, and ant(4_)-Ia genes,28 macrolides (the ermA, ermB, ermC, ereA, ereB,
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msrA/B, mphA, and mefA genes),29 and lincosamide and streptogramin A (lsa gene)30,31
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were also investigated.
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Determination of the role of efflux pumps in biocide resistance. Gram-
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positive strains showing high-resistance phenotype were inspected for restored
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sensitivity to the compounds in the presence of 25 µg/ml reserpine (Sigma-Aldrich) as
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efflux pump inhibitor.32
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Determination of growth capacity. Growth capacity of wildtype and biocide-
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adapted strains was determined on 96-well microtiter plates inoculated in triplicate with
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approx. 104 CFU/ml of each tested strain as described previously.12 The corresponding
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sterility controls were included.
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Determination of heat tolerance. Bacterial cell suspensions (approx. 106
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CFU/ml in 2 ml peptone water) in triplicate were incubated for 5 min at 60, 65 and 70
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°C.12,33 After heating, samples were serially diluted and surface-plated on TSA to
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determine viable cell concentrations.
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Determination of resistance to simulated gastric fluid and bile salts.
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Wildtype and biocide-adapted cells (approx. 106 CFU/ml in TSB) were tested in
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triplicate for survival in simulated gastric acid fluid and bile salts according to Kim et
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al.34 and Gadea et al.12 Viable cell counts were determined at time zero and after
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incubations by serial dilution and surface plating on TSA.
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Fluorescent anisotropy measurements. Membrane fluidity of intact whole
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cells was measured by fluorescence anisotropy with the hydrophobic probe 1,6-
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diphenyl-1,3,5-hexatriene (DPH) (Sigma-Aldrich, Madrid, Spain) as described by
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Alonso-Hernando, Alonso-Calleja, and Capita (2010).36 The determinations of
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fluorescence anisotropy (Cary Eclipse Fluorescence Spectrophotometer; Varian Inc.,
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CA, USA) were optimised as described previously12.
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Statistical analysis. Data on heat tolerance, growth capacity and fluorescence
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anisotropy were compared with a Student t-test (Statgraphics Centurion Version XVII-
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X64; StatPoint Technologies, Inc., USA) in order to detect statistically significant
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differences. Principal component analysis (PCA) with Pearson correlation coefficient (r)
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was used to analyze biocide tolerance data after exposure to CE or CH (MATLAB
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R2011b 7.13 version; MathWorks, USA). Significant correlations (P
