APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Sept. 1991, p. 2651-2655 Vol. 57, No.

9
0099-2240/91/092651-05$02.00/0
Copyright © 1991, American Society for Microbiology

Use of the Polymerase Chain Reaction in Detection of Culturable

and Nonculturable Vibrio vulnificus Cells
LAURA A. BRAUNS, MICHAEL C. HUDSON, AND JAMES D. OLIVER*
Department of Biology, University of North Carolina at Charlotte,
Charlotte, North Carolina 28223
Received 26 March 1991/Accepted 5 July 1991

Vibrio vulnificus is a human pathogen associated with consumption of raw oysters. During the colder months
the organism apparently enters a viable but nonculturable state and thus cannot be cultured by ordinary
bacteriological methods. For this reason, another means of detecting this bacterium is necessary. In the present
study we utilized the polymerase chain reaction (PCR) to detect V. vulnificus DNA, thus eliminating the
problem of nonculturability. DNA from both culturable and nonculturable cells of V. vulnificus was amplified
by PCR with primers flanking a 340-bp fragment of the cytotoxin-hemolysin gene. As little as 72 pg of DNA
from culturable cells and 31 ng of DNA from nonculturable cells could be detected. Fifty cycles of a two-step
reaction (30 s [each] at 94 and 65°C) were found to be optimal as well as more time efficient than the three-step
PCR. The total procedure from the point of DNA extraction to observation on a gel required less than 8 h.
Possible reasons for the difficulties encountered in amplifying DNA from nonculturable cells, e.g., gene
rearrangement or loss of the hemolysin gene, are discussed.

Vibrio vulnificus is a human pathogen which has been
implicated in both primary septicemia and wound infections
(13). The organism is indigenous to estuarine environments
(10, 11, 26, 27), and primary septicemia generally results
from ingestion of raw oysters harboring the bacterium.
People with suppressed immune responses or those with
certain underlying chronic diseases involving elevated iron
levels in serum are especially vulnerable to infection (13, 23).
Primary septicemia infections are characterized by a rapid
progression to secondary cutaneous lesions and necrotic
ulcers of the extremities, with mortality exceeding 50% (23).
Wound infections may result from exposure of wounds to
seawater harboring the bacterium. Localized edema and
rapidly evolving cellulitis result and may require tissue
debridement or limb amputation; 20% of such wound infec-
tions are fatal (23).
By using standard culture techniques, cells of V. vulnificus
can be isolated from the marine environment only during
those months when water temperatures are warm (10, 11,
28). A similar inability to culture estuarine vibrios when
water column temperatures are low has been reported for
Vibrio parahaemolyticus (9, 12), Vibrio mimicus (4), and
Vibrio cholerae (3). However, it is likely that such bacteria
are not absent but are in an apparently dormant state (31,
37), possibly representing a survival mechanism against cold
temperatures. Indeed, in laboratory studies, cells exposed to
such temperatures have been shown to remain viable but not
to be culturable by standard bacteriological methods. This
phenomenon, which has been referred to as the viable but
not culturable state (31), has been reported for a variety of
bacteria, including V. cholerae (5, 40), Salmonella spp. (14,
32), Campylobacter jejuni (30), Escherichia coli (40), and
more recently, V. vulnificus (16, 25, 38).
In 1990, Colwell et al. (6) reported that nonculturable cells
of V. cholerae, when ingested by human volunteers, caused
clinical symptoms consistent with non-O1 cholera infection
and that V. cholerae could be recovered from stool speci-
*
Corresponding author.
2651
mens. More recently, Nilsson et al. (20) described the
resuscitation of V. vulnificus from the nonculturable state.
Thus, the potential public health hazard presented by such
vibrios existing in the nonculturable state may be significant.
One obvious difficulty in elucidating this potential hazard is
the inability to detect such cells in the natural environment
by employing routine bacteriological methods. Further, any
detection method that is employed must ultimately be capa-
ble of detecting the often low numbers of the organism under
investigation against a large background of other procaryotic
and eucaryotic cells, as well as organic detrital material
which may be present in the sample. One possible method of
detecting such nonculturable cells is the polymerase chain
reaction (PCR). This technique allows a specific segment of
DNA to be amplified by a factor of 106 or more within hours
(34), thus potentially permitting the detection of cells present
in low numbers. Further, it has recently been demonstrated
that DNA extraction and PCR techniques can be employed
with extremely complex, organically rich environmental
samples such as river sediments (36). Finally, PCR method-
ology depends only on the presence of target DNA and not
culturable cells, and thus PCR is potentially able to detect
the presence of viable but nonculturable cells.
It has recently been reported that PCR can be used to
detect culturable cells of V. vulnificus present in artificially
contaminated oyster homogenates (7). In the present study,
we report for the first time the detection of viable but
nonculturable cells, as well as culturable cells, of this
important human pathogen. We employed as the target DNA
sequence a 388-bp region of the hemolysin gene previously
shown (18) to be unique to V. vulnificus.
MATERIALS AND METHODS
Preparation of culturable and nonculturable cells. For
culturable V. vulnificus (strain C7184) and other bacteria
(Table 1), cells were harvested from cultures grown over-
night on heart infusion agar (Difco, Detroit, Mich.) at 370C.
Total viable counts were performed on heart infusion agar in
triplicate to determine cell numbers. Nonculturable cells of
2652 BRAUNS ET AL.
TABLE 1. Strains examined by PCR
Organism Strain
Vibrio alginolyticus .................. ATCC 17749
Vibrio cholerae .................. CVD a N16961
Vibrio mimicus .................. ATCC 33653
Vibrio parahaemolyticus .................. ATCC 27519
Vibrio proteolyticus .................. ATCC 15338
Enterobacter aerogenes .................. Laboratory strain
Escherichia coli .................. Laboratory strain
Proteus vulgaris .................. Laboratory strain
Pseudomonas aeruginosