Detection of antibiotic resistant bacteria inhabiting the sand of non-recreational marine beach Zbigniew Mudryk, Piotr Perlin ´ ski, Piotr Skórczewski * Department of Experimental Biology, Pomeranian Academy, Arciszewskiego 22 B, 76-200 Słupsk, Poland article info Keywords: Marine beach Heterotrophic bacteria Antibiotic resistance abstract The present study examined the antibiotic resistance of heterotrophic bacteria, which were isolated from the sand of the beach located in the National Park of the southern Baltic Sea coast. The bacteria demon- strated low levels of antibiotic resistance. These microorganisms were the most resistant to cefaclor and clindamycin and the most sensitive to clarithromycin, doxycycline, gentamycin and oxytetracycline. The majority of bacteria inhabiting the sand of the studied beach were resistant to only one antibiotic out of 18 tested antibiotics in this study. The bacteria inhabiting the middle part of the beach and the dune were more antibiotic resistant than bacteria isolated from the seawater and the shoreline-seawater contact zone. Generally, there was no significant difference in antibiotic resistance between bacteria isolated from the surface and the subsurface sand layers. The bacterial antibiotic resistance level depends on the chemical structure of antibiotics. Ó 2009 Elsevier Ltd. All rights reserved. 1. Introduction In the past few decades the uncontrolled and extensive use of pharmaceutical substances mainly antibiotics in human and veter- inary medicine, animal husbandry, agriculture and aquaculture has caused the increased introduction of those antimicrobial agents in the aquatic environment (Hirsch et al., 1999; Metcalfe et al., 2003; Dang et al., 2008). The presence of antibiotics and their metabolites was observed in various aquatic ecosystems including rivers, lakes, estuaries, coastal area and sea (Le and Munekage, 2004; Costanzo et al., 2005; Alpay-Karaoglu et al., 2007; Gulkowska et al., 2007). The concentration of antibiotics in different water basins ranges from 0.01 to 12.9 lg dm À3 (Backhaus and Grimme, 1999; Batt et al., 2006). The primary source of antibiotics in natural environ- ments is the extraction of incompletely metabolized antibiotics by humans and animals. Depending on the type of antibiotic, be- tween 30% and 90% of an administered dose of most antibiotics gi- ven to humans and animals are extracted in the urine or feces as the active substance and introduced to the sewage system, soil and water basins (Biyela et al., 2004; Costanzo et al., 2005). The results of investigations with the use of test systems indicate that a number of antibiotics is poorly or not biodegradable in the water basins and those antimicrobial agents showed direct toxic effects against many aquatic organisms (Kümmerer et al., 2000; Le and Munekage, 2004; Gulkowska et al., 2007; Tamtam et al., 2008). As a consequence of the widespread use of antibiotics by hu- mans, dramatic and global increase is observed in aquatic environ- ments in reference to the number of antibiotics resistant bacteria, multiple antibiotic resistance, pathogenic bacteria resistance and reduced efficacy of antibiotic treatment for diseases caused by resistant pathogens (Meirelles-Pereira et al., 2002; Park et al., 2003; Schwartz et al., 2003; Dang et al., 2006). The simultaneous increased concentration of antibiotics in aquatic ecosystems gener- ated new selective pressures on natural bacterial populations (Kümmerer, 2004; Alpay-Karaoglu et al., 2007). The rapid increase in multiple antibiotic resistant aquatic bac- teria is due, partially to the ability of those bacteria to transfer from antibiotic resistance markers among the bacterial popula- tion by cell to cell contact. The prolonged exposure to low doses of antibiotics leads to the selective proliferation of resistant bac- teria, which could horizontally transfer genes to other bacterial species (Schwartz et al., 2003; Alanis, 2005; Batt et al., 2006; Aminov and Mackie, 2007). The agent responsible for the transfer of resistance is an extrachromosomal genetic element, termed an R-factor or R-plasmid with the size of 0.90–158.8 kb (Liu et al., 2006). Bacteria containing R-plasmids isolated from humans and animals demonstrate high frequency of occurrence. It is known that those bacteria can transfer resistance plasmids in situ to indigenous microflora. The potential of the plasmid transfer is especially significant in view of the fact that many bacteria con- taining R-plasmids, which possession is associated with antibiotic resistance, exhibit higher rates of survival in aquatic environ- ments (Baya et al., 1986). There are also other ways in which bac- teria can become resistant to antibiotics. They may develop 0025-326X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.marpolbul.2009.09.025 * Corresponding author. Tel.: +48 59 84 05 335; fax: +48 59 84 24 275. E-mail address: psps@poczta.fm (P. Skórczewski). Marine Pollution Bulletin 60 (2010) 207–214 Contents lists available at ScienceDirect Marine Pollution Bulletin journal homepage: www.elsevier.com/locate/marpolbul