Insusceptibility to disinfectants in bacteria from animals, food and humans – is there a link to antimicrobial resistance?

ORIGINAL RESEARCH ARTICLE published: 18 March 2014 doi: 10.3389/fmicb.2014.00088 Insusceptibility to disinfectants in bacteria from animals, food and humans—is there a link to antimicrobial resistance? Karin Schwaiger 1† , Katrin S. Harms 1 , Meike Bischoff 1 , Petra Preikschat 2 , Gabriele Mölle 2 , Ilse Bauer-Unkauf 2 , Solveig Lindorfer 1 , Sandra Thalhammer 1 , Johann Bauer 1 and Christina S. Hölzel 1* † 1 2 Animal Hygiene, Wissenschaftszentrum Weihenstephan für Ernährung, Landnutzung und Umwelt, Technische Universität München, Freising, Germany Bavarian Health and Food Safety Authority (LGL), Erlangen, Germany Edited by: Mark Montforts, National Institute for Public Health and the Environment, Netherlands Reviewed by: Atte Von Wright, University of Eastern Finland, Finland Rob De Jonge, National Institute for Public Health and the Environment, Netherlands Sara V. Lundstrom, University of Gothenburg, Sweden *Correspondence: Christina S. Hölzel, Animal Hygiene, Wissenschaftszentrum Weihenstephan für Ernährung, Landnutzung und Umwelt, Technische Universität München, Weihenstephaner Berg 3, 85354 Freising, Germany e-mail: christina.hoelzel@ wzw.tum.de † Present address: Karin Schwaiger, Food Safety, Department of Veterinary Science, Faculty of Veterinary Medicine, LMU München, Oberschleißheim, Germany; Christina S. Hölzel, Hygiene and Technology of Milk, Department of Veterinary Science, Faculty of Veterinary Medicine, LMU München, Oberschleißheim, Germany Enterococcus faecalis (n = 834) and Enterococcus faecium (n = 135) from blood and feces of hospitalized humans, from feces of outpatients and livestock and from food were screened for their susceptibility to a quaternary ammonium compound (didecyldimethylammoniumchloride, DDAC) and to 28 antibiotics by micro-/macrodilution. The maximum DDAC-MIC in our field study was 3.5 mg/l, but after adaptation in the laboratory, MIC values of 21.9 mg/l were observed. Strains for which DDAC had MICs > 1.4 mg/l (“non-wildtype,” in total: 46 of 969 isolates/4.7%) were most often found in milk and dairy products (14.6%), while their prevalence in livestock was generally low (0–4%). Of human isolates, 2.9–6.8% had a “non-wildtype” phenotype. An association between reduced susceptibility to DDAC, high-level-aminoglycoside resistance and aminopenicillin resistance was seen in E. faecium (p < 0.05). No indications for a common source of non-wildtype strains were found by RAPD-PCR; however, several non-wildtype E. faecalis shared the same variant of the emeA-gene. In addition, bacteria (n = 42) of different genera were isolated from formic acid based boot bath disinfectant (20 ml of 55% formic acid/l). The MICs of this disinfectant exceeded the wildtype MICs up to 20-fold (staphylococci), but were still one to three orders of magnitude below the used concentration of the disinfectant (i. e., 1.1% formic acid). In conclusion, the bacterial susceptibility to disinfectants still seems to be high. Thus, the proper use of disinfectants in livestock surroundings along with a good hygiene praxis should still be highly encouraged. Hints to a link between antibiotic resistance and reduced susceptibility for disinfectants—as seen for E. faecium—should be substantiated in further studies and might be an additional reason to confine the use of antibiotics. Keywords: disinfectant, aminoglycoside biocide, INTRODUCTION Prevention of zoonoses—including the spread of antimicrobial resistant bacteria—is mainly a question of how to reduce the prevalence of contagious microorganisms. Whenever actions are taken to reduce the spread of bacteria in livestock, effective disinfection is crucial. Antimicrobial resistance is basically increasing whenever resistant bacteria are selected by antimicrobial use (Bronzwaer et al., 2002; Lipsitch and Samore, 2002; Livermore, 2005). A certain antibiotic agent might directly select for resistance directed against itself, might indirectly (cross-)select for cross-resistance to chemically related agents and/or might (co-)select for coresistance to unrelated substances (Shah, 2005), the latter based e.g., on co-transfer of resistance genes due to genetic linkage www.frontiersin.org antiseptic, antimicrobial resistance, QAC, formic acid, enterococci, on mobile elements. Besides co-selection posed by antibiotic agents, diverse other co-selectors are discussed, e.g., heavy metal ions (Berg et al., 2005; Baker-Austin et al., 2006; Hölzel et al., 2012), pesticides (Bordas et al., 1997), or disinfectants (Levy, 2000). For several bacterial species, including methicillin resistant Staphylococcus aureus, a link between resistance against antibiotics and reduced susceptibility for disinfectants has been described in the past (Heir et al., 1999; Bjorland et al., 2001, 2005; Sidhu et al., 2002a). At the same time, other studies did not find clear indications for co-resistance against antibiotics and disinfectants (Suller and Russell, 2000; Loughlin et al., 2002). Enterococci—apart from VRE—have rarely been investigated for this correlation up to now, despite the fact that enterococci are emerging—meanwhile maybe better called: March 2014 | Volume 5 | Article 88 | 1 Schwaiger et al. Link of biocide insusceptibility and antimicrobial resistance faecium (n = 130) from diverse sources (hospital/food industry/farm animals) for their susceptibility to a disinfectant (didecydimethylammoniumchlorid, DDAC) and 22 antibiotic agents. The distribution of strains with DDAC-MICs > or < 1.4 mg/l within antibiotic resistant and susceptible strains was assessed. In case of elevated DDAC-MIC-values strains were investigated for clonal relationship using amplicon-typing (RAPD-PCR). In several isolates, the emeA-genotype, coding for a multidrug efflux transporter, was further investigated by melting curve analysis and sequencing. The higher the initial MIC of strains the more rapidly they might be adapted to rising concentrations of disinfectants, as shown by Sidhu et al. (2002b). Therefore, we performed additionally an exploratory adaptation test with the DSM 2570 reference strain and three strains with DDAC_MICs > 1.4 mg/l. To have a first glance on real life conditions, different bacterial species were additionally isolated from the disinfectant fluid of boot baths and tested for their MIC-values for DDAC, formic acid and antibiotics. “emerged”—nosocomial pathogens (Tailor et al., 1993; Willems and van Schaik, 2009) with a high recombination potential (Aarestrup et al., 2002; Leavis et al., 2006; Palmer and Gilmore, 2010; Schwaiger et al., 2011, 2012). Acquired insusceptibility to disinfectants can be conferred by newly acquired genes (like qac-genes, Poole, 2002) or by mutations, e.g., of intrinsic multidrug transporters (like norA, emeA, Kaatz et al., 1993; Ng et al., 1994; Jonas et al., 2001) or of target structures (e.g., cell membranes), although the latter is described to be a rare event with biocides (Poole, 2002). To slight (...truncated)