Antibiotic and Heavy Metal Resistant Bacteria Isolated from Aegean Sea

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https://doi.org/10.1595/205651320X15953337767424 Johnson Matthey Technol. Rev., 2020, 64, (4), 507–525

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Antibiotic and Heavy Metal Resistant Bacteria Isolated from Aegean Sea Water and Sediment in Güllük Bay, Turkey Quantifying the resistance of identified bacteria species with potential for environmental remediation applications

Gülşen Altuğ* in sediment and seawater samples taken from Department of Marine Biology, Faculty the Aegean Sea, Turkey, between 2011 and 2013. of Aquatic Sciences, Istanbul University, Bioindicator bacteria in seawater samples were Balabanağa Mahallesi Ordu Caddesi No 8, Laleli, tested using the membrane filtration technique. The Fatih, Istanbul, 34134, Turkey spread plate technique and VITEK® 2 Compact 30 micro identification system were used for Mine Çardak heterotrophic aerobic bacteria in the samples. The Department of Fisheries Technology, Faculty of minimum inhibition concentration method was Çanakkale Applied Sciences, Çanakkale Onsekiz used for heavy metal-resistant bacteria. Antibiotic- Mart University, Terzioğlu Campus, Çanakkale, resistant bacteria were tested using the disk diffusion 17020, Turkey method. All bacteria isolated from sediment samples showed 100% resistance to rifampicin, sulfonamide, Pelin Saliha Çiftçi Türetken tetracycline and ampicillin. 98% of isolates were Department of Marine Biology, Faculty resistant against nitrofurantoin and oxytetracycline. of Aquatic Sciences, Istanbul University, Higher antibiotic and heavy metal resistance was Balabanağa Mahallesi Ordu Caddesi No 8, Laleli, recorded in bacteria isolated from sediment than Fatih, Istanbul, 34134, Turkey seawater samples. The highest levels of bacterial metal resistance were recorded against copper Samet Kalkan (58.3%), zinc (33.8%), lead (32.1%), chromium Department of Marine Biology, Faculty of (31%) and iron (25.2%). The results show that Fisheries and Aquatic Sciences, Recep Tayyip antibiotic and heavy metal resistance in bacteria Erdoğan University, Zihni Derin Campus, Rize, from sediment and seawater can be observed as 53100, Turkey responses to environmental influences including pollution in marine areas. Sevan Gürün Department of Marine Biology, Faculty 1. Introduction of Aquatic Sciences, Istanbul University, Balabanağa Mahallesi Ordu Caddesi No 8, Laleli, In the era of Industry 4.0, with global climate change, Fatih, Istanbul, 34134, Turkey increasing population and developing technology, the spread of heavy metal pollutants in aquatic *Email: [email protected] areas is increasing. Bacterial resistance and metal accumulation capability are common phenomena that can be exploited for the bioremediation of Heavy metal and antibiotic-resistant bacteria the environment, hence these resistant bacteria have potential for environmental bioremediation may be potential candidates for biotechnological applications. Resistant bacteria were investigated applications. Despite the risks caused by antibiotic-

507 © 2020 Johnson Matthey https://doi.org/10.1595/205651320X15953337767424 Johnson Matthey Technol. Rev., 2020, 64, (4) resistant bacteria, heavy metal-resistant bacteria persistence of antibiotic resistance in bacterial can be used in detoxification processes to convert pathogens is a threat and a source of considerable a toxic form to a harmless form of a substance concern to public health (8–15). It is known that by developing biotransformation mechanisms. environmental factors such as overpopulation, Bioremediation studies have been carried out to livestock farming, insufficient drainage and identify candidate species (1–4). sanitation infrastructure may provide hotspots In recent years, the increase in pollution by toxic for environmental antibiotic-resistant bacteria compounds and heavy metals in marine areas makes transmission (16). it increasingly important to study the relationships In aquatic environments, antibiotic-resistant between bacteria and toxic compounds. Studies bacteria can be accompanied by heavy metal- related to the transformation of compounds into resistant bacteria that are often induced by the different forms via bacterial metabolic processes presence of metal caused by anthropogenic activities for the removal of toxic substances from the and environmental factors (16, 17). Heavy metals environment have gained importance. Detection are introduced into the marine environment in of bacteria that are resistant to heavy metals in different ways. Accumulation in sediment can affect natural environments constitutes the first step to aquatic life negatively for a long time. Bacteria provide data for remediation studies. that will take part in the transformation of heavy Bacteria are some of the most important metal salts into harmless forms must be resistant components in marine ecosystems. Since bacteria to the heavy metals. Bacteria that cannot adapt to adapt to new conditions created by environmental the changes metabolically will be eliminated and variables around them, knowledge of bacteria therefore various pollution inputs accumulated provides data in terms of defining environmental in the sediment will affect the composition of factors, public health status and ecosystem microbial diversity. Sediments containing harmful, function. Marine areas are exposed to domestic and inorganic or organic particles are relatively industrial wastes depending on local technology heterogeneous in terms of physical, chemical and levels and population. Many xenobiotic micro biological properties and are an important source pollutants, antibiotic derivatives and metabolites of heavy metal contamination (11). It has been reach the sea from human activity. This concerning reported that microplastics mediate the spread of issue is considered an important factor for global metal- and antibiotic-resistant pathogens due to health with respect to the evolution and detection their ability to adsorb various pollutants (18, 19). of antibiotic resistance in bacterial pathogens (5). Bacteria resistant to heavy metals in marine areas Since the spread of antimicrobial resistance is not have developed various resistance mechanisms restricted by phylogenetic, geographic or ecological to counteract heavy metal stress. Only bacteria borders, studies describing regional status of that can withstand the current heavy metal bacterial resistance in natural areas are important. concentration can survive in these areas. Antibiotic resistance can spread rapidly among Heavy metals accumulate in biota via food chains bacterial species (6). It is known that the and are transferred between organisms in marine occurrence of antibiotic-resistant bacteria in environments. This cumulative process, named natural environments reduces the effectiveness of biomagnification, is higher in the sea than in antibiotics in the treatment of infectious diseases. terrestrial environments (15, 20) and this implies Due to the increasing global resistance of bacteria significant effects of heavy metal pollution in against antibiotics, humanity is constantly being marine areas. On the other hand, heavy metal- forced to develop new antibiotic derivatives. resistant bacteria can play a role in detoxification by This vicious circle is one of the most important converting a toxic form into a harmless form through problems of our age and poses a threat for the biotransformation mechanisms that develop in future. Thus, it is important to know the resistance natural environments. These mechanisms include levels of bacteria and to produce regional antibiotic the formation and sequestration of heavy metals resistance profiles in natural areas. Aquatic in complexes and the reduction of a metal to a less environments constitute a way to disseminate toxic species (21, 22). not only antibiotic-resistant bacteria but also the Metal-resistant bacteria have developed very resistant genes in natural bacterial habitats (7). efficient and varying mechanisms for tolerating It has been well documented that the aquatic high levels of toxic metals and thus they carry an environment is a potential reservoir of antibiotic- important potential for controlling heavy metal resistant bacteria, furthermore the prevalence and pollution (23). In many prokaryotes, it has been

508 © 2020 Johnson Matthey https://doi.org/10.1595/205651320X15953337767424 Johnson Matthey Technol. Rev., 2020, 64, (4) shown that the mechanism for resisting heavy 2. Material and Methods metals develops over time. This process has 2.1 Sampling Area been studied in species such as Escherichia coli and Staphylococcus aureus. It is reported that Güllük Bay is an important location due to its many different species of Pseudomonas, Bacillus, natural resources. The region is open to different Enterobacter, Providencia and Chryseobacterium environmental influences and inputs due to are efficient for reducing heavy metals (1–4). tourism, port activities, marine transportation, It is known that the occurrence of bacteria domestic and industrial wastes and fish farms. resistant to antibiotics and heavy metal salts in The bay is also affected by the presence of Sarıçay the sea is related to the pollutants present in the Creek, Kazıklı Port, Güllük Port and Akbük Port environment. For the reasons highlighted above, it (24–26). Fish farms were operated in Güllük Bay is important to determine the profile of antibiotic until 2008. Although they have been relocated and heavy metal-resistant bacteria in marine away from the coastal regions to an offshore area, environments. Marine areas which have different the indirect effects of this long-time pollution may environmental inputs present novel media for have contributed to the sediment. bacterial studies. The export of feldspar and bauxite from the region For the present study, the Güllük Bay of the has been conducted from ports within the borders Aegean Sea, Turkey, was chosen since it is a of Güllük Bay. The port is mainly used by dry cargo dynamic area due to marine transportation, and other cargo-type ships. It is reported that an seasonal population growth depending on tourism, annual average of 800,000 tonnes of ballast water aquaculture, recreational and agricultural activities is transported to the bay from 157 different ports. and terrestrial pollution inputs transported from The amount of ballast water carried is reported rivers. as: 68% from the Mediterranean, 21% from the Probable faecal source analysis conducted in Aegean Sea, 7% from the Sea of Marmara, 2% Güllük Bay showed that the primary source of the from the Atlantic Ocean and 1% from the Black detected bacteriological pollution is anthropogenic Sea and Red Sea, respectively (37). The operation (24). A significant part of domestic wastewater of many tourism-oriented boats in Güllük Bay is in the region collects in sealed septic tanks. It also among the possible polluters of the bay due is possible for the wastewater to reach the sea to bilge water and wastewater. More than half of by mixing the sedimentary septic tanks with Turkey’s sea bream and sea bass production was groundwater. Chemical and biological studies in farms operating in the coastal areas of the (24– 33) confirm that regional pollutants have Güllük Bay for many years. These farms have reached Güllük Bay. been operating in the offshore areas of the region It is well known that sewage transported via for the past 10 years. The domestic wastewater domestic wastewater carries antibiotics to marine of the human population, reaching approximately environments. This has an effect on metabolic 50,000 around the region in the summer months, capabilities of bacteria in marine environments. and the wastes of small industrial establishments For example, β-lactam antibiotic derivatives used such as yogurt, yeast and olive oil producers that for human infection treatment may enter marine directly reach streams are the other main sources environments via domestic wastewater. Bacteria of pollution in Güllük Bay. The population of the may obtain resistance via intercellular contact Bodrum peninsula, which is 25,000 in winter, can mostly using a conjugation mechanism (34). reach 1,500,000 in summer (27). The change in The existence of antibiotic-resistant bacteria is the population between the seasons was among an indicator of domestic pollution. Furthermore, the biggest pollution sources according to the antibiotic-resistant bacteria may cause a vicious terrestrial bioindicator bacteria distribution in cycle. This problem has grown in recent years due coastal areas in the region (24, 26). to systematic use of antibiotics in animal husbandry Sampling stations were selected to represent and overuse of antibiotics (35, 36). tourist areas (G1, G5, G7, G8); harbours (G4, G6); The frequencies of heavy metal-resistant bacteria fresh water entry-exit points of the Sarıçay Creek and antibiotic-resistant bacteria were investigated (G9); fish farms (G11, G12, G13); and the deepest in seawater and sediment samples collected from point in the bay as a reference station (G14). Güllük Bay in the period between May 2011 and Figure 1 shows the location of Güllük Bay and the February 2013. sampling stations.

Code Mid point, m Max. depth, m

G1 25 50

G1 G4 25 50

G5 10 20 G4 G6 11 22 G7 14 28 G6 G14 G5 G8 G8 8 16

G7 G9 G9 4 8

G10 18 37 G10 Black Sea G11 5 10 G13 Marmara Sea G12 25 50 G11 Aegean TURKEY G12 Sea G13 12 25

G14 33 66 Mediterranean Sea

Fig. 1. Location of Güllük Bay and seawater (0–30 cm surface, mid-point and bottom-point) and sediment sampling stations

2.2 Sampling 2.3 Bacterial Isolation and Identification Seawater and sediment samples were collected from 12 different sampling stations in Güllük Bay Bacterial heavy metal and antibiotic resistance between May 2011 and February 2013. Three were tested in heterotrophic aerobic bacteria units of seawater samples were taken from each isolated from seawater and sediment samples. station at surface (0–30 cm), mid-point and Heavy metal and antibiotic resistance of indicator bottom-point water (Figure 1). In each sampling bacteria (faecal coliform, coliform and faecal process covering 12 stations, 36 seawater Streptococcus) isolated from the seawater samples samples were collected. In the spring and summer were also tested. months monthly, at other times seasonally, a total of 432 seawater samples were collected in the 2.3.1 Seawater Samples period between May 2011 and February 2013. The seawater samples were collected using a Indicator bacteria and heterotrophic aerobic bacteria Nansen bottle that was cleaned with acid (10% analyses were performed on the seawater samples. HCl in distilled water), sterilised with alcohol The membrane filtration technique was used to (50:50, v/v) and rinsed with sterile water. The detect indicator bacteria. A sample containing seawater samples were then transferred into 300 ml seawater was diluted serially (10–5 dilution) brown sterile glass bottles and transported to the and filtered through membrane filters (0.45 µm, laboratory as a cold chain. Sartorius AG, Göttingen, Germany). The filters Surface sediment samples were collected using were placed on m-Endo, m-FC and m-Azide media Ekman grab (HYDRO-BIOS Apparatebau GmbH, (Sartorius AG). The plates were incubated for 24 h Germany, 15 × 15) from the sampling stations (at 37 ± 0.1°C; at 44 ± 0.1°C for m-FC). Brown‑red which have various depths from 8 m to 66 m colonies growing on the azide medium were (Figure 1). A total of 144 units of sediment considered as suspicious faecal Streptococcus, blue samples were collected during the two-year study colonies growing on the m-FC medium as suspicious from 12 stations (one from each station). The faecal coliform and yellow-green colonies with yellow- sediment samples were transferred into sterile zip metallic gloss on the m-Endo medium as suspicious seal bags from Ekman grab and transferred in the coliform. Cytochrome oxidase test (API® 20 Strep, cold chain to the laboratory. bioMérieux, France) was performed on suspicious

510 © 2020 Johnson Matthey https://doi.org/10.1595/205651320X15953337767424 Johnson Matthey Technol. Rev., 2020, 64, (4) coliform colonies and oxidase negative colonies were Further processes related to heterotrophic bacteria evaluated numerically. Cytochrome oxidase (API® 20 identification were continued by using ®VITEK 2 Strep, bioMérieux) and indole tests were performed Compact 30 similarly to the seawater samples on the suspicious colonies of faecal coliform. described above. Colonies with oxidase negative and indole positive results were evaluated as faecal coliform. Suspicious 2.4 Bacterial Resistance Against Streptococcus colonies, to which the catalase test Antibiotics was applied (1 ml, 3% H2O2), were incubated on Bile Esculin Agar (BEA) for 18 h at 37°C for esculin The antibiotic resistance of the isolates was hydrolysis and 40% bile resistance control. Blackening examined by the Kirby–Bauer method with slight in the medium and the formation of black shadow modifications. Two or three colonies of each isolate around the colony, positive of esculin hydrolysis, were suspended with 5 ml of DifcoTM Marine Broth and the number of colonies showing growth in the 2216 and diluted with sterile water against the medium were evaluated as 40% bile resistant, and 0.5 McFarland turbidity standard to approximately catalase negative and breeding colonies in BEA 106 cells ml–1 and swabbed as 2 ml on DifcoTM were evaluated as faecal Streptococcus. Counted Marine Agar 2216. Antibiotic discs (Oxoid, UK) colonies were multiplied by the 10–5 dilution factor to containing ampicillin (10 µg), nitrofurantoin determine the number of colony forming units (CFU) (300 µg), oxytetracycline (30 µg), sulfonamide 100 ml–1 in the original sample (38). (300 µg), rifampicin (2 µg), tetracycline (10 µg) The spread plate technique was used for and tetracycline (30 µg) were incubated for two to heterotrophic aerobic bacteria analyses in seawater. three days at 37°C. The results were interpreted Seawater samples 0.1 ml with 10–5 dilution were according to the guidelines of the Clinical Laboratory used for duplicate spreading on the DifcoTM Marine Standard Institute (CLSI) (41). All isolates that Agar 2216 (Becton, Dickinson and Company, USA) showed resistance were classified as ‘resistant’. and the plates were incubated for five days at Other isolates that did not show resistance were 22 ± 0.1°C. At the end of the incubating period, classified as ‘sensitive’ or ‘susceptible’. counted colonies were multiplied by the 10–5 –1 dilution factor to determine the number of CFU ml 2.4.1 Multiple Antibiotic Resistance in the original sample. An average of 10 different colonies were picked and restreaked several times The multiple antibiotic resistance (MAR) index of to obtain pure cultures. The pure isolates were a given sample was calculated by the equation: Gram-stained. For identification of spore-forming a/ (bc), where a represents the aggregate antibiotic bacilli, the isolates were stained with Indian ink resistance score of all isolates from a sample; b is according to the negative staining technique the total number of isolates; and c is the number of and were evaluated using a light microscope isolates from a sample (42). Bacterial isolates that (Nikon E110, Nikon, Japan). The isolates were displayed resistance to three or more antibiotic then tested using Gram‑negative fermenting agents were designated as multiple antibiotic and non‑fermenting bacilli (GN), Gram-positive resistant (ranging from two to 10). cocci and non-spore-forming bacilli (GP) and Gram‑positive spore-forming bacilli (BCL) cards in 2.5 Bacterial Resistance Against the automated micro identification system VITEK® Heavy Metal Salts 2 Compact 30 (bioMérieux) (39). Different concentrations (50 μg ml–1, 100 μg ml–1, –1 –1 –1 2.3.2 Sediment Samples 150 μg ml , 200 μg ml and 250 μg ml ) of heavy metal salts (FeSO4, ZnSO4, CuSO4, Cr2(SO4)3 and

The spread plate technique was used for Pb(NO3)2) were used to test the bacteria resistivity heterotrophic aerobic bacteria analyses in sediment against iron, zinc, copper, chromium and lead. samples. Each sediment sample was mixed and The microdilution method was followed with minor homogenised. Then 1 g sample was taken from modifications to determine the resistance of isolates each and serially diluted with sterile commercial to heavy metals (43). Stock solutions of metal seawater. 0.1 ml samples of 10–5 dilutions were salts prepared in distilled water were sterilised by taken and spread on DifcoTM Marine Agar 2216. The filtration (0.20 μm). In U-well microtiter plates, plates were incubated for five days at 22 ± 0.1°C. serial dilutions of heavy metals were prepared Growing colonies were evaluated as CFU g–1 (40). and then each well was inoculated with bacteria

511 © 2020 Johnson Matthey https://doi.org/10.1595/205651320X15953337767424 Johnson Matthey Technol. Rev., 2020, 64, (4) inoculation. The OxoidTM Turbidometer (Thermo Shewanella algae and Vibrio parahaemolyticus Fisher Scientific Inc, USA) provides the inoculum isolates from the sediment samples showed density standardisation for 0.5 McFarland which is resistance to all antibiotics (Table I). necessary to ensure accurate reproducible results. The highest number of antibiotic-resistant Before the addition of bacterial inoculation, no bacteria were detected from the sediment precipitation was seen. The plates were incubated samples. The frequency of resistant bacteria (%) at 37°C for 24 h and then examined for visual to oxytetracycline (30 µg), nitrofurantoin (300 µg), turbidity. The lowest concentration of the metal rifampicin (2 µg), tetracycline (10 µg), tetracycline salt, at which growth was inhibited (indicated (30 µg), sulfonamide (300 µg) and ampicillin by lack of turbidity), was taken as the minimum (10 µg) from the seawater and sediment samples inhibitory concentration (MIC) (44) Samples of are shown in Figure 2. The frequencies of antibiotic 10 μl were drawn from each well without turbidity resistance in bacteria species from seawater and and were subcultured on agar plates to determine sediment samples are shown in Figure 3. bactericidal concentration. A total of 258 and 158 isolates were tested Reference strains of Escherichia coli (ATCC® against antibiotics from seawater and sediment 25922TM), Salmonella enterica (ATCC® 2577TM) samples, respectively. The frequencies of and Staphylococcus epidermidis (ATCC® 12228TM) resistance against seven antibiotics in bacteria which are susceptible to Cu2+, Zn2+, Pb2+, Cr2+ and species isolated from the seawater samples Fe3+ and metal-free plates were used in the control were recorded as 49% in Gammaproteobacteria, tests to evaluate the viability of the strains and 22% in Αlphaproteobacteria, 3% in culture media. All of the experiments were carried Betaproteobacteria, 14% in Bacilli, 8% in out in triplicate. Flavobacteriia and 4% in Actinomycetales. The resistance frequencies against seven antibiotics 3. Results in bacteria isolated from the sediment samples were recorded as 43% in Gammaproteobacteria, 3.1 Bacterial Resistance Against 34% in Bacilli, 7% in Αlphaproteobacteria, 7% Antibiotics in Betaproteobacteria, 7% in Flavobacteriia and 2% in Actinomycetales. Table I shows the antibiotic-resistant, intermediate or susceptible bacteria species isolated from the 3.2 Multiple Antibiotic Resistance seawater and sediment samples in this study. Indexes Bacterial species isolated from the seawater samples showed considerable resistance to rifampicin (98%), The MAR index was calculated for each of the sulfonamide (98%) and ampicillin (76%) and antibiotic-resistant bacteria. If the MAR index considerable sensitivity to tetracycline-30 µg (52%), is lower than 0.2, it shows a non-point based tetracycline-10 µg (39%) and oxytetracycline source of pollution and if it is higher than 0.2 it (33%). Almost all the bacterial species isolated from shows point‑based pollution and a high risk of sediment samples showed resistance to rifampicin contamination by excessive antibiotic presence (100%), sulfonamide (100%), ampicillin (100%), (23). Table II shows the MAR indexes. nitrofurantoin (98%), tetracycline-30 µg (100%), The MAR indexes of the study showed possible tetracycline-10 µg (100%) and oxytetracycline exposure of these bacterial isolates to the tested (98%) while they showed almost no sensitivity antibiotics. The MAR index of bacteria isolated to antibiotics except nitrofurantoin (2%) and from all stations around fish farm areas (0.0576) oxytetracycline (2%). Pseudomonas aeruginosa was 2.6 times greater than the MAR index for the (24%) and Sphingomonas paucimobilis (20%), combined non-fish farm areas (0.022). isolated from seawater samples, showed higher resistance to antibiotics than did Raoultella 3.3 Bacterial Resistance Against oxytica, Staphylococcus xylosus, Kocuria kristinae, Heavy Metals Aeromonas salmonicida and Proteus vulgaris strains. On the contrary, Aeromonas caviae, The frequencies of heavy metal resistance in the Alicyclobacillus acidoterrestris, Brevundimonas bacteria species isolated from the seawater samples diminuta, Chryseobacterium indologenes, were recorded as 76.72% in Gammaproteobacteria, Lactococcus garvieae, Neisseria animaloris, 71.82% in Αlphaproteobacteria, 80.01% in Pseudomonas aeruginosa, Serratia marcescens, Bacilli, 56.92% in Flavobacteriia and 75% in

512 © 2020 Johnson Matthey https://doi.org/10.1595/205651320X15953337767424 Johnson Matthey Technol. Rev., 2020, 64, (4) OT (30 µg) R: 66.7% I: 33.3% S: 0.0% R: 25% I: 0.0% S: 75% R: 26.31% I: 34.21% S: 39.47% R: 25% I: 25% S: 50% R: 0.0% I: 0.0% S: 100% R: 100% I: 0.0% S: 0.0% R: 100% I: 0.0% S: 0.0% R: 0.0% I: 100% S: 0.0% R: 0.0% I: 50% S: 50% R: 50% I: 0.0% S: 50% R: 0.0% I: 100% S: 0.0% R: 0.0% I: 0.0% S: 100% F/M (300 µg) R: 66.7% I: 0.0% S: 33.3% R: 50% I: 0.0% S: 50% R: 60.52% I: 0.0% S: 39.47% R: 75% I: 0% S: 25% R: 0.0% I: 0.0% S: 100% R: 100% I: 0.0% S: 0.0% R: 100% I: 0.0% S: 0.0% R: 100% I: 0.0% S: 0.0% R: 50% I: 0.0% S: 50% R: 50% I: 0.0% S: 50% R: 100% I: 0.0% S: 0.0% R: 100% I: 0.0% S: 0.0% RD (2 µg) R: 100% I: 0.0% S: 0.0% R: 100% I: 0.0% S: 0.0% R: 97.36% I: 0.0% S: 2.63% R: 100% I: 0.0% S: 0.0% R: 100% I: 0.0% S: 0.0% R: 100% I: 0.0% S: 0.0% R: 100% I: 0.0% S: 0.0% R: 100% I: 0.0% S: 0.0% R: 50% I: 0.0% S: 50% R: 100% I: 0.0% S: 0.0% R: 100% I: 0.0% S: 0.0% R: 100% I: 0.0% S: 0.0% TE (10 µg) R: 33.3% I: 33.3% S: 33.3% R: 50% I: 0.0% S: 50% R: 42.10% I: 7.89% S: 50% R: 0% I: 25% S: 75% R: 0.0% I: 0.0% S: 100% R: 100% I: 0.0% S: 0.0% R: 100% I: 0.0% S: 0.0% R: 0.0% I: 0.0% S: 100% R: 50% I: 0.0% S: 50% R: 50% I: 0.0% S: 50% R: 0.0% I: 0.0% S: 100% R: 0.0% I: 0.0% S: 100% R: 97.38% I: 0.0% S: 2.63% R: 100% I: 0.0% S: 0.0% R: 100% I: 0.0% S: 0.0% R: 100% I: 0.0% S: 0.0% R: 100% I: 0.0% S: 0.0% R: 100% I: 0.0% S: 0.0% S (300 µg) R: 100% I: 0.0% S: 0.0% R: 100% I: 0.0% S: 0.0% R: 100% I: 0.0% S: 0.0% R: 100% I: 0.0% S: 0.0% R: 100% I: 0.0% S: 0.0% R: 100% I: 0.0% S: 0.0% TE (30 µg) R: 33.3% I: 0.0% S: 66.7% R: 50% I: 0.0% S: 50% R: 31.57% I: 5.26% S: 63.15% R: 0.0% I: 0.0% S: 100% R: 0.0% I: 0.0% S: 100% R: 100% I: 0.0% S: 0.0% R: 100% I: 0.0% S: 0.0% R: 0.0% I: 0.0% S: 100% R: 50% I: 0% S: 50% R: 50% I: 0.0% S: 50% R: 0.0% I: 0.0% S: 100% R: 0.0% I: 0.0% S: 100% a R: 71.05% I: 2.63% S: 26.31% R: 50% I: 0.0% S: 50% R: 100% I: 0.0% S: 0.0% R: 50% I: 0.0% S: 50% R: 100% I: 0.0% S: 0.0% R: 100% I: 0.0% S: 0.0% Antibiotics AM (10 µg) R: 66.7% I: 33.3% S: 0.0% R: 75% I: 0.0% S: 25% R: 100% I: 0.0% S: 0.0% R: 100% I: 0.0% S: 0.0% R: 100% I: 0.0% S: 0.0% R: 100% I: 0.0% S: 0.0%

(3) (3) (38) (4) (4) (4) (3) Bacterial isolates tested (n) Brevundimonas diminuta Brevundimonas vesicularis Sphingomonas paucimobilis Sphingomonas thalpophilum Burkholderia cepacia (3) Burkholderia mallei (3) Neisseria animaloris (3) Acinetobacter lwoffii (3) Aeromonas hydrophila (4) Aeromonas salmonicida Aeromonas sobria Aeromonas veronii Antibiotic Resistant, Intermediate or Susceptible Bacteria Species Isolated from Seawater and Sediment from Species Isolated Susceptible Bacteria or Resistant, Intermediate Antibiotic

Order/class tested (%) Proteobacteria/ Alpha proteobacteria (27%) Proteobacteria/ Beta proteobacteria (%) Proteobacteria/ Gamma proteobacteria (53%)

Sample r Seawate Table I

513 © 2020 Johnson Matthey https://doi.org/10.1595/205651320X15953337767424 Johnson Matthey Technol. Rev., 2020, 64, (4) R: 100% I: 0.0% S: 0.0% R: 100% I: 0.0% S: 0.0% R: 33.3% I: 66.6% S: 0.0% R: 0.0% I: 0.0% S: 100% R: 0.0% I: 0.0% S: 100% R: 50% I: 50% S: 0% R: 75% I: 0.0% S: 25% R: 100% I: 0.0% S: 0.0% R: 0.0% I: 0.0% S: 100% R: 100% I: 0.0% S: 0.0% R: 90.91% I: 0.0% S: 9.09% R: 0.0% I: 0.0% S: 100% R: 100% I: 0.0% S: 0.0% OT (30 µg) R: 100% I: 0.0% S: 0.0% R: 100% I: 0.0% S: 0.0% R: 33.3% I: 0.0% S: 66.6% R: 100% I: 0.0% S: 0.0% R: 100% I: 0.0% S: 0.0% R: 100% I: 0.0% S: 0.0% R: 100.0% I: 0.0% S: 0.0% R: 100% I: 0.0% S: 0.0% R: 0.0% I: 0.0% S: 100% R: 100% I: 0.0% S: 0.0% R: 90.91% I: 0.0% S: 9.09% R: 0.0% I: 0.0% S: 100% R: 100% I: 0.0% S: 0.0% F/M (300 µg) R: 100% I: 0.0% S: 0.0% R: 100% I: 0.0% S: 0.0% R: 100% I: 0.0% S: 0.0% R: 100% I: 0.0% S: 0.0% R: 100% I: 0.0% S: 0.0% R: 100% I: 0.0% S: 0.0% R: 100.0% I: 0.0% S: 0.0% R: 100% I: 0.0% S: 0.0% R: 100% I: 0.0% S: 0.0% R: 100% I: 0.0% S: 0.0% R: 100% I: 0.0% S: 0.0% R: 100% I: 0.0% S: 0.0% R: 100% I: 0.0% S: 0.0% RD (2 µg) R: 100% I: 0.0% S: 0.0% R: 100% I: 0.0% S: 0.0% R: 66.6% I: 0.0% S: 33.3% R: 100% I: 0.0% S: 0.0% R: 100% I: 0.0% S: 0.0% R: 100% I: 0.0% S: 0.0% R: 89.3% I: 0.0% S: 10.7% R: 100% I: 0.0% S: 0.0% R: 0.0% I: 0.0% S: 100% R: 100% I: 0.0% S: 0.0% R: 90.91% I: 0.0% S: 9.09% R: 0.0% I: 0.0% S: 100% R: 100% I: 0.0% S: 0.0% R: 100% I: 0.0% S: 0.0% R: 100% I: 0.0% S: 0.0% R: 0.0% I: 100% S: 0.0% R: 92.9% I: 3.5% S: 3.5% R: 100% I: 0.0% S: 0.0% R: 100% I: 0.0% S: 0.0% R: 100% I: 0.0% S: 0.0% R: 100% I: 0.0% S: 0.0% R: 100% I: 0.0% S: 0.0% TE (10 µg) R: 100% I: 0.0% S: 0.0% R: 66.6% I: 0.0% S: 33.3% R: 100% I: 0.0% S: 0.0% R: 100% I: 0.0% S: 0.0% S (300 µg) R: 100% I: 0.0% S: 0.0% R: 100% I: 0.0% S: 0.0% R: 33.3% I: 0.0% S: 66.6% R: 0.0% I: 100% S: 0.0% R: 0.0% I: 0.0% S: 100% R: 50% I: 0.0% S: 50% R: 71.4% I: 10.7% S: 17.8% R: 100% I: 0.0% S: 0.0% R: 0.0% I: 0.0% S: 100% R: 100% I: 0.0% S: 0.0% R: 90.91% I: 0.0% S: 9.09% R: 0.0% I: 0.0% S: 100% R: 100% I: 0.0% S: 0.0% R: 0.0% I: 0.0% S: 100% R: 100% I: 0.0% S: 0.0% R: 0.0% I: 50% S: 50% R: 78.5% I: 10.7% S: 10.7% R: 100% I: 0.0% S: 0.0% R: 100% I: 0.0% S: 0.0% R: 100% I: 0.0% S: 0.0% R: 100% I: 0.0% S: 0.0% R: 100% I: 0.0% S: 0.0% Antibiotics TE (30 µg) R: 100% I: 0.0% S: 0.0% R: 33.3% I: 33.3% S: 33.3% R: 100% I: 0.0% S: 0.0% R: 100% I: 0.0% S: 0.0%

(4) (3) group (3) (3) (3) (30) (3) (3) subsp. (13) (6) (4)

ozaenae dissolvens Bacterial isolates tested (n) AM (10 µg) Citrobacter sedlakii (3) Cronobacter dublinensis lausannensis Enterobacter aerogenes Enterobacter cloacae subsp. Enterobacter cloacae (4) Enterobacter cloacae complex Escherichia coli Klebsiella pneumoniae subsp. Pasteurella canis Proteus vulgaris Proteus penneri Pseudomonas aeruginosa Raoultella ornithinolytica Raoultella ytica

Order/class tested (%)

