Distribution of antibiotic resistance and the presence of vancomycin-resistance genes (vanA and vanB) in Enterobacteriaceae isolated from the Sea of Marmara, the Canakkale Strait and the Istanbul Strait, Turkey

Antibiotics are commonly used in human and veterinary medicine to control bacterial infections (Sarmah et al. 2006). However, due to their poor absorbance in human and animal intestines, the majority of antibiotics are excreted unchanged in feces and urine and eventually find their way into the environment (Schlusener 2006). Through a range of biochemical and physiological mechanisms, antibiotic residues cause development of resistance in bacteria and the antimicrobial resistance has recently been recognized as a worldwide ecological problem (Nordmann et al. 2005; Kaszanyitzky et al. 2007; Kemper 2008). High antibiotic loads rapidly accumulate and persist in aquatic environments (Hirsch et al. 1999) and as a result, a dramatic and global increase has been observed in the number of antibiotic-resistant bacteria, along with the multiple antibiotic resistance and pathogenic bacteria resistance. Antibiotic resistance can originate from gene mutations or horizontal transfer between phylogenetically diverse bacteria. In addition, newly acquired resistance genes may be maintained in new populations in the absence of antibiotic selection pressure. The presence of antibiotic-resistant genes, such as tet genes, van genes and sul genes, has been reported in wastewater, surface water and sediments (Pei et al. 2006; Ram et al. 2007).

The widespread use of vancomycin in treating gram positive bacterial infections throughout the world has resulted in a rapid increase in vancomycin-resistant enterococci (Riberio et al. 2006). The rapid spread of vancomycin resistance among bacteria is due to the location of van genes in movable genetic structures, such as plasmids, transposons and integrons. Therefore, vancomycin resistance has been used as a key determinant in natural environments, such as rivers, lakes and seawater (Guardabassi et al. 2000). Different vancomycin-resistance determinants have been demonstrated and classified in categories A, B, C, D, E, G. The most frequently detected vancomycin-resistance genes among the Enterobacteriaceae family are vanA and vanB, and these are the most globally widespread and prevalent phenotypes.

Although antibiotic-resistance of bacterial isolates has been studied in clinical isolates, there are limited studies on the presence of antibiotic-resistant bacteria in Turkish waters. The purpose of this study was to investigate the distribution of multiple antibiotic resistance in isolates of the Enterobacteriaceae family from the Sea of Marmara, the Canakkale Strait and the Istanbul Strait. The study was limited to gram negative bacteria, Enterobacteriaceae, due to our preliminary results that indicated only <2% of isolates belonged to Enterococcaceae and none of Enterococci isolates were resistant to vancomycin. The presence of vancomycin-resistance genes (vanA and vanB) in isolates of Enterobacteriaceae were also investigated due to the observed resistance to vancomycin.

Coastal regions around the world are characterized by higher counts of indicator bacteria (Ramaiah et al. 2004; Kalkan & Altuğ 2015) and our findings are consistent with those of other scientists. In this study, the highest level of Enterobacteriaceae species was determined in the Sea of Marmara, as compared to the Istanbul and Canakkale Straits. This result is in accordance with the previous finding that characterizes the Sea of Marmara as having higher levels of bacterial loads compared to those of the Çnakkale Strait (Çrdak et al. 2015). The Sea of Marmara is an intercontinental basin receiving discharges from the northeast, mainly through the Istanbul Strait and other river discharges which are under the influence of the most populated and developed socio-economic region in Turkey. Previous research also reported that most of the pathogenic species detected in the Sea of Marmara belonged to the Enterobacteriaceae family (Altuğ et al. 2012). In the present study, however, the levels of indicator bacteria were higher than that reported by Altuğ et al. (2012), indicating the increased deterioration of the marine environment in the region.

In this study, isolates showing resistance to the greatest number of antibiotics were identified from E. coli isolates. The level of antibiotic resistance found in the E. coli isolates was similar to that previously described in E. coli isolates from the Sea of Marmara, where 71.4% of the isolates showed resistance to one or more antibiotics. The level of E. coli β-lactam antibiotics resistance is in agreement with that described previously for GN bacteria isolated from beach sediment samples (Mudryk 2005). Significant levels of antibiotic resistance were also found in Enterobacter, Salmonella and Enterococcus isolates. Kimberly (2008) reported that in the U.S. state of Oklahoma, antibiotic resistance frequency of Salmonella isolates was higher than that of E. coli. This may be explained by the enhanced ability of Salmonella to survive in marine environments (Wait & Sobsey 2001). The higher resistance of E. coli isolates in the present study may be due to the continuous input of anthropological pollution into the Sea of Marmara. However, localities with lower levels of fecal indicators may also have a large number of resistant isolates (Altuğ & Balkis 2009). This may be due to differential transport of nutrients by deep sea discharges that creates a nutrient-limited zone in surface waters where the growth of fecal indicators is limited but antibiotic-resistant isolates persist.

