- Email: [email protected]
Seasonal shifts in population structure of Vibrio vulnificus in an estuarine environment as revealed by partial 16S ribosomal DNA sequencing
FEMS Microbiology Ecology 45 (2003) 23^27
www.fems-microbiology.org
Seasonal shifts in population structure of Vibrio vulni¢cus in an estuarine environment as revealed by partial 16S ribosomal DNA sequencing Meilan Lin 1 , John R. Schwarz
Department of Marine Biology, Texas ApM University at Galveston, 5007 Avenue U, Galveston, TX 77551, USA Received 7 August 2002; received in revised form 24 March 2003; accepted 26 March 2003 First published online 16 April 2003
Abstract The partial sequence (600 bp) containing the most variable region of Vibrio vulnificus 16S ribosomal DNA (rDNA) was determined for 208 randomly selected V. vulnificus strains isolated from Galveston Bay, TX, USA between June 2000 and June 2001. A comparative analysis of the determined partial 16S rDNA sequences revealed the existence of two different partial 16S rDNA sequences (type A and type B, 1.3% base substitutions) among the 208 V. vulnificus isolates. A higher proportion of 16S rDNA type A strains was isolated in June and July while a considerably higher proportion of type B strains was isolated in September. In addition, after no V. vulnificus strains were detected during the winter months (December^February), only type A strains were isolated during the following months (March^May). The results suggest that the relative abundance of type A and type B V. vulnificus strains in Galveston Bay varies with the season and that the differences between the two 16S rDNA types may affect the viability of these organisms in the natural environment. 7 2003 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords : Partial 16S rDNA sequence; 16S rDNA types; Temperature; Salinity ; Population structure ; Vibrio vulni¢cus
1. Introduction Vibrio vulni¢cus is an autochthonous bacterium found in estuarine and marine environments of temperate and tropical climates [1^3]. It is an opportunistic human pathogen known to cause primary septicemia, particularly in individuals with underlying chronic diseases such as diabetes, impaired liver function and/or compromised immune systems. Infection in these persons is associated with consumption of raw shell¢sh, seawater contamination of open wounds or contact with raw shell¢sh [4^6]. V. vulni¢cus has been isolated from a wide range of environmental sources including seawater, plankton, sediment, shell¢sh and ¢sh [7^9]. The presence of the organism is favored by relatively high water temperatures ( s 20‡C) and low-to-moderate salinities (5^25 ppt) [2,10,11]. The
* Corresponding author. Tel. : +1 (409) 740 4453; Fax : +1 (409) 740 4787. E-mail address : [email protected] (J.R. Schwarz). 1
Present address: Houston Department of Health and Human Services, 1115 S. Braeswood, Houston, TX 77030, USA.
bacterium is readily isolated from water and shell¢sh during the warmer months, but the organism is di⁄cult to isolate during the colder months [9,11,12]. This decrease in culturability may be due to the entry of the organism into a viable but non-culturable state, a survival response by V. vulni¢cus to low-temperature stress [13,14]. The 16S rRNA gene (rDNA) is ubiquitous among cellular organisms and is highly conserved [15]. A comparative analysis by Aznar et al. of the complete 16S rDNA sequences of 21 V. vulni¢cus strains revealed two di¡erent 16S rDNA sequences that divide these strains into two di¡erent groups: type A and type B [16]. In a separate study, 40 V. vulni¢cus strains isolated o¡ the southern coast of Korea in August 1999 were found to be 35% 16S rDNA type A and 65% type B strains [17]. Little, however, is known about temporal changes in the relative abundance of di¡erent 16S rDNA types of V. vulni¢cus in the natural environment. In this study, internal fragments (600 bp) of the 16S rDNAs from 208 randomly selected V. vulni¢cus strains isolated from Galveston Bay, TX, USA over a 13-month period were sequenced and compared to investigate : (1) the existence of di¡erent 16S rDNA sequences among the
0168-6496 / 03 / $22.00 7 2003 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. doi:10.1016/S0168-6496(03)00091-6
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M. Lin, J.R. Schwarz / FEMS Microbiology Ecology 45 (2003) 23^27
Water and oyster samples were collected between June 2000 and June 2001 at ¢ve Galveston Bay sites (private oyster leases): Lease 301 (29‡27P57QN, 94‡42P03QW), Lease 414 (29‡29P19QN, 94‡47P56QW), Lease 433 (29‡31P53QN, 94‡48P12QW), Lease 410 (29‡29P43QN, 94‡51P33QW) and Lease 426 (29‡29P00QN, 94‡54P02QW). Samples were collected biweekly during the spring and summer months and monthly during the fall and winter months. 125 ml of seawater was collected aseptically 0.5 m below the surface and 15 oysters were harvested by dredging at each site. Water temperature and salinity at each sample site were determined using a YSI model 30 salinity meter (YSI, Yellow Springs, OH, USA). The water and oyster samples were placed into an insulated container with frozen cool packs and transported to the Seafood Safety Laboratory at Texas ApM University at Galveston, TX, USA, for bacteriological analysis. Samples were analyzed within 12 h of collection. The procedure used for isolation and identi¢cation of V. vulni¢cus is described in the USA Food and Drug Administration’s Bacteriological Analytical Manual [18]. The identities of suspect colonies were con¢rmed by enzyme immunoassay which employed a monoclonal antibody speci¢c for V. vulni¢cus [19]. Two to three identi¢ed V. vulni¢cus colonies were randomly picked from each sample that was positive for V. vulni¢cus and a total of 208 V. vulni¢cus strains isolated from Galveston Bay water and oysters were included in this study.
