APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Mar. 1994, p. 908-912
Vol. 60, No. 3
0099-2240/94/$04.00+O Copyright ©) 1994, American Society for Microbiology
Distribution of Tetracycline Resistance Determinants among Gram-Negative Bacteria Isolated from Polluted and Unpolluted Marine Sediments SIGRID RITA ANDERSEN1 AND RUTH-ANNE SANDAA2* Department of General Microbiology, University of Copenhagen, DK-1307 Copenhagen K, Denmark,' and Department of Microbiology, University of Bergen, N-5020 Bergen, Norway2 Received 6 July 1993/Accepted 31 December 1993
Tetracycline-resistant gram-negative bacteria were isolated from four different marine sediments in Scandinavia and analyzed with DNA probes for the determinant classes A to E. Colony hybridizations of 429 isolates revealed that class E is the dominating resistance determinant in these marine sediments. Comparison of fecally polluted and unpolluted sediments showed few determinant classes in unpolluted sediment and a complex composition of several determinant classes in polluted sediment. Total DNA extraction and analysis with DNA probes for determinant classes A to E resulted in no hybridization signal, because of the low number of gram-negative tetracycline-resistant bacteria. Identification of class E isolates revealed that this determinant is present not only in Aeromonas hydrophila, Escherichia coli, and Vibrio salmonicida but also in additional strains.
Solheimsviken (Bergen), close to 12 sewage outlets with untreated hospital and domestic sewage. These outlets correspond to 66,000 person equivalents. Fonnosen (Stord, Norway) was chosen as the sampling site for the unpolluted sediments as this inlet, situated directly on the North Sea is considered undisturbed by human activity. Both Norwegian sampling sites were located on the west coast of Norway. In Denmark, samples from both polluted and unpolluted marine sediments were collected from 0resund. The sewage outlet of a major treatment plant, Lynetten, was chosen as the sampling site for polluted sediment. The treatment plant has a capacity of 1.6 million person equivalents. The volume of treated sewage discharges in the water body is 230,000 m3/day. The unpolluted samples were collected from Holltenderdybet, a separate branch of 0resund, which, because of the marine currents, has no direct connection to the water polluted with sewage. Since the whole 0resund contains intermediate levels of pollution, we recognize that these unpolluted samples cannot be regarded as totally pristine. Two samples from each site were collected with a grab and transported immediately to the laboratory for analysis. Portions of the samples, applied later for total DNA extraction, were frozen at - 20°C. Bacterial count and isolation. Sediment (10 g) was homogenized at low speed for 1 min in a Waring commercial blender with 90 ml of dilution medium. Dilution medium consisted of 70% seawater autoclaved twice with an interval of 2 days. Serial dilutions in 90 ml of 70% seawater were spread in triplicate on tryptone soya agar (TSA), which contained, per liter, 30 g of tryptone soya broth (Oxoid Ltd., London United Kingdom), 10 g of NaCl, and 15 g of agar (Difco Laboratories, Detroit, Mich.), for determination of the total number of CFU. Aliquots of dilutions were also spread on TSA containing 5 ,ug of tetracycline (Sigma) per ml for selection of tetracyclineresistant strains. The plates were incubated at 15°C for 4 to 7 days. Colonies from TSA plates were replicated to MacConkey plates (MacConkey agar no. 3; Oxoid) to determine the frequencies of presumptive gram-negative isolates. The number of lactose-fermenting coliforms were identified as red colonies on MacConkey agar. Colonies from TSA plates with 5 ,ug of tetracycline per ml were replicated to TSA plates
High levels of antibiotic-resistant microorganisms in natural environments are a direct consequence of the extensive use of antibiotics for medical and veterinary purposes during the past 40 years. Several studies have focused on the incidence of antibiotic-resistant bacteria in marine environments (5, 11) and the horizontal transfer of genes in marine sediments (29, 30). Tetracycline resistance is widely disseminated among various bacterial species (17). In 1980, Mendez et al. (19) grouped naturally occurring plasmid-encoded tetracycline resistance determinants into four classes. They concluded that the frequencies of these resistance determinants result from the use of tetracyclines for treatment of humans, animals, and plants. At present five classes of genetically distinguishable tetracycline resistance determinants, designated A through E, have been described among aerobic enteric gram-negative rods (17, 19). The frequencies of tetracycline resistance determinant classes have been examined in coliforms from human or animal fecal specimens (18) and in fish pathogenic bacteria (3, 8). In the present study we attempt to characterize samples from natural environments by examination of randomly selected tetracycline-resistant isolates. We group the sediment isolates by using the five probes (A to E) for tetracycline resistance determinants. The objectives of this report were to examine (i) the distribution of tetracycline resistance determinants among gram-negative, aerobic bacteria in marine sediments, (ii) the difference between occurrence of tetracycline resistance determinants in polluted and unpolluted environments, and (iii) the association of genera with tetracycline resistance determinant E in the open environment.
MATERIALS AND METHODS Sampling. Samples of marine sediments with and without direct human fecal pollution were collected in June 1992. In Norway, the polluted marine sediments were collected from *
Corresponding author. Fax: 47 55 32 39 62. Electronic mail address:
[email protected]. 908
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TETRACYCLINE RESISTANCE DETERMINANTS IN MARINE SAMPLES
TABLE 1. DNA probes constructed and used in hybridization experiments Tetracycline
E.
determinant
strain
TetA TetB TetC TetD
JM83 HBlOl DO-7 C600
TetE
HBIO
coli
Restriction fragment used as DNA probe
750-bp SinaI fragment of pSLI8 1,275-bp Hinzcll fragment of pRTI 1 929-bp BstNI fragment of pBR322 3,050-bp HiindIII-Pstl fragment of pSL106 2,500-bp ClaI-PvuI fragment of pSL1504
Refer-
ence
18 7 18 18
17
containing 25 plg of tetracycline per ml, incubated further for 4 to 7 days, and replicated to MacConkey plates containing 25 ,ug of tetracycline per ml. Colonies growing on medium with 25 ,g of tetracycline per ml were considered tetracycline resistant. In Norway, a total of 204 isolates from polluted (112 isolates) and unpolluted (92 isolates) samples were randomly picked from MacConkey plates containing tetracycline. Likewise, in Denmark, a total of 225 isolates from polluted (150 isolates) and unpolluted (75 isolates) samples were randomly picked. Tetracycline-resistant isolates were analyzed by the KOH method for Gram reaction (6). Colony hybridization. Colony blots were prepared from the tetracycline-resistant isolates. The colonies were transferred to nylon membrane filters (BA85; Schleicher & Schuell, Dassel, Germany) with sterile Q-tips. Colony blotting was performed on the basis of the method of Grunstein and Hogness (12), modified by Sambrook et al. (24). After colony blotting the filters were dried and baked for 2 min in a microwave oven. The DNA probes for the five tetracycline resistance determinant classes, A to E, were prepared from different Esclherichia coli strains harboring plasmids carrying the different types of tetracycline resistance determinants (Table 1) (kindly provided by Stuart B. Levy, Department of Molecular Biology and Microbiology, Tufts University, Boston, Mass.). Plasmids were prepared by using the Qiagen kit for midi-plasmid preparation (Qiagen, Inc., Chatsworth, Ga.). Restriction enzyme assays were performed according to the manufacturer's instruction (Boehringer GmbH, Mannheim, Germany). The purified restriction DNA fragments were labeled by using nonradioactive random primed DNA labelling and the digoxigenin luminescence detection kit (Boehringer GmbH), following the manufacturer's instructions. The filters were hybridized with the nonradioactive tetracycline resistance probe under stringent conditions in 10 ml of hybridization buffer (50% [vol/vol] formamide, 5 x SSC [20 x SSC is 3 M NaCl plus 0.3 M sodium citrate] [pH 7.0], 2% blocking reagent [Boehringer GmbH], 0.1% N-lauroylsarcosine, 0.02% sodium dodecyl sulfate [SDS]) for 6 h at 42°C. After hybridization, the filters were washed two times for 5 min (each) in 2 x SSC-0.1% SDS at room temperature and twice for 15 min (each) in 0.1 x SSC-0.1% SDS at 68°C. The detection was performed by using a chemiluminescent alkaline phosphatase substrate, AMPPD [3-(2'spiroadamantane)-4-methoxy-4-(3"-phosphoryloxy)phenyl-1 ,2dioxetane, disodium salt], following the manufacturer's instructions. The filters were visualized by exposure of the membrane filters to X-ray film for I h. As positive and negative controls, the tetracycline resistance determinants A to E were included on each filter. Extraction of total DNA. Two methods were applied for extracting total DNA from the sediments collected from Solheimsviken. Total DNA from sediment samples was iso-
9(9
lated by the direct extraction method described by Ogram et al. (20), with some modifications. Samples (10 g) were suspended in 20 ml of 1.0 mM NaPO4 (pH 7.0). SDS was added to a final concentration of 1.0%. Samples were heated to 70°C for 40 min, with occasional mixing. Five grams of both large glass beads (1.0-mm diameter) and small glass beads (0.2-mm diameter) was added to the mixture, and the mixture was incubated on a vortexer for 30 min at room temperature. The pellet was cleared by centrifugation at 8,512 x g for 15 min at 10°C, and the supernatant was transferred to a fresh tube. KCl was added to a final concentration of 1 M, and the mixture was shaken for 10 min. The samples were incubated on ice for 30 min to precipitate the SDS. The lysate was cleared by centrifugation at 8,512 x g for 15 min at 10°C and transferred to a clean tube. DNA was extracted twice with equal volumes of phenol equilibrated to pH >7.8 (24) and once with equal volumes of chloroform-isoamyl alcohol (24:1 [vol/vol]). After each extraction the mixture was centrifuged at 12,000 x g for 15 min at room temperature. The aqueous phase recovered from the extraction was precipitated with isopropanol overnight at room temperature and centrifuged for 40 min at 12,000 x g at 40C. The DNA pellet was washed with cold 75% ethanol. Dried pellets were resuspended in 2 ml of sterile distilled water. Total DNA from four 10-g sediment samples, collected from Solheimsviken, was also isolated by the method for bacterial fractionation and lysis as described by Torsvik et al. (31). The bacterial fractionation procedure was performed with the following modifications (30a). Ultramarine solution (Waterlife Research Industries Ltd., West Drayton, Middlesex, England) was used instead of Winogradsky's salt solution, and 1.5% (wt/vol) NaCl was added to the Crombach buffer. Analysis of total DNA. One hundred microliters of the DNA samples and dilutions of E. coli D22-14 with the tetracycline resistance determinant E was slot blotted onto a nylon filter (Schleicher & Schuell) with Minifold I (Schleicher & Schuell), according to the manufacturer's instructions. Before the samples were applied to the manifold, 0.1 volume of 3 M NaOH was added the samples, and the tubes were incubated for I h at 65°C. The suspension was then cooled to room temperature, and 6 x SSC was added. The samples were then applied to the manifold. After slot blotting, the filters were dried and baked for 2 min in a microwave oven. Hybridization was performed with the determinant classes A to E at 42°C for 6 h by using the stringency conditions described for colony hybridization. Identification. Isolates that responded to the class E probe or none of the five probes were subject to further examinations. These isolates were tested for Gram reaction, oxidase test, oxidation and fermentation of glucose in Hugh & Leifson medium, and motility and cell morphology by microscopy of liquid cultures of tryptone soya broth. From each sampling site, a number of isolates representing all the different phenotypes were selected for further identification with API20E and API2ONE (api Bio-Merieux SA, Marcy l'Etoile, France). Two isolates were further analyzed by using the GN Microplate (BIOLOG Inc., Hayward, Calif.) system for growth on 95 different carbon sources. RESULTS The total bacterial counts on TSA and MacConkey agar and the frequencies of Gram-negatives and tetracycline-resistant colonies are shown in Table 2. The two samples from each of the Norwegian sites were treated separately, while the Danish samples were pooled and mixed thoroughly. The frequencies of CFU able to grow on MacConkey agar varied from 8.9 to
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TABLE 4. Identification of isolates belonging to tetracycline resistance determinant class E
TABLE 2. Total number of CFU (TSA), number of red CFU indicating lactose-fermenting coliforms, and percentage of CFU cultivable on MacConkey agar and demonstrating tetracycline resistance in unpolluted and polluted marine sediments CFU/g (wet wt) Sampling site
TSA
Norway Unpolluted Unpolluted Polluted Polluted
Red
Cultivable on
MacConkey agar
Showing tetracycline resistance
9.7 1.2 4.5 1.2
NDa ND
0.29 0.05 0.80 5.50
0.28 0.11
104 105
x 105 x 107
4 x 104 8 x 104
10.3 10.8 8.9 83.3
5.4 x 103 4.1 x 105
7.9 x 10 2.8 x 104
15.4 18.0
x x
No. of isolates
site
identified/
Norway Polluted Unpolluted Denmark Polluted
10/70 11/88
19b/66
Unpolluted
7/13
% of total CFU
colonies on MacConkey
Total
Sampling
Denmarkb
Unpolluted Polluted a
ND, not detected. Two sediment samples from each site
were
pooled before analysis.
Species (no. of isolates)
Aeromonas hydrophila (10) Marine Alcaligenes Sp.a (1)
Aeromonas hydrophila (6) Aeromonas hydrophila or Aeromonas caviae (2) Aeromonas salmonicidac (1) Aeromonas sp. (low discrimination) (2) Escherichia coli (1) Serratia marcescens (1) Providencia stuartii (1) Pseudomonas maltophilia (6) Vibrio fluvialis (1)
No responses with API20NE; one isolate was characterized with BIOLOG. b Five isolates could not be identified by genus and species. c No response with the Aeromonas salmonicida antibody test.
a
83.3%. The highest percentage of tetracycline-resistant isolates in the Norwegian samples was found in the polluted samples, while the highest percentage of tetracycline-resistant isolates in the Danish samples was seen in the unpolluted sample. Examination of all isolates for hybridization to the class A to E probes revealed that class E was the most frequent of the five tetracycline resistance determinants in all samples (Table 3). All five tetracycline resistance determinant classes were detected in the Danish samples from polluted sediment. Statistical analysis of the hybridization results (Table 3) was performed by analysis of variance by using a general linear model (26). By testing the variance between the Norwegian and Danish samples, the unpolluted and polluted samples, and the eight categories of resistance determinants found, significant difference was found only between the different resistance determinant classes (P < 0.0002). Furthermore, Duncan's multiple range test for differences in means revealed that class E deviated significantly from the other classes. In both cases the significance level was 5%. Samples from Solheimsviken exhibited the highest number of tetracycline-resistant bacteria (Table 2) and was chosen for total DNA extraction by two procedures. Total DNA gave no signals when probed with the tetracycline determinant probes A to E. The detection limit,
TABLE 3. Distribution of tetracycline resistance determinant classes in polluted and unpolluted samples No. of isolatesa Class
Norway Polluted
Denmark
Unpolluted
A
6 (5.3)
0
B
0 0
0 0
C
D
4 (3.6) 70 (62.5) 1(0.9) 29 (25.9) 2 (1.8)
E AE DE None
Total
no.
112
0 88 (95.7) 0 0 4 (4.3)
92
Polluted
Unpolluted
7 (4.7) 5 (3.3) 20 (13.3) 15 (10.0) 66 (44.0) 2 (1.3) 14 (9.4) 21(14.0)
0 0 6 (8.0) 6 (8.0) 13 (17.3) 0 6 (8.0) 44 (58.7)
150
75
Numbers in the parentheses indicate the frequency of each determinant class expressed as a percentage of the total number of isolates. a
with the nonradioactively labelled probe for the tetracycline determinant E, was 2.5 x 105 cells per ml. The identification of isolates responding to the class E probe are listed in Table 4. From the Norwegian samples, all isolates from each sampling site belonged to a single phenotype, indicating that a single species was the dominating carrier of the tetracycline resistance genes in this environment. From the Norwegian polluted site, all class E isolates were Aeromonas hydrophila. Similarly, according to the preliminary tests, all isolates from the unpolluted site in Norway had identical phenotypes. These isolates gave no positive reactions with the API2ONE or API20E identification kit. Alternatively, a single representative isolate was tested with the BIOLOG system. These test results corresponded well with the description of the genus Alcaligenes. Fifty-three BIOLOG characters were relevant for the identification of different Alcaligenes species. The best correspondence was obtained for Alcaligenes aestus (6 of the 53 C sources gave a deviating response). Since this marine species is considered to be species incertae sedis (13), we will denote these isolates as marine Alcaligenes spp. The Danish samples seemed to have a more complex distribution of the tetracycline resistance determinants among different bacterial species. Whereas Aeromonas hydrophila and other Aeromonas species had a dominating role, we also identified E. coli, Serratia marcescens, and Providencia stuartii among the class E isolates in the Danish polluted samples. One isolate was identified with the API20 NE as Aeromonas salmonicida. This isolate was tested by immunofluorescence microscopy, with monoclonal antibodies specific to Aeromonas salmonicida outer lipopolysaccharides (9). No reaction was observed, confirming that this isolate most probably was not Aeromonas salmonicida. Five isolates, representing three different phenotypes, could not be identified with the API system. In the Danish unpolluted samples, Vibrio fluvialis was represented together with Pseudomonas maltophilia. The results of identification of isolates with tetracycline resistance determinants belonging to none of the five classes are shown in Table 5. These isolates had a pattern of species somewhat similar to that of the class E isolates. Again the isolates from Norwegian polluted and unpolluted sites were identified as Aeromonas hydrophila and marine Alcaligenes
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TETRACYCLINE RESISTANCE DETERMINANTS IN MARINE SAMPLES
TABLE 5. Identification of isolates from the category of tetracycline-resistant bacteria belonging to none of the five tetracycline resistance determinant classes Sampling site
No. of
isolaites
Species
identified/
(no. of isolates)
Norway
Polluted Unpolluted Denmark Polluted
2/2 4/4
9'/2 1
Aeromoncas hldrophila (2) Marine Alcaligenies sp." (I)
Aeromoncas hydrophila (3) Aerornonas hlydrophila or Aeronronias sobria (I) Aeromoonas sobria (1)
Unpolluted
5'/44
Pseudionionas fllorescens 1 (1) Pseuidomonas fluorescenis 1 (3) Pseludornonas maltophilia (I)
No responses with API2()NE; one isolate was characterized with BIOLOG. Three isolates could not be identified by genus and species. One isolate could not be identified by genus and specics.
spp., respectively. Also in the Danish polluted site, Aeromonlas hydrophila seemed to dominate. One isolate was identified as Pseuclomonasflluorescens 1, and three isolates were not identified. In the Danish unpolluted site we found Pseudomonias flluorescents 1, Pselidomonas maltophilia, and one isolate that was not identified with the API system. The four unidentified isolates had identical phenotypes and seemed to represent a single bacterial species.