Sample Seawater

514 © 2020 Johnson Matthey https://doi.org/10.1595/205651320X15953337767424 Johnson Matthey Technol. Rev., 2020, 64, (4) OT (30 µg) R: 66.6% I: 0.0% S: 33.4% R: 54.54% I: 36.36% S: 9.06% R: 20% I: 0.0% S: 80% R: 0.0% I: 50% S: 50% R: 100% I: 0.0% S: 0.0% R: 0.0% I: 100% S: 0.0% R: 60% I: 20% S: 20% R: 66.7% I: 0.0% S: 33.3% R: 100% I: 0.0% S: 0.0% R: 0.0% I: 0.0% S: 0.0% R: 33.3% I: 0.0% S: 66.7% R: 45.45% I: 18.18% S: 36.36% R: 66.6% I: 0.0% S: 33.3% F/M (300 µg) R: 100% I: 0.0% S: 0.0% R: 63.63% I: 0.0% S: 36.36% R: 100% I: 0.0% S: 0.0% R: 0.0% I: 0.0% S: 100% R: 100% I: 0.0% S: 0.0% R: 100% I: 0.0% S: 0.0% R: 100% I: 0.0% S: 0.0% R: 66.7% I: 0.0% S: 33.3% R: 100% I: 0.0% S: 0.0% R: 100% I: 0.0% S: 0.0% R: 100% I: 0.0% S: 0.0% R: 54.54% I: 0.0% S: 45.45% R: 66.6% I: 0.0% S: 33.3% RD (2 µg) R: 100% I: 0.0% S: 0.0% R: 100% I: 0.0% S: 0.0% R: 100% I: 0.0% S: 0.0% R: 100% I: 0.0% S: 0.0% R: 100% I: 0.0% S: 0.0% R: 100% I: 0.0% S: 0.0% R: 100% I: 0.0% S: 0.0% R: 100% I: 0.0% S: 0.0% R: 100% I: 0.0% S: 0.0% R: 100% I: 0.0% S: 0.0% R: 33.3% I: 66.7% S: 0.0% R: 100% I: 0.0% S: 0.0% R: 100% I: 0.0% S: 0.0% TE (10 µg) R: 66.6% I: 0.0% S: 33.4% R: 72.72% I: 0.0% S: 27.27% R: 20% I: 50% S: 50% R: 0.0% I: 0.0% S: 100% R: 100% I: 0.0% S: 0.0% R: 100% I: 0.0% S: 0.0% R: 80% I: 20% S: 0.0% R: 100% I: 0.0% S: 0.0% R: 100% I: 0.0% S: 0.0% R: 100% I: 0.0% S: 0.0% R: 66.7% I: 0.0% S: 33.3% R: 45.45% I: 0.0% S: 54.54% R: 66.6% I: 0.0% S: 33.3% R: 100% I: 0.0% S: 0.0% R: 100% I: 0.0% S: 0.0% R: 100% I: 0.0% S: 0.0% R: 100% I: 0.0% S: 0.0% R: 100% I: 0.0% S: 0.0% R: 100% I: 0.0% S: 0.0% R: 100% I: 0.0% S: 0.0% S (300 µg) R: 100% I: 0.0% S: 0.0% R: 100% I: 0.0% S: 0.0% R: 100% I: 0.0% S: 0.0% R: 100% I: 0.0% S: 0.0% R: 0.0% I: 100% S: 100% R: 66.7% I: 0.0% S: 33.3% TE (30 µg) R: 66.6% I: 0.0% S: 33.4% R: 45.45% I: 18.18% S: 36.36% R: 40% I: 0.0% S: 60% R: 0.0% I: 0.0% S: 100% R: 100% I: 0.0% S: 0.0% R: 0.0% I: 0.0% S: 100% R: 60% I: 0.0% S: 40% R: 66.7% I: 0.0% S: 33.3% R: 100% I: 0.0% S: 0.0% R: 0.0% I: 0.0% S: 100% R: 33.4% I: 0.0% S: 66.7% R: 36.36% I: 0.0% S: 63.63% R: 66.6% I: 0.0% S: 33.3% R: 81.81% I: 0.0% S: 18.18% R: 50% I: 0.0% S: 50% R: 0.0% I: 100% S: 0.0% R: 100% I: 0.0% S: 0.0% R: 66.7% I: 0.0% S: 33.3% R: 54.54% I: 18.18% S: 27.27% R: 100% I: 0.0% S: 0.0% Antibiotics AM (10 µg) R: 100% I: 0.0% S: 0.0% R: 80% I: 0.0% S: 20% R: 100% I: 0.0% S: 0.0% R: 100% I: 0.0% S: 0.0% R: 0.0% I: 0.0% S: 100% R: 33.3% I: 0.0% S: 66.7% (5) (4) (7) (5) (3) . (13) (13) (7) spp (5) (3) (3) Bacterial isolates tested (n) Serratia marcescens (5) Shewanella putrefaciens Stenotrophomonas maltophilia Vibrio vulnificus Enterococcus faecium (3) Alicyclobacillus acidocaldarius Bacillus cereus Bacillus pumilus Staphylococcus xylosus Staphylococcus aureus Staphylococcus warneri Chryseobacterium indologenes Myroides

Order/class tested (%) Firmicutes/ Bacilli (9%) Bacteroidetes/ (8%) Flavobacteriia

Sample r Seawate

515 © 2020 Johnson Matthey https://doi.org/10.1595/205651320X15953337767424 Johnson Matthey Technol. Rev., 2020, 64, (4) OT (30 µg) R: 0.0% I: 0.0% S: 100% R: 50% I: 0.0% S: 50% R: 0.0% I: 1000% S: 0.0% R: 0.0% I: 100% S: 0.0% R: 100% I: 0.0% S: 0.0% R: 100% I: 0.0% S: 0.0% R: 100% I: 0.0% S: 0.0% R: 100% I: 0.0% S: 0.0% R: 100% I: 0.0% S: 0.0% R: 100% I: 0.0% S: 0.0% R: 100% I: 0.0% S: 0.0% R: 100% I: 0.0% S: 0.0% R: 100% I: 0.0% S: 0.0% F/M (300 µg) R: 0.0% I: 0.0% S: 100% R: 100% I: 0.0% S: 0.0% R: 0.0% I: 0.0% S: 100% R: 50% I: 0.0% S: 50% R: 100% I: 0.0% S: 0.0% R: 100% I: 0.0% S: 0.0% R: 100% I: 0.0% S: 0.0% R: 100% I: 0.0% S: 0.0% R: 100% I: 0.0% S: 0.0% R: 100% I: 0.0% S: 0.0% R: 100% I: 0.0% S: 0.0% R: 100% I: 0.0% S: 0.0% R: 100% I: 0.0% S: 0.0% RD (2 µg) R: 100% I: 0.0% S: 0.0% R: 100% I: 0.0% S: 0.0% R: 100% I: 0.0% S: 0.0% R: 100% I: 0.0% S: 0.0% R: 100% I: 0.0% S: 0.0% R: 100% I: 0.0% S: 0.0% R: 100% I: 0.0% S: 0.0% R: 100% I: 0.0% S: 0.0% R: 100% I: 0.0% S: 0.0% R: 100% I: 0.0% S: 0.0% R: 100% I: 0.0% S: 0.0% R: 100% I: 0.0% S: 0.0% R: 100% I: 0.0% S: 0.0% TE (10 µg) R: 0.0% I: 0.0% S: 100% R: 50% I: 0.0% S: 50% R: 100% I: 0.0% S: 0.0% R: 50% I: 0.0% S: 50% R: 100% I: 0.0% S: 0.0% R: 100% I: 0.0% S: 0.0% R: 100% I: 0.0% S: 0.0% R: 100% I: 0.0% S: 0.0% R: 100% I: 0.0% S: 0.0% R: 100% I: 0.0% S: 0.0% R: 100% I: 0.0% S: 0.0% R: 100% I: 0.0% S: 0.0% R: 100% I: 0.0% S: 0.0% R: 100% I: 0.0% S: 0.0% R: 100% I: 0.0% S: 0.0% R: 100% I: 0.0% S: 0.0% R: 100% I: 0.0% S: 0.0% R: 100% I: 0.0% S: 0.0% R: 100% I: 0.0% S: 0.0% R: 100% I: 0.0% S: 0.0% R: 100% I: 0.0% S: 0.0% S (300 µg) R: 100% I: 0.0% S: 0.0% R: 100% I: 0.0% S: 0.0% R: 100% I: 0.0% S: 0.0% R: 100% I: 0.0% S: 0.0% R: 100% I: 0.0% S: 0.0% TE (30 µg) R: 0.0% I: 0.0% S: 100% R: 100% I: 0.0% S: 0.0% R: 0.0% I: 0.0% S: 100% R: 50% I: 0.0% S: 50% R: 100% I: 0.0% S: 0.0% R: 100% I: 0.0% S: 0.0% R: 100% I: 0.0% S: 0.0% R: 100% I: 0.0% S: 0.0% R: 100% I: 0.0% S: 0.0% R: 100% I: 0.0% S: 0.0% R: 100% I: 0.0% S: 0.0% R: 100% I: 0.0% S: 0.0% R: 100% I: 0.0% S: 0.0% R: 0.0% I: 0.0% S: 100% R: 100% I: 0.0% S: 0.0% R: 0.0% I: 100% S: 0.0% R: 50% I: 0.0% S: 50% R: 100% I: 0.0% S: 0.0% R: 100% I: 0.0% S: 0.0% R: 100% I: 0.0% S: 0.0% R: 100% I: 0.0% S: 0.0% Antibiotics AM (10 µg) R: 100% I: 0.0% S: 0.0% R: 100% I: 0.0% S: 0.0% R: 100% I: 0.0% S: 0.0% R: 100% I: 0.0% S: 0.0% R: 100% I: 0.0% S: 0.0% (1) (1) (4) (3) (4) (3) (1) (1) (1) (1) (1) Bacterial isolates tested (n) Dermacoccus nishinomiyaensis Kocuria kristinae Kocuria varians Micrococcus luteus Brevundimonas diminuta Sphingomonas paucimobilis Sphingomonas thalpophilum Neisseria animaloris (3) Chromobacterium violaceum Aeromonas caviae Aeromonas sobria Pseudomonas aeruginosa Serratia marcescens (5)

Order/class tested (%) Actinobacteria/ Actinomycetales (3%) Proteobacteria/ Alpha proteobacteria (7%) Proteobacteria/ Beta proteobacteria (7%) Proteobacteria/ Gamma proteobacteria (43%)

Sample r Seawate Sediment

516 © 2020 Johnson Matthey https://doi.org/10.1595/205651320X15953337767424 Johnson Matthey Technol. Rev., 2020, 64, (4) OT (30 µg) R: 100% I: 0.0% S: 0.0% R: 100% I: 0.0% S: 0.0% R: 66.7% I: 0.0% S: 33.3% R: 100% I: 0.0% S: 0.0% R: 100% I: 0.0% S: 0.0% R: 100% I: 0.0% S: 0.0% R: 100% I: 0.0% S: 0.0% R: 100% I: 0.0% S: 0.0% R: 100% I: 0.0% S: 0.0% R: 100% I: 0.0% S: 0.0% R: 100% I: 0.0% S: 0.0% R: 100% I: 0.0% S: 0.0% R: 100% I: 0.0% S: 0.0% F/M (300 µg) R: 100% I: 0.0% S: 0.0% R: 100% I: 0.0% S: 0.0% R: 66.7% I: 0.0% S: 33.3% R: 100% I: 0.0% S: 0.0% R: 100% I: 0.0% S: 0.0% R: 100% I: 0.0% S: 0.0% R: 100% I: 0.0% S: 0.0% R: 100% I: 0.0% S: 0.0% R: 100% I: 0.0% S: 0.0% R: 100% I: 0.0% S: 0.0% R: 100% I: 0.0% S: 0.0% R: 100% I: 0.0% S: 0.0% R: 100% I: 0.0% S: 0.0% RD (2 µg) R: 100% I: 0.0% S: 0.0% R: 100% I: 0.0% S: 0.0% R: 100% I: 0.0% S: 0.0% R: 100% I: 0.0% S: 0.0% R: 100% I: 0.0% S: 0.0% R: 100% I: 0.0% S: 0.0% R: 100% I: 0.0% S: 0.0% R: 100% I: 0.0% S: 0.0% R: 100% I: 0.0% S: 0.0% R: 100% I: 0.0% S: 0.0% R: 100% I: 0.0% S: 0.0% R: 100% I: 0.0% S: 0.0% R: 100% I: 0.0% S: 0.0% TE (10 µg) R: 100% I: 0.0% S: 0.0% R: 100% I: 0.0% S: 0.0% R: 100% I: 0.0% S: 0.0% R: 100% I: 0.0% S: 0.0% R: 100% I: 0.0% S: 0.0% R: 100% I: 0.0% S: 0.0% R: 100% I: 0.0% S: 0.0% R: 100% I: 0.0% S: 0.0% R: 100% I: 0.0% S: 0.0% R: 100% I: 0.0% S: 0.0% R: 100% I: 0.0% S: 0.0% R: 100% I: 0.0% S: 0.0% R: 100% I: 0.0% S: 0.0% R: 100% I: 0.0% S: 0.0% R: 100% I: 0.0% S: 0.0% R: 100% I: 0.0% S: 0.0% R: 100% I: 0.0% S: 0.0% R: 100% I: 0.0% S: 0.0% R: 100% I: 0.0% S: 0.0% R: 100% I: 0.0% S: 0.0% R: 100% I: 0.0% S: 0.0% R: 100% I: 0.0% S: 0.0% R: 100% I: 0.0% S: 0.0% S (300 µg) R: 100% I: 0.0% S: 0.0% R: 100% I: 0.0% S: 0.0% R: 100% I: 0.0% S: 0.0% TE (30 µg) R: 100% I: 0.0% S: 0.0% R: 100% I: 0.0% S: 0.0% R: 100% I: 0.0% S: 0.0% R: 100% I: 0.0% S: 0.0% R: 100% I: 0.0% S: 0.0% R: 100% I: 0.0% S: 0.0% R: 100% I: 0.0% S: 0.0% R: 100% I: 0.0% S: 0.0% R: 100% I: 0.0% S: 0.0% R: 100% I: 0.0% S: 0.0% R: 100% I: 0.0% S: 0.0% R: 100% I: 0.0% S: 0.0% R: 100% I: 0.0% S: 0.0% R: 100% I: 0.0% S: 0.0% R: 100% I: 0.0% S: 0.0% R: 100% I: 0.0% S: 0.0% R: 100% I: 0.0% S: 0.0% R: 100% I: 0.0% S: 0.0% R: 100% I: 0.0% S: 0.0% R: 100% I: 0.0% S: 0.0% R: 100% I: 0.0% S: 0.0% R: 100% I: 0.0% S: 0.0% R: 100% I: 0.0% S: 0.0% Antibiotics AM (10 µg) R: 100% I: 0.0% S: 0.0% R: 100% I: 0.0% S: 0.0% R: 100% I: 0.0% S: 0.0% (15) (11) (11) (11) (23) (11) (11) (12) . (11) (12) spp Bacterial isolates tested (n) Shewanella algae Shewanella putrefaciens Vibrio alginolyticus (14) Vibrio fluvialis Vibrio parahaemolyticus (13) Vibrio vulnificus Alicyclobacillus acidoterrestris Bacillus cereus Bacillus pumilus Lactococcus garvieae (13) Chryseobacterium indologenes Myroides Micrococcus lylae

Order/class tested (%) Firmicutes/ Bacilli (34%) Bacteroidetes/ Flavobacteriia (7%) Actinobacteria/ Actinomycetales (2%)

Sample t Sedimen Ampicillin (AM, 10 µg), nitrofurantoin (F/M, 300 µg), oxytetracycline (OT, 30 µg), sulfonamide (S, 300 µg), rifampicin (RD, 2 µg), tetracycline (TE, 10 µg) and tetracycline (TE, 30 µg). Resistant (TE, 30 µg). Resistant (TE, 10 µg) and tetracycline 2 µg), tetracycline 300 µg), rifampicin (RD, 30 µg), sulfonamide (S, (OT, (F/M, 300 µg), oxytetracycline Ampicillin (AM, 10 µg), nitrofurantoin a (R), intermediate (I) or susceptible (S)

Sediment Seawater Fig. 2. The frequency of bacteria resistant to Ampicillin, 10 µg specific antibiotics (%) Sulfonamide, 300 µg in the seawater and sediment samples Oxytetracycline, 30 µg Tetracycline, 30 µg Tetracycline, 10 µg Rifampicin, 2 µg Nitrofurantoin, 300 µg

0 20 40 60 80 100 Resistance, %

(a) (b) Betaproteobacteria 3% Actinomycetales 2% Actinomycetales 4% Flavobacteriia 7% Flavobacteriia 8% Betaproteobacteria 7% Bacilli 14% Alphaproteobacteria 7% Alphaproteobacteria 22% Bacilli 34%

Gammaproteobacteria 49% Gammaproteobacteria 43%

0 20 40 60 0 10 20 30 40 50 Resistance, % Resistance, % Fig 3. The frequencies of antibiotic resistance in bacteria species from (a) seawater samples and (b) sediment samples