amplify a 996-bp fragment containing the most variable region of the V. vulni¢cus 16S rDNA. These two primers were selected from two highly conserved regions of the 16S rDNA based on the alignment of the published V. vulni¢cus 16S rDNA sequences, i.e. GenBank accession nos. X76333, X76334 [16], X74726, X74727 [20], and X56582 [21]. PCR was conducted in a 50-Wl reaction mixture containing 1.0 WM (each) primer, 1.5 mM MgCl2 , 0.2 mM (each) deoxynucleoside triphosphate (Applied Biosystems, Foster City, CA, USA), 2.5 U of AmpliTaq Gold DNA polymerase (Applied Biosystems), 5% (vol/ vol) dimethyl sulfoxide (Stratagene, La Jolla, CA, USA), and 0.5^0.6 Wg of template DNA. The reaction mixture was ¢rst subjected to an initial denaturation at 95‡C for 10 min and then 35 cycles of 94‡C for 30 s, 55‡C for 30 s and 72‡C for 75 s, followed by a ¢nal extension step at 72‡C for 10 min. Ampli¢cation was performed on a 9700 thermal cycler (Perkin-Elmer). PCR products were analyzed by electrophoresis on 1% agarose gels containing ethidium bromide (0.1 Wg ml31 ) (Pierce, Rockford, IL, USA). PCR amplicons were puri¢ed with the ExoSAP-IT kit (USB, Cleveland, OH, USA) according to the manufacturer’s instructions. After puri¢cation, a 2-Wl aliquot was used as sequencing template. The ampli¢ed fragments were directly sequenced on both strands by using the same PCR primers. Cycle sequencing reaction mixture was prepared by using the ABI Prism BigDye Terminator Cycle Sequencing v2.0 Ready Reaction kit (Applied Biosystems) according to the manufacturer’s instructions. Excessive terminators were removed by using the RapXtract Dye Terminator Removal kit (Prolinx, Bothell, WA, USA) before the samples were prepared and loaded onto the ABI Prism 310 Genetic Analyzer (Applied Biosystems) according to the manufacturer’s protocol. Sequences were automatically analyzed and corrected manually.
2.2. DNA extraction
2.4. Sequence analysis
A single colony of each strain was inoculated into heart infusion broth (Difco Laboratories, Detroit, MI, USA) supplemented with 0.5% NaCl and incubated overnight at 37‡C. Chromosomal DNA was extracted from the overnight cultures by using the QIAamp DNA mini kit (Qiagen, Valencia, CA, USA) according to the manufacturer’s instructions. The quantity and quality of the DNA were determined by UV light absorbance at 260 and 280 nm with a MBA 2000 spectrophotometer (Perkin-Elmer, Norwalk, CT, USA).
The resulting 16S rDNA sequences were aligned with the previously published V. vulni¢cus 16S rDNA sequences (GenBank accession nos. X76333, X76334 [16], X74726, X74727 [20], and X56582 [21]) by using the program BioEdit (version 5.0.9; available at http://jwbrown.mbio.ncsu.edu/BioEdit/bioedit.html). The 16S rDNA sequence of V. vulni¢cus ATCC 27562 (GenBank accession no. X76333 [16]) was used as a reference. Only the homologous regions of the aligned sequences were included in sequence comparisons, with the low-quality sequences on the ends and the sequences close to the ends with ambiguous bases excluded from the comparisons.