In this study, the highest frequency of bacterial resistance was to kanamycin, followed by vancomycin (glycopeptide) and ampicillin (β-lactam). The lowest frequency of resistance was to ceftazidime (20%) and gentamicin (25%). Similarly, β-lactam ampicillin- and penicillin-resistant bacterial isolates have been isolated from various marine environments (Hermansson et al. 1987; Mudryk 2005; Altuğ et al. 2010). Results on the frequencies of bacterial resistance to a given antibiotic differ considerably and frequency values of 29% for kanamycin (Algeria), 68% for vancomycin (Portugal) and 63% for ampicillin (China) have been reported (Alouache et al. 2012; Novais et al. 2005; Tao et al. 2010). Although such dif erences in the percentage of bacterial resistance to various antibiotics may reflect regional history of antibiotic use, the presence of bacterial resistance in the marine environment pose a threat to human health. A temporal change in bacterial resistance to dif erent antibiotics has also been shown; for example, the previous study found the highest frequency of resistance to ampicillin in GN isolates from the Golden Horn Estuary (Istanbul Strait) and the Sea of Marmara (Altuğ et al. 2007). In addition, in the present study, the isolates that showed the highest resistance to kanamycin, vancomycin and ampicillin also displayed the lowest antibiotic resistance against gentamicin, ceftazidime and cefotaxime. The differences in the frequencies of resistance among various antimicrobial agents have been attributed to the presence of antibiotic-resistant plasmids in terrestrial bacteria entering into seawater (Vignesh et al. 2012).

The MAR index is commonly used to identify the level of bacterial resistance in a given population exposed to multiple sources of antimicrobial agents. The presence of multidrug-resistant isolates is alarming because infection with such isolates leads to a higher fatality rate than infection with antibiotic-sensitive isolates (Manjusha et al. 2005). The MAR index value >0.2 indicates the exposure to contamination sources having high risk levels of antibiotics, whereas values ≤;0.2 indicate a low risk contamination (Pontes et al. 2009). In the present study, the MAR index of isolates from the Sea of Marmara (0.306-0.343) was higher compared to isolates obtained from the Canakkale Strait (0.29-0.316), reflecting higher contamination of antibiotic residues, possibly due to the discharges of wastewater into the Sea of Marmara. Multiple antibiotic resistance were also found in isolates of Enterobacteriaceae isolated from marine environments and ranged within 0.44-0.52 in Bangladesh, 0.19-0.45 in China and 0.4-0.8 in Egypt (Matyar 2012; Zahid et al. 2009; Tao et al. 2010).

In this study, resistance to vancomycin, the vanA gene has been detected in 5 different isolates. However, no resistance to the vanB gene was detected. It seems that bacteria have not been brought into contact with any vanB resistant isolates. This is in agreement with other reports showing that vanA resistance is more common in the environment than vanB resistance (Riberio et al. 2007). This is the first report confirming the presence of the vanA gene in dif erent isolates from the aquatic environment in Turkey. In 2013, Turkey was the largest user of antibiotics in Europe, and vancomycin accounted for approximately 12% of all antibiotics used (TUIK, 2012).

In conclusion, our results indicated a temporal increase in the number of resistant isolates isolated from the seawater samples from the Sea of Marmara, the Istanbul Strait and the Çnakkale Strait. Bacterial pollution increases the levels of β-lactam antibiotic resistance among members of Enterobacteriaceae in the Sea of Marmara. We have found higher levels of kanamycin, vancomycin and amoxicillin/ clavulanic acid resistance in isolates from the Sea of Marmara due to the extensive use of antibiotics for the treatment of bacterial infections. The increase in resistant isolates and coliform bacteria counts is associated with the deteriorating environmental conditions, possibly due to the increased rates of untreated wastewater discharges through rivers or surface runoff. Further research is required to determine potential steps aimed at reducing the input of antibiotic residues and to establish regulatory standards to control their dissemination into the marine environment.