Table II Multiple Antibiotic Resistance Indexes and Resistance Ratios Number of antibiotics to which bacteria show MAR Index Resistance, % p-value resistance 1 0.0052 0.75187 0.3635 2 0.0104 18.0451 0.6513 3 0.0022 12.7819 0.1323 4 0.0294 12.7819 0.1325 5 0.048 9.7744 0.3635 6 0.0576 14.2857 0.4084 7 0.0208 31.57894 0.1234

Actinomycetales. The frequencies of resistance to and Fe2+ were detected as an average of 33.3%, Cu2+, Zn2+, Pb2+, Cr2+ and Fe2+ were detected as 30.3%, 25.5%, 35.3% and 28.4% respectively in an average of 58.3%, 33.8%, 32.1%, 31.0% and 158 strains isolated from the sediment samples. 25.2% respectively in 258 bacterial strains isolated The frequencies of heavy metal-resistant bacteria from seawater samples. isolated from sediment samples were higher than The frequencies of heavy metal resistance in the frequencies of heavy metal-resistant bacteria bacteria species isolated from the sediment samples isolated from the seawater samples. Table III were recorded as 100% in Αlphaproteobacteria, shows the heavy metal resistance in bacteria 100% in Betaproteobacteria, 97.5% in isolates from seawater and sediment in Güllük Bay. Flavobacteriia, 95% in Gammaproteobacteria, The MICs of the isolates ranged from 0.004 mM 72.5% in Bacilli and 66.6% in Actinomycetales. The to 2.5 mM. The isolates from sediment samples frequencies of resistance to Cu2+, Zn2+, Pb2+, Cr2+ obtained from stations close to fish farms showed

Table III Heavy Metal Resistance in Bacteria Species from Seawater and Sediment in Güllük Bay, Turkey Resistant Metal concentrations, µg ml–1 Isolates Heavy Sampling isolates metals sides 0.8 1.6 3.1 6.5 12.5 25 50 100 200 >200 n n % Seawater 3 3 3 7 8 9 10 11 6 – 258 149 58.3 Cu2+ Sediment 7 4 9 3 6 17 17 29 11 – 158 53 33.3 Seawater 3 6 4 9 8 7 11 8 2 – 258 86 33.8 Zn2+ Sediment 4 2 2 8 4 19 36 22 6 – 158 48 30.3 Seawater 1 8 2 7 7 10 11 12 2 – 258 82 32.1 Pb2+ Sediment 1 9 4 4 3 18 13 23 17 – 158 40 25.5 Seawater 3 2 7 4 6 7 12 6 4 16 258 79 31.0 Cr2+ Sediment 3 4 5 8 4 9 11 10 27 32 158 56 35.3 Seawater 1 2 – – 9 24 15 6 – – 258 67 25.2 Fe2+ Sediment 3 13 3 5 9 15 24 29 – – 158 45 28.4 Total number of tested isolates 416 higher frequency of resistance against chromium, in another study (48). For example, there were copper and zinc than other stations. The highest significant increases in numbers of bacteria resistant resistance (MIC value: 2.5 mM) was displayed to oxytetracycline, oxolinic acid and florfenicol against Cr+ by all isolates. Bacillus isolates showed a in sediments from an aquaculture site compared higher resistance to chromium, lead and copper than with those from a non-aquaculture control site. Pseudomonas isolates, and Vibrio isolates showed Interestingly, in another study a similar number higher resistance to zinc, copper and chromium than of antibiotic-resistant bacteria were isolated from Escherichia coli. Tolerance to the maximum MIC aquaculture and non-aquaculture sites (49). Gram- (>2.5 mM) for chromium was 10.1% for Bacillus negative bacteria (predominantly Plesiomonas and 0.8% for Pseudomonas isolates. Bacillus isolates shigelloides and Aeromonas hydrophila) were from sediment samples showed higher resistance to isolated from aquaculture ponds in the south- chromium, lead, iron and copper than Klebsiella spp. eastern USA and it was reported that antibiotic and Escherichia coli strains from seawater samples. resistance to tetracycline, oxytetracycline, Similarly, Shewanella spp. and Serratia spp. strains chloramphenicol, ampicillin and nitrofurantoin from the sediment samples also showed higher were higher in antibiotic-treated ponds compared resistance than the species mentioned above. to non-treated rivers (50). It was determined that bacteria isolated from Sopot Beach, Poland, were 4. Discussion resistant to ampicillin (51). A high percentage of bacteria were reported as resistant to streptomycin Indicator bacteria levels reported in Güllük Bay (100%), cefazolin (89.8%), ampicillin (83.7%) and and the presence of pathogenic bacteria (25, 26) trimethoprim-sulfamethoxazole (69.4%), whereas support the relationship between the resistance data a low percentage of bacteria were resistant to detected in the current study with bacteriological cefepime (12.3%) and meropenem (14.3%) in the pollution levels. In the present study, bioindicator aquaculture region of İskenderun Bay, Turkey (52). bacteria showing human-induced pollution input In the current study, higher numbers of sulfonamide, isolated from seawater had the highest frequency rifampicin and ampicillin-resistant bacteria were of resistance against nitrofurantoin (100%) and recorded in the stations around aquaculture areas sulfonamide (95%). Sulfonamides were the first than other stations. Sphingomonas paucimobilis, antibiotics developed for clinical use. Sulfonamides Escherichia coli and Enterobacter cloacae isolated have been widely used to treat bacterial and from both seawater and sediment at the stations protozoan infections in humans, domestic animals around aquaculture areas had the highest levels and fish since their introduction to clinical practice of antibiotic resistance. The development of in 1935 (45–47). The results of higher resistance resistant pathogens in aquaculture environments against sulfonamide in the present study were is well documented (53, 54) and evidence of similar to the findings of sulfonamide resistance transfer of resistance encoding plasmids between

519 © 2020 Johnson Matthey https://doi.org/10.1595/205651320X15953337767424 Johnson Matthey Technol. Rev., 2020, 64, (4) aquaculture environments and humans has been was found to be 2.6 times greater in the stations presented recently (55). It has been reported that around fish farm areas (0.057) than the other antibiotic‑resistant bacteria are present in a seafood stations (0.022). ecosystem where antibiotics have never been used Marine sediments offer more informative results (56). This is interesting in terms of showing that than seawater about environmental pollution aquaculture areas may be adversely affected by due to the accumulation of various pollutants the presence of environmental antibiotic-resistant at the bottom of the sea, therefore analysis of bacteria. sediments is widely used in tests. The association In the present study, a high percentage of the of microorganisms with sediment particles is one bacteria Sphingomonas paucimobilis were isolated, of the primary factors in assessing microbial fate which was especially prevalent in Güllük Bay. The in aquatic systems. In this study, the bacteria natural habitat of Sphingomonas has not been isolated from sediment in all samples showed a defined, but it is widely distributed in the natural higher resistance rate than bacteria isolated from environment especially in water and soil (57). The seawater. Detection of higher antibiotic resistance second most prevalent species were Escherichia in sediment bacteria than bacteria isolated from coli and Enterobacter cloacae. Escherichia coli is seawater showed that sediment bacteria were an indicator of faecal contamination in aquatic exposed to more antibiotics. Natural ecosystems environments. Enterobacter cloacae is the most containing high concentrations of heavy metals frequent species associated with nosocomial are also frequent. Heavy metal resistance infections along with Klebsiella pneumoniae that genes are commonly found in environmental is a growing problem in human healthcare. The bacteria (71). The resistance to seven highest number of Bacillus cereus was isolated heavy metals has been reported in the order from the sediment underneath fish farms. A few Cu > Mn > Ni > Zn > Pb > Cd > Fe for seawater Bacilli of marine origin have been reported to bacteria isolated from the Golden Horn, Istanbul, produce unusual metabolites different from those Turkey (17). Heavy metal resistance in bacteria isolated from terrestrial bacteria (58). Due to the found in seawater from the Mediterranean has ubiquity and ability of the Bacillus species to survive been reported as Cd > Cu > Cr = Pb > Mn; in under difficult circumstances, Bacillus strains Karataş, Turkey Cd > Cu > Cr = Mn > Pb; and are considered to be species of certain habitats İskenderun Bay, Cu > Cd > Mn > Cr > Pb (72). (59, 60). In the current study, Bacillus pumilus, In the present study, resistance to five different B. thuringiensis, B. mycoides and B. cereus were heavy metals (Zn2+, Pb2+, Cu2+, Cr3+ and isolated from the sediment samples of the stations Fe3+) were investigated for all isolates. Trends around fish farms. in heavy metal resistance vary depending on The high frequency of resistance among bacterial the sample sites: Güllük Bay, fish farm water isolates in the present study confirms the earlier column: Cu > Zn > Pb > Cr > Fe; sediment: reports regarding the role of antimicrobial use that Cr > Cu> Zn > Fe > Pb. Frequency of bacteria plays a role in selecting antibiotic-resistant bacteria resistance to heavy metals shows the direct in water and aquatic sediments (46–52). Many effects of metal pollution. Neisseria animaloris, previous studies have shown that the increases in Aeromonas caviae and Bacillus cereus isolated antibiotic resistance in human medicine, agriculture from sediment samples were the most tolerant and aquaculture are directly related to the amounts of all the heavy metal salts. Chryseobacterium of antimicrobials used (61–65). indologenes displayed the highest degree of Infections caused by antibiotic-resistant bacteria sensitivity to all metal salts while Lactococcus are one of the most important public health garvieae showed the highest degree of sensitivity concerns worldwide. Currently, MARs have been to Zn2+, Pb2+, Cu2+ and Fe3+. Kocuria kristinae, reported in a wide range of human pathogenic Escherichia coli and Acinetobacter lwoffii, which or opportunistic bacteria such as Vibrio sp. (66), were isolated from the seawater underneath the Klebsiella pneumoniae (67), Salmonella sp. (68), fish farm, displayed similar sensitivities to all Pseudomonas aeruginosa and also in pathogens tested heavy metal salts. Resistances to heavy (69, 70). Reservoirs of antibiotic resistance can metals for Aeromonas and Pseudomonas isolates interact between different ecological systems and were similar to those from İskenderun Bay, with potential transfer of resistant bacteria or resistant cadmium, 35.0% and 56.5%; copper, 98.3% and genes from animals to humans may occur through 75.4%; chromium, 38.3% and 31.9%; lead, 1.7% the food chain (70). In the current study, the and 7.2%; manganese, 43.3% and 44.9%; and MAR index of multiple antibiotic-resistant bacteria zinc 35.0% and 41.3%, respectively (72).