V. vulni¢cus strains, and (2) the temporal changes of V. vulni¢cus population structure in terms of 16S rDNA type.
2. Materials and methods 2.1. Isolation of V. vulni¢cus
2.3. PCR ampli¢cation and 16S rDNA sequencing Polymerase chain reaction (PCR) primers 209f (5PTCTCGCGTCAGGATATGCCCAGGTG-3P) and 1204r (5P-GGGCCATGATGACTTGACGTCGTCC-3P) (Sigma-Genosys, The Woodlands, TX, USA) were used to
3. Results and discussion This study’s comparison of the determined high-quality
FEMSEC 1515 2-6-03
M. Lin, J.R. Schwarz / FEMS Microbiology Ecology 45 (2003) 23^27
shown in Fig. 2. The relative abundance of type A and type B V. vulni¢cus strains exhibited considerable variation over the 13-month period. Only V. vulni¢cus type A strains were detected during the March^May time period, with type A strains persisting as the predominant strain through July, after which their abundance decreased markedly. On the other hand, type B strains were only ¢rst detected in June, but by September had become the predominant strain. However, both types of V. vulni¢cus strains decreased rapidly with the arrival of autumnal cold fronts, becoming non-detectable by the December sampling. In a limited prior study, Kim and Jeong [17] reported that 40 V. vulni¢cus isolates recovered in late August o¡ the southern coast of Korea (water temperature of 22‡C and salinity of 32 ppt) consisted of 35% 16S rDNA type A and 65% type B strains. It is of particular interest to note that after no V. vulni¢cus was detected during the winter months (December^ February), only type A strains and no type B strains were isolated during the subsequent spring months of March^ May. The inability to isolate V. vulni¢cus during the cold winter months has been thought to be due to the entrance of the bacterium into a viable but non-culturable state [13,14]. It has been suggested that there are two phases of the viable but non-culturable state : (i) a loss of culturability while still maintaining cellular integrity and intact RNA and DNA, and (ii) a gradual degradation of RNA and DNA, ultimately leading to total loss of viability [22]. Both laboratory [23] and ¢eld [24] studies have indicated that V. vulni¢cus cells are able to resuscitate from the viable but non-culturable state and become fully culturable following a temperature upshift. Other reports suggest that the reappearance of culturable populations is a result of regrowth of one or more cold-resistant, culturable cells which had not been detected when the population was initially assayed [25,26]. In either case, the fact that this
partial 16S rDNA sequences (ca. 600 bp, corresponding to nucleotides 290^890 of reference strain ATCC 27562 sequence, GenBank accession no. X76333 [16]) of the 208 V. vulni¢cus strains isolated from Galveston Bay revealed two di¡erent partial 16S rDNA sequences. These two di¡erent 16S rDNA sequences separate the V. vulni¢cus strains into two groups: type A, partial 16S rDNA sequences identical to the corresponding sequence of V. vulni¢cus ATCC 27562 (GenBank accession no. X76333 [16]); and type B, partial 16S rDNA sequences identical to the corresponding sequence of V. vulni¢cus C7184 (GenBank accession no. X76334 [16]). The di¡erences between the two types of partial 16S rDNA sequences were eight bases (1.3% substitutions) within the variable region (Fig. 1). These results are similar to the ¢ndings of Aznar et al. [16], who compared the di¡erences between the complete sequences of 16S rDNA type A and type B strains (17 bases, 1.1% substitutions). Seasonal variations in water temperature, salinity and the relative number of 16S rDNA type A and type B strains of V. vulni¢cus isolated from Galveston Bay are
Relative Number of Isolates
35
16S rDNA type A strain 16S rDNA type B strain Water temperature Salinity
25
30 25
20
20 15 15 10
10
5 0 June
Temperature (oC) Salinity (ppt)
Fig. 1. Base di¡erences between type A and type B partial 16S rDNA sequences of V. vulni¢cus strains isolated from Galveston Bay (only the variable portions are shown). Numbers on the left indicate the base positions corresponding to nucleotides 431^490 of reference strain ATCC 27562 16S rDNA sequence (GenBank accession no. X76333 [1]). Asterisks indicate that the bases are conserved in both types of sequences and periods indicate that the bases vary between the two types of sequences.
30
25
5
July
Aug
Sept
2000
Oct
Nov
Dec
Jan
Month
Feb
Mar
April May
0 June
2001
Fig. 2. Seasonal variation in water temperature, salinity and the relative numbers of 16S rDNA type A and type B V. vulni¢cus strains isolated from Galveston Bay.
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M. Lin, J.R. Schwarz / FEMS Microbiology Ecology 45 (2003) 23^27
study detected only 16S rDNA type A strains during these spring months suggests that the di¡erences between the two 16S rDNA types may a¡ect the viability of these organisms following extended periods of cold winter temperature. This is in direct opposition to the statement by Kim and Jeong [17], in which they suggest ‘that the di¡erences in 16S rDNA type might not a¡ect the viability or be the determining factor of the dominant strain of these organisms in the marine environment’. Water temperature and salinity are primary factors controlling the distribution of V. vulni¢cus in the environment [10,11]. Both parameters varied seasonally in this study, with temperature lowest in January and peaking in July^ August and salinity levels lowest in February to early June and highest in summer to mid-fall (Fig. 2). The rise in water temperature from winter minima and the relatively low salinities recorded in March (ca. 17‡C and 7 ppt) appear to trigger the detectable outgrowth of V. vulni¢cus populations from their winter ‘dormancy’. While this ¢nding is not new, the fact that all of the V. vulni¢cus detected during the ¢rst 3 months of outgrowth (March^May) are of type A has not been previously reported. Additionally, higher salinities appear to favor the continued presence of type B (August^September). However, it is yet to be determined exactly how these two parameters contribute to our observed seasonal variation in the relative abundance of type A and type B V. vulni¢cus strains and if these major factors a¡ect the two 16S rDNA types di¡erently. Further studies on these topics may also lead to a better understanding of the signi¢cance of the viable but nonculturable state of V. vulni¢cus from a molecular point of view. In summary, the results of this study reveal the existence of two di¡erent partial 16S rDNA sequences among the V. vulni¢cus strains isolated from Galveston Bay and the seasonal variation in the relative abundance of these two 16S rDNA types in the V. vulni¢cus population.
[2]
[3]
[4] [5] [6] [7]
[8]
[9]
[10]
[11]
[12]
[13] [14]
[15] [16]
[17]
Acknowledgements We thank Jaime R. Alvarado for his technical advice. We are grateful to the Texas Department of Health for assistance in sample collection and shipping. The technical assistance of Mona Hochman, Stephen Burkett, Karen Juntunen and Justin Weems is also greatly appreciated. This work was supported in part by grants (0102980012b-1997 and 010298-0002-1999) from the State of Texas THECB Advanced Technology Program.
References
[18]
[19]
[20]
[21]
[1] Oliver, J.D., Warner, R.A. and Cleland, D.R. (1982) Distribution and ecology of Vibrio vulni¢cus and other lactose-fermenting marine
[22]
vibrios in coastal waters of the southern United States. Appl. Environ. Microbiol. 44, 1404^1414. Tamplin, M.L., Rodrick, G.E., Blake, N.J. and Cuba, T. (1982) Isolation and characterization of Vibrio vulni¢cus from two Florida estuaries. Appl. Environ. Microbiol. 44, 1466^1470. Wright, A.C., Hill, R.T., Johnson, J.A., Roghman, M-C., Colwell, R.R. and Morris Jr., J.G. (1996) Distribution of Vibrio vulni¢cus in Chesapeake Bay. Appl. Environ. Microbiol. 62, 717^724. Hlady, W.G. (1997) Vibrio infections associated with raw oyster consumption in Florida, 1981-1994. J. Food Prot. 60, 353^357. Linkous, D.A. and Oliver, J.D. (1999) Pathogenesis of Vibrio vulni¢cus. FEMS Microbiol. Lett. 174, 207^214. Oliver, J.D. (1989) Vibrio vulni¢cus. In: Food-born Bacterial Pathogens (Doyle, M.P., Ed.), pp. 570^600. Marcel Dekker, New York. DePaola, A., Capers, G.M. and Alexander, D. (1994) Densities of Vibrio vulni¢cus in the intestines of ¢sh from the U.S. Gulf Coast. Appl. Environ. Microbiol. 60, 984^988. Montanari, M.P., Pruzzo, C., Pane, L. and Colwell, R.R. (1999) Vibrios associated with plankton in a coastal zone of the Adriatic Sea (Italy). FEMS Microbiol. Ecol. 29, 241^247. Vanoy, R.W., Tamplin, M.L. and Schwarz, J.R. (1992) Ecology of Vibrio vulni¢cus in Galveston Bay oysters, suspended particular matter, sediment and seawater: detection by monoclonal antibody-immunoassay-most probable number procedure. J. Ind. Microbiol. 9, 219^ 223. Kelly, M.T. (1982) E¡ect of temperature and salinity on Vibrio (Beneckkea) vulni¢cus occurrence in a Gulf Coast environment. Appl. Environ. Microbiol. 44, 820^824. Motes, M.L., Depaola, A., Cook, D.W., Veazey, J.E., Hunsucker, J.C., Garthright, W.E., Blodgett, R.J. and Chirtel, S.J. (1998) In£uence of water temperature and salinity on Vibrio vulni¢cus in Northern Gulf and Atlantic Coast oysters (Crassostrea virginica). Appl. Environ. Microbiol. 64, 1459^1465. O’Neill, K.R., Jones, S.H. and Grimes, D.J. (1992) Seasonal incidence of Vibrio vulni¢cus in the Great Bay estuary of New Hampshire and Maine. Appl. Environ. Microbiol. 58, 3257^3262. Oliver, J.D. (1995) The viable but non-culturable state in the human pathogen Vibrio vulni¢cus. FEMS Microbiol. Lett. 133, 203^208. Wolf, P.W. and Oliver, J.D. (1992) Temperature e¡ects on the viable but nonculturable state of Vibrio vulni¢cus. FEMS Microbiol. Ecol. 101, 33^39. Woese, C.R. (1987) Bacterial evolution. Microbiol. Rev. 51, 221^271. Aznar, R., Ludwig, W., Amann, R.I. and Schleifer, K.H. (1994) Sequence determination of rRNA gene of pathogenic Vibrio species and whole-cell identi¢cation of Vibrio vulni¢cus with rRNA-targeted oligonucleotide probes. Int. J. Syst. Bacteriol. 44, 330^337. Kim, M.S. and Jeong, H.D. (2001) Development of 16S rRNA targeted PCR methods for the detection and di¡erentiation of Vibrio vulni¢cus in marine environments. Aquaculture 193, 199^211. Elliot, E.L., Kaysner, C.A., Jackson, L. and Tamplin, M.L. (1995) Vibrio cholerae, V. parahaemolyticus, V. vulni¢cus, and other Vibrio spp. In: U.S. Food and Drug Administration Bacteriological and Analytical Manual, 8th ed., pp. 9.01^9.27. Association of O⁄cial Analytical Chemists (AOAC), Gaithersburg, MD. Tamplin, M.L., Martin, A.L., Ruple, A.D., Cook, D.W. and Kaspar, C.W. (1991) Enzyme immunoassay for identi¢cation of Vibrio vulni¢cus in seawater, sediment and oysters. Appl. Environ. Microbiol. 57, 1235^1240. Ruimy, R., Breittmayer, V., Elbaze, P., Lafay, B., Boussemart, O., Gauthier, M. and Christen, R. (1994) Phylogenetic analysis and assessment of the genera Vibrio, Photobacterium, Aeromonas, and Plesiomonas deduced from small-subunit rRNA sequences. Int. J. Syst. Bacteriol. 44, 416^426. Dorsch, M., Lane, D. and Stackebrandt, E. (1992) Towards a phylogeny of the genus Vibrio based on 16S rRNA sequences. Int. J. Syst. Bacteriol. 42, 58^63. Weichart, D., McDougald, D., Jacobs, D. and Kjelleberg, S. (1997)
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M. Lin, J.R. Schwarz / FEMS Microbiology Ecology 45 (2003) 23^27 In situ analysis of nucleic acids in cold-induced nonculturable Vibrio vulni¢cus. Appl. Environ. Microbiol. 63, 2754^2758. [23] Whitesides, M.D. and Oliver, J.D. (1997) Resuscitation of Vibrio vulni¢cus from the viable but nonculturable state. Appl. Environ. Microbiol. 63, 1002^1005. [24] Oliver, J.D., Hite, F., McDougald, D., Andon, N.L. and Simpson, L.M. (1995) Entry into, and resuscitation from, the viable and nonculturable state by Vibrio vulni¢cus in an estuarine environment. Appl. Environ. Microbiol. 61, 2624^2630.
27
[25] Bogosian, G., Aardema, N.D., Bourneuf, E.V., Morris, P.J.L. and O’Neil, J.P. (2000) Recovery of hydrogen peroxide-sensitive culturable cells of Vibrio vulni¢cus gives the appearance of resuscitation from viable but nonculturable state. J. Bacteriol. 182, 5070^5075. [26] Weichart, D., Oliver, J.D. and Kjelleberg, S. (1992) Low temperature induced nonculturability and killing of Vibrio vulni¢cus. FEMS Microbiol. Lett. 100, 205^210.
FEMSEC 1515 2-6-03
www.fems-microbiology.org
Seasonal shifts in population structure of Vibrio vulni¢cus in an estuarine environment as revealed by partial 16S ribosomal DNA sequencing Meilan Lin 1 , John R. Schwarz
Department of Marine Biology, Texas ApM University at Galveston, 5007 Avenue U, Galveston, TX 77551, USA Received 7 August 2002; received in revised form 24 March 2003; accepted 26 March 2003 First published online 16 April 2003
Abstract The partial sequence (600 bp) containing the most variable region of Vibrio vulnificus 16S ribosomal DNA (rDNA) was determined for 208 randomly selected V. vulnificus strains isolated from Galveston Bay, TX, USA between June 2000 and June 2001. A comparative analysis of the determined partial 16S rDNA sequences revealed the existence of two different partial 16S rDNA sequences (type A and type B, 1.3% base substitutions) among the 208 V. vulnificus isolates. A higher proportion of 16S rDNA type A strains was isolated in June and July while a considerably higher proportion of type B strains was isolated in September. In addition, after no V. vulnificus strains were detected during the winter months (December^February), only type A strains were isolated during the following months (March^May). The results suggest that the relative abundance of type A and type B V. vulnificus strains in Galveston Bay varies with the season and that the differences between the two 16S rDNA types may affect the viability of these organisms in the natural environment. 7 2003 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords : Partial 16S rDNA sequence; 16S rDNA types; Temperature; Salinity ; Population structure ; Vibrio vulni¢cus
1. Introduction Vibrio vulni¢cus is an autochthonous bacterium found in estuarine and marine environments of temperate and tropical climates [1^3]. It is an opportunistic human pathogen known to cause primary septicemia, particularly in individuals with underlying chronic diseases such as diabetes, impaired liver function and/or compromised immune systems. Infection in these persons is associated with consumption of raw shell¢sh, seawater contamination of open wounds or contact with raw shell¢sh [4^6]. V. vulni¢cus has been isolated from a wide range of environmental sources including seawater, plankton, sediment, shell¢sh and ¢sh [7^9]. The presence of the organism is favored by relatively high water temperatures ( s 20‡C) and low-to-moderate salinities (5^25 ppt) [2,10,11]. The
* Corresponding author. Tel. : +1 (409) 740 4453; Fax : +1 (409) 740 4787. E-mail address : [email protected] (J.R. Schwarz). 1
Present address: Houston Department of Health and Human Services, 1115 S. Braeswood, Houston, TX 77030, USA.
bacterium is readily isolated from water and shell¢sh during the warmer months, but the organism is di⁄cult to isolate during the colder months [9,11,12]. This decrease in culturability may be due to the entry of the organism into a viable but non-culturable state, a survival response by V. vulni¢cus to low-temperature stress [13,14]. The 16S rRNA gene (rDNA) is ubiquitous among cellular organisms and is highly conserved [15]. A comparative analysis by Aznar et al. of the complete 16S rDNA sequences of 21 V. vulni¢cus strains revealed two di¡erent 16S rDNA sequences that divide these strains into two di¡erent groups: type A and type B [16]. In a separate study, 40 V. vulni¢cus strains isolated o¡ the southern coast of Korea in August 1999 were found to be 35% 16S rDNA type A and 65% type B strains [17]. Little, however, is known about temporal changes in the relative abundance of di¡erent 16S rDNA types of V. vulni¢cus in the natural environment. In this study, internal fragments (600 bp) of the 16S rDNAs from 208 randomly selected V. vulni¢cus strains isolated from Galveston Bay, TX, USA over a 13-month period were sequenced and compared to investigate : (1) the existence of di¡erent 16S rDNA sequences among the
0168-6496 / 03 / $22.00 7 2003 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. doi:10.1016/S0168-6496(03)00091-6
FEMSEC 1515 2-6-03
24
M. Lin, J.R. Schwarz / FEMS Microbiology Ecology 45 (2003) 23^27
Water and oyster samples were collected between June 2000 and June 2001 at ¢ve Galveston Bay sites (private oyster leases): Lease 301 (29‡27P57QN, 94‡42P03QW), Lease 414 (29‡29P19QN, 94‡47P56QW), Lease 433 (29‡31P53QN, 94‡48P12QW), Lease 410 (29‡29P43QN, 94‡51P33QW) and Lease 426 (29‡29P00QN, 94‡54P02QW). Samples were collected biweekly during the spring and summer months and monthly during the fall and winter months. 125 ml of seawater was collected aseptically 0.5 m below the surface and 15 oysters were harvested by dredging at each site. Water temperature and salinity at each sample site were determined using a YSI model 30 salinity meter (YSI, Yellow Springs, OH, USA). The water and oyster samples were placed into an insulated container with frozen cool packs and transported to the Seafood Safety Laboratory at Texas ApM University at Galveston, TX, USA, for bacteriological analysis. Samples were analyzed within 12 h of collection. The procedure used for isolation and identi¢cation of V. vulni¢cus is described in the USA Food and Drug Administration’s Bacteriological Analytical Manual [18]. The identities of suspect colonies were con¢rmed by enzyme immunoassay which employed a monoclonal antibody speci¢c for V. vulni¢cus [19]. Two to three identi¢ed V. vulni¢cus colonies were randomly picked from each sample that was positive for V. vulni¢cus and a total of 208 V. vulni¢cus strains isolated from Galveston Bay water and oysters were included in this study.