DISCUSSION Our results show that the tetracycline resistance determinant E was the most dominating resistance determinant in the samples both from the polluted and unpolluted marine sediments in Norway and Denmark. Although local differences can be detected, the class E seems to be the indigenous tetracycline resistance determinant for microorganisms in these marine sediments. The actual number of tetracycline resistance determinants in the marine environment may be higher considering that a high percentage of the bacteria in the environment may exist in a viable but not culturable state (23). The unpolluted sediments from Norway contained only class E determinants, whereas the other sediments hosted a variety of tetracycline resistance determinant classes. The Danish polluted sediment had the most complex composition, with eight categories of resistance determinants represented. The composition of tetracycline resistance determinants among selected gram-negative bacterial species has previously been investigated with the five probes. Among lactose-fermenting, tetracycline-resistant coliforms isolated from fecal samples, class B was found most frequently (73.3%), while classes A and C occurred less frequently (18). One isolate was later demonstrated to contain the class E determinant (17). Tetracycline resistance determinant class D is rarely found in lactose-fermenting bacteria (18) but is common in fish pathogens in Japan (4). In Norway, the tetracycline resistance determinant class E has been detected in the fish pathogenic bacteria Vibrio salmonicida (28), Aercornonas salmonicida, and Vibrio anguillaruim (27a). A later study of A. hydrophila, from an aquaculture environment in the United States, revealed that 50% of the isolates were class E, 35% were class A, one isolate contained both determinants, and four isolates were neither class (8). In a recent study by Lee et al. it was demonstrated that class B was the most predominant type of tetracycline
911
resistance determinant among strains isolated from pigs exposed to antimicrobial agents, whereas classes C and E were the most common tetracycline resistance determinants for isolates from pigs in a herd not exposed to antimicrobial agents (14). Although class E was the most prevalent of the five determinant classes among the sediment bacteria, a number of resistant isolates from each sample did not hybridize with any of the available probes. This was especially pronounced in the Danish samples. Similar findings have previously led to the discovery of new tetracycline resistance genes (2, 3, 21), suggesting that more tetracycline resistance determinants among gram-negative bacteria are yet to be identified. Using the DNA probes for the determinant classes (A to E) in the total DNA extraction resulted in no hybridization signal. Most probably the number of gram-negative tetracyclineresistant bacteria were below the detection limit for the nonradioactively labelled probe. It has been shown that certain of the resistance determinants may be more prevalent in some genera (15). For example, class E, originally isolated from E. coli (17), appears to be frequent also in Aeromonas hydrophila (8, 16). In our study the class E determinant was found in at least six other species besides Aeromonas hydrophila and E. coli. Since gene transfer does occur in marine sediments (22, 25), it would be natural to assume that the tetracycline resistance determinant genes spread between different bacterial species in the marine sediment. The species composition of the tetracycline-resistant population may therefore be variable. In Norway, the total consumption of tetracycline for human and veterinarian purposes in 1991 was 8.6 metric tons (statistics provided by The Norwegian Medicinal Depot, Oslo, Norway). In Denmark, information about consumption of tetracycline is not available to the public. However, the total consumption of tetracycline was estimated to be approximately 24.0 metric tons per year, on the basis of expenditures for tetracycline agents. Not surprisingly, marine environments are the major recipients for antibiotic residues and antibioticresistant microorganisms. A recent study of the outlet of antibiotic-resistant coliforms from a mechanical-biological sewage treatment plant into 0resund showed fluctuations in the frequencies of resistant organisms in effluent-treated sewage. Tetracycline-resistant bacteria constituted between 0 and 9.9% of the total coliforms in treated sewage expelled into 0resund (1). The significance of marine sediments as reservoirs of human pathogenic bacteria and viruses has long been recognized (11). It is well-known that bacteria survive longer in a mixture of seawater and sediments than in seawater alone (10). Many studies have demonstrated that R plasmids occur naturally in sediments (11, 27) and that horizontal transfer may occur (22, 25). The ecological consequences of the disposal of fecally polluted water in marine environments may be, along with the spread of antibiotic-resistant microorganisms, a genetic pollution with resistance determinants that are foreign to the indigenous bacteria, resulting in altered distribution of resistance determinants. ACKNOWLEDGMENTS We thank Lena Bj0rn Johansson, Lise 0vreas, and Tonie Estman for isolation and hybridization of all the tetracycline resistant strains. We also thank Henrik Skovg'ard Pedersen for assistance with the statistical analysis and S. B. Levy for gifts of bacterial strains carrying the cloned tetracycline resistance determinants. This work was supported by the Council of Nordic Ministers.
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