Both Gram-positive and Gram-negative bacteria pesticides and heavy metals might encourage can resist heavy metals (73). Resistance to selection and result in antibiotic and heavy metal toxic metals in bacteria probably reflects the resistance. Marine environmental conditions are level of environmental contamination with extremely dynamic compared to the terrestrial these substances and it may be related to the environment, allowing bacteria to bring resistance concentration of bacteria (74). The present project mechanisms they have developed together while found heavy metal pollution in Güllük Bay sediment being adapted to the varying conditions. This samples at all stations. In the sediment samples, makes the isolation of various bacteria useful to the heavy metal contents were reported at varying assess environmental pollution and provides a rates: between 1 μg g–1 and 209 μg g–1 for lead; pathway to possible solutions to remove pollution 10 μg g–1 and 259 μg g–1 for zinc; 1 μg g–1 and from marine environments. For bacteria to take 59 μg g–1 for copper; 0.1 μg g–1 and 46 μg g–1 part in the transformation of any heavy metal salt for chromium; <0.01 μg g–1 and 2.8 μg g–1 for into a harmless form, those bacteria must firstly cadmium; <0.01 μg g–1 and 0.4 μg g–1 for arsenic; be resistant to the heavy metal; thus the data and 0.6% and 5.9% for aluminium, respectively. related to frequency of metal resistant bacteria can The region was defined according to cadmium, lead provide knowledge on the continual accumulation and zinc levels as moderately polluted. Recorded or transformation of heavy metals in the marine high metal values were evaluated as an indicator of environment. domestic and industrial inputs, carried via Sarıçay The findings of the current study provide Creek, port operations and tourism activities data regarding the distribution of heavy metal- within Güllük Bay (75). In the current study, the and antibiotic-resistant bacteria in seawater high frequencies of heavy metal-resistant bacteria and sediment samples of Güllük Bay, Aegean detected in the sediment samples support this Sea, Turkey. As a result, preliminary data on data. Bacterial heavy metal resistance detected candidate bacteria will offer opportunities for in the study may depend on many factors. A further studies on the elimination of heavy metal possible explanation for differences in heavy metal contamination by the detection of heavy metal- resistance is the proximity of Güllük Bay to iron-steel resistant bacteria. factories. Additionally, Güllük Harbour is a serious pollution source. It was reported that 2862‑unit 5. Conclusions ships carried 4.8 million tonnes of ballast water to Güllük Harbour during 2007–2012 (37). Another Analyses of the presence of antibiotic resistance potential source of increased resistance may be the in bacteria provide knowledge on pollution sources discharge of thermal power plants located 107 km, such as septic systems on regional ecosystems. 46 km and 39 km away from Güllük Bay. The Since antibiotic-resistant bacteria can affect effects of thermal power plant discharge on the pathogen virulence, these pollution sources can accumulation of heavy metals have been reported induce pathogens and can create health risks in other studies (29, 75). for both humans and the ecosystem. In the The association between antibiotic resistance present study, bacteria resistant to antibiotics and resistance to heavy metals is quite common and heavy metals in seawater and sediment were in the same organism. The increasing numbers investigated. The bacterial information obtained of antibiotic and heavy metal-resistant bacteria provides essential data for identifying the regional could be a result of gene transfer activities distribution of resistant bacteria. Levels of demonstrating that industrial pollution most resistance against heavy metals and antibiotics in likely selects for antibiotic resistance and vice bacteria isolated from seawater and sediments of versa (58). In this study, similarly, the most the Aegean Sea were quantified. Bacteria isolated antibiotic-resistant bacteria such as Sphingomonas from Güllük Bay sediment were resistant to all paucimobilis, Escherichia coli and Enterobacter antibiotics tested and exhibited higher resistance cloacae were also resistant to heavy metals. than those isolated from seawater. The frequency Metal‑resistant isolates from Güllük Bay also of antibiotic-resistant bacteria was higher around showed high resistance to sulfonamide, rifampicin fish farms and near the exit of Sarıçay Creek. The and ampicillin. Bacteria from different sources widespread resistances of indicator bacteria to such as humans, animals and soil can transfer or antibiotics suggest the presence of anthropogenic exchange their resistance genes. At the same time, influences due to domestic waste and maritime water contaminated with antibiotics, disinfectants, transport.

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The Authors

Gülşen Altuğ is a professor in the Department of Marine Biology of the Faculty of Aquatic Science at Istanbul University, Turkey. Her research focuses on marine bacteriology, including bacterial diversity and micro-geographical variations, clinical, industrial and ecological uses of marine isolates, bacterial pollution, epibiotic bacterial communities and anti-bacterial characteristics, bacterial remediation (oil degrading capacity of marine isolates) and resistant bacterial isolates against heavy metals and antibiotics. She is also the inventing founder of the biotechnology company named BIYOTEK15 R&D Training and Consulting Industry and Trade Ltd Company in Entertech of Istanbul University Technocity.

Mine Çardak is an associate professor at Çanakkale Onsekiz Mart University, School of Çanakkale Applied Sciences, Department of Fisheries Technology, Turkey. Her researches focus on marine bacteriology, bacterial resistance against heavy metals and antibiotics, bacterial pollution and biotechnology. She has worked as a scientist since 2000.

Pelin Saliha Çiftçi Türetken is a researcher at İstanbul University, Faculty of Aquatic Sciences, Department of Marine Biology. Her research focus on marine bacteriology, bacterial remediation, bacterial resistance and biotechnology. She has a PhD degree in marine biology. She has worked as an academic at university since 2005.

Samet Kalkan has a PhD degree from Istanbul University, Institute of Graduate Studies in Science and Engineering, Department of Marine Biology. He currently works as a doctor scientist at Recep Tayyip Erdogan University- Faculty of Fisheries, Department of Marine Biology, Turkey. He has worked as academic at university since 2010. His main researches focus on marine bacteria, bacterial diversity, bacterial pollution, resistant bacteria against heavy metals-antibiotics, also marine biotechnology. He has scientific abroad experiences in Italy and Portugal.

Sevan Gürün graduated with a degree in Biology from Istanbul University. He has a PhD degree from Istanbul University, Institute of Graduate Studies in Science and Engineering, Department of Marine Biology. He worked as a researcher in various scientific projects. He has been working as a researcher in a private company since 2016. His expertise focuses on bacterial diversity, marine bacteria, bacterial pollution, bacterial biotechnology, resistant bacteria against heavy metals and antibiotics.