Difloxacin (DIF) belongs to the class of fluoroquinolones, a group of synthetic antibiotics commonly used in human and veterinary medicine for the treatment of both Gram-negative and Gram-positive infections (Aarestrup, 2005; Cabello, 2006; Adriaenssens et al., 2011). They inhibit the essential bacterial enzymes DNA gyrase and DNA topoisomerase IV (Drlica & Zhao, 1997). In recent years, the resistance against fluoroquinolones has increased and limited the treatment of bacterial infections (Dalhoff, 2012). The use of fluoroquinolones in animal husbandry is assumed to contribute to the emergence and spread of antimicrobial resistance (Collignon et al., 2009), and recently, a potential transfer of plasmid-mediated quinolone resistance genes from wastewater of swine feedlots to adjacent fields was described (Li et al., 2012). After oral application to the pigs, only a minor part of DIF is metabolized by the animal, and the main part of the parent compound is excreted in bioactive concentrations (Sukul et al., 2009). In manure, DIF is very stable during storage (Lamshöft et al., 2010) and reaches agricultural fields where it may affect the structure and function of the microbial community and may lead to an increased abundance and transfer of antibiotic resistance genes. Recently, in the field experiment reported here and in other trials, it was shown that DIF was hardly (bio) accessible and rapidly formed nonextractable residues, which might limit its effect on the microbial community (Rosendahl et al., 2012). In another experiment, 14C-labeled DIF was applied with manure or water to soil, and the mineralization of 14C-DIF was shown to be low (below 0.2%), while nonextractable residues of 14C-DIF increased to 70–74% after 56 and 120 days (Junge et al., 2012). Nevertheless, in a microcosm experiment, effects of DIF on the microbial biomass, respiration, potential denitrification, and ratio of bacteria/fungi were observed up to eight days after application by manure (Kotzerke et al., 2011). To date, five different transferable mechanisms of quinolone resistance (TMQR) are described in the literature including quinolone target protection (qnr genes), enzymatic inactivation (ACC(6′)-Ib-cr), plasmid-mediated effects on slow growth, plasmid-encoded efflux pumps, and exogenous exchange of DNA (reviewed by Ruiz et al., 2012b). Qnr genes have been found in a large variety of plasmids (Strahilevitz et al., 2009). However, DNA sequences in the vicinity of qnr genes were reported to be rather similar and usually integrated in complex sul1-type integrons and associated with insertion sequence common region 1 (ISCR1) (Nordmann & Poirel, 2005; Garnier et al., 2006). Integrons can be used by Gram-positive and Gram-negative bacteria to stockpile and express different exogenous open reading frames including resistance genes and are considered to play a central role in the worldwide problem of antibiotic resistance (Mazel, 2006; Gillings et al., 2008a). The presence of antibiotics such as fluoroquinolones can lead to the activation of the SOS response in bacteria, which results in integrase overexpression and correspondingly in the raise of recombination events of gene cassettes (Stalder et al., 2012). Therefore, the abundance of class 1 integrons and related sul genes in a microbial community might be used as an estimate of potential coselection of resistance gene cassettes. In addition, an induction of natural competence in response to fluoroquinolone antibiotics was described (Charpentier et al., 2012), which might contribute to the spread of resistance genes in bacterial populations via transformation. Therefore, we hypothesize that despite the reported rapid formation of nonextractable residues of DIF in soil (Rosendahl et al., 2012), the application of manure from DIF-treated pigs will affect the structure of the bacterial communities in field soil as well as the abundance of resistance genes and mobilizing elements such as integrons compared with soil treated with manure from unmedicated pigs, which might contribute to the public health risk of antibiotic resistance.
The aim of this field study was to assess the effects of manure from DIF-treated pigs on the bacterial community structure in bulk soil and rhizosphere of maize by denaturing gradient gel electrophoresis (DGGE) of 16S rRNA gene fragments amplified from total community (TC) DNA. Detection of the quinolone resistance genes qnrA, qnrB, and qnrS1/qnrS2 was performed by PCR from TC DNA with subsequent Southern blot hybridization. Sulfadiazine resistance genes sul1 and sul2 as well as class 1 integron-integrase genes (intI1) and traN specific for LowGC-type plasmids, which were recently described to play an important role in dissemination of resistance against sulfonamides in manure and manure-treated soils (Heuer et al., 2009), were quantified in TC DNA by real-time PCR as marker for a potential coselection and transferability of resistance genes.