amplify a 996-bp fragment containing the most variable region of the V. vulni¢cus 16S rDNA. These two primers were selected from two highly conserved regions of the 16S rDNA based on the alignment of the published V. vulni¢cus 16S rDNA sequences, i.e. GenBank accession nos. X76333, X76334 [16], X74726, X74727 [20], and X56582 [21]. PCR was conducted in a 50-Wl reaction mixture containing 1.0 WM (each) primer, 1.5 mM MgCl2 , 0.2 mM (each) deoxynucleoside triphosphate (Applied Biosystems, Foster City, CA, USA), 2.5 U of AmpliTaq Gold DNA polymerase (Applied Biosystems), 5% (vol/ vol) dimethyl sulfoxide (Stratagene, La Jolla, CA, USA), and 0.5^0.6 Wg of template DNA. The reaction mixture was ¢rst subjected to an initial denaturation at 95‡C for 10 min and then 35 cycles of 94‡C for 30 s, 55‡C for 30 s and 72‡C for 75 s, followed by a ¢nal extension step at 72‡C for 10 min. Ampli¢cation was performed on a 9700 thermal cycler (Perkin-Elmer). PCR products were analyzed by electrophoresis on 1% agarose gels containing ethidium bromide (0.1 Wg ml31 ) (Pierce, Rockford, IL, USA). PCR amplicons were puri¢ed with the ExoSAP-IT kit (USB, Cleveland, OH, USA) according to the manufacturer’s instructions. After puri¢cation, a 2-Wl aliquot was used as sequencing template. The ampli¢ed fragments were directly sequenced on both strands by using the same PCR primers. Cycle sequencing reaction mixture was prepared by using the ABI Prism BigDye Terminator Cycle Sequencing v2.0 Ready Reaction kit (Applied Biosystems) according to the manufacturer’s instructions. Excessive terminators were removed by using the RapXtract Dye Terminator Removal kit (Prolinx, Bothell, WA, USA) before the samples were prepared and loaded onto the ABI Prism 310 Genetic Analyzer (Applied Biosystems) according to the manufacturer’s protocol. Sequences were automatically analyzed and corrected manually.
2.2. DNA extraction
2.4. Sequence analysis
A single colony of each strain was inoculated into heart infusion broth (Difco Laboratories, Detroit, MI, USA) supplemented with 0.5% NaCl and incubated overnight at 37‡C. Chromosomal DNA was extracted from the overnight cultures by using the QIAamp DNA mini kit (Qiagen, Valencia, CA, USA) according to the manufacturer’s instructions. The quantity and quality of the DNA were determined by UV light absorbance at 260 and 280 nm with a MBA 2000 spectrophotometer (Perkin-Elmer, Norwalk, CT, USA).
The resulting 16S rDNA sequences were aligned with the previously published V. vulni¢cus 16S rDNA sequences (GenBank accession nos. X76333, X76334 [16], X74726, X74727 [20], and X56582 [21]) by using the program BioEdit (version 5.0.9; available at http://jwbrown.mbio.ncsu.edu/BioEdit/bioedit.html). The 16S rDNA sequence of V. vulni¢cus ATCC 27562 (GenBank accession no. X76333 [16]) was used as a reference. Only the homologous regions of the aligned sequences were included in sequence comparisons, with the low-quality sequences on the ends and the sequences close to the ends with ambiguous bases excluded from the comparisons.
V. vulni¢cus strains, and (2) the temporal changes of V. vulni¢cus population structure in terms of 16S rDNA type.
2. Materials and methods 2.1. Isolation of V. vulni¢cus
2.3. PCR ampli¢cation and 16S rDNA sequencing Polymerase chain reaction (PCR) primers 209f (5PTCTCGCGTCAGGATATGCCCAGGTG-3P) and 1204r (5P-GGGCCATGATGACTTGACGTCGTCC-3P) (Sigma-Genosys, The Woodlands, TX, USA) were used to
3. Results and discussion This study’s comparison of the determined high-quality
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M. Lin, J.R. Schwarz / FEMS Microbiology Ecology 45 (2003) 23^27
shown in Fig. 2. The relative abundance of type A and type B V. vulni¢cus strains exhibited considerable variation over the 13-month period. Only V. vulni¢cus type A strains were detected during the March^May time period, with type A strains persisting as the predominant strain through July, after which their abundance decreased markedly. On the other hand, type B strains were only ¢rst detected in June, but by September had become the predominant strain. However, both types of V. vulni¢cus strains decreased rapidly with the arrival of autumnal cold fronts, becoming non-detectable by the December sampling. In a limited prior study, Kim and Jeong [17] reported that 40 V. vulni¢cus isolates recovered in late August o¡ the southern coast of Korea (water temperature of 22‡C and salinity of 32 ppt) consisted of 35% 16S rDNA type A and 65% type B strains. It is of particular interest to note that after no V. vulni¢cus was detected during the winter months (December^ February), only type A strains and no type B strains were isolated during the subsequent spring months of March^ May. The inability to isolate V. vulni¢cus during the cold winter months has been thought to be due to the entrance of the bacterium into a viable but non-culturable state [13,14]. It has been suggested that there are two phases of the viable but non-culturable state : (i) a loss of culturability while still maintaining cellular integrity and intact RNA and DNA, and (ii) a gradual degradation of RNA and DNA, ultimately leading to total loss of viability [22]. Both laboratory [23] and ¢eld [24] studies have indicated that V. vulni¢cus cells are able to resuscitate from the viable but non-culturable state and become fully culturable following a temperature upshift. Other reports suggest that the reappearance of culturable populations is a result of regrowth of one or more cold-resistant, culturable cells which had not been detected when the population was initially assayed [25,26]. In either case, the fact that this
partial 16S rDNA sequences (ca. 600 bp, corresponding to nucleotides 290^890 of reference strain ATCC 27562 sequence, GenBank accession no. X76333 [16]) of the 208 V. vulni¢cus strains isolated from Galveston Bay revealed two di¡erent partial 16S rDNA sequences. These two di¡erent 16S rDNA sequences separate the V. vulni¢cus strains into two groups: type A, partial 16S rDNA sequences identical to the corresponding sequence of V. vulni¢cus ATCC 27562 (GenBank accession no. X76333 [16]); and type B, partial 16S rDNA sequences identical to the corresponding sequence of V. vulni¢cus C7184 (GenBank accession no. X76334 [16]). The di¡erences between the two types of partial 16S rDNA sequences were eight bases (1.3% substitutions) within the variable region (Fig. 1). These results are similar to the ¢ndings of Aznar et al. [16], who compared the di¡erences between the complete sequences of 16S rDNA type A and type B strains (17 bases, 1.1% substitutions). Seasonal variations in water temperature, salinity and the relative number of 16S rDNA type A and type B strains of V. vulni¢cus isolated from Galveston Bay are
Relative Number of Isolates
35
16S rDNA type A strain 16S rDNA type B strain Water temperature Salinity
25
30 25
20
20 15 15 10
10
5 0 June
Temperature (oC) Salinity (ppt)
Fig. 1. Base di¡erences between type A and type B partial 16S rDNA sequences of V. vulni¢cus strains isolated from Galveston Bay (only the variable portions are shown). Numbers on the left indicate the base positions corresponding to nucleotides 431^490 of reference strain ATCC 27562 16S rDNA sequence (GenBank accession no. X76333 [1]). Asterisks indicate that the bases are conserved in both types of sequences and periods indicate that the bases vary between the two types of sequences.
30
25
5
July
Aug
Sept
2000
Oct
Nov
Dec
Jan
Month
Feb
Mar
April May
0 June
2001
Fig. 2. Seasonal variation in water temperature, salinity and the relative numbers of 16S rDNA type A and type B V. vulni¢cus strains isolated from Galveston Bay.
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26
M. Lin, J.R. Schwarz / FEMS Microbiology Ecology 45 (2003) 23^27
study detected only 16S rDNA type A strains during these spring months suggests that the di¡erences between the two 16S rDNA types may a¡ect the viability of these organisms following extended periods of cold winter temperature. This is in direct opposition to the statement by Kim and Jeong [17], in which they suggest ‘that the di¡erences in 16S rDNA type might not a¡ect the viability or be the determining factor of the dominant strain of these organisms in the marine environment’. Water temperature and salinity are primary factors controlling the distribution of V. vulni¢cus in the environment [10,11]. Both parameters varied seasonally in this study, with temperature lowest in January and peaking in July^ August and salinity levels lowest in February to early June and highest in summer to mid-fall (Fig. 2). The rise in water temperature from winter minima and the relatively low salinities recorded in March (ca. 17‡C and 7 ppt) appear to trigger the detectable outgrowth of V. vulni¢cus populations from their winter ‘dormancy’. While this ¢nding is not new, the fact that all of the V. vulni¢cus detected during the ¢rst 3 months of outgrowth (March^May) are of type A has not been previously reported. Additionally, higher salinities appear to favor the continued presence of type B (August^September). However, it is yet to be determined exactly how these two parameters contribute to our observed seasonal variation in the relative abundance of type A and type B V. vulni¢cus strains and if these major factors a¡ect the two 16S rDNA types di¡erently. Further studies on these topics may also lead to a better understanding of the signi¢cance of the viable but nonculturable state of V. vulni¢cus from a molecular point of view. In summary, the results of this study reveal the existence of two di¡erent partial 16S rDNA sequences among the V. vulni¢cus strains isolated from Galveston Bay and the seasonal variation in the relative abundance of these two 16S rDNA types in the V. vulni¢cus population.
[2]
[3]
[4] [5] [6] [7]
[8]
[9]
[10]
[11]
[12]
[13] [14]
[15] [16]
[17]
Acknowledgements We thank Jaime R. Alvarado for his technical advice. We are grateful to the Texas Department of Health for assistance in sample collection and shipping. The technical assistance of Mona Hochman, Stephen Burkett, Karen Juntunen and Justin Weems is also greatly appreciated. This work was supported in part by grants (0102980012b-1997 and 010298-0002-1999) from the State of Texas THECB Advanced Technology Program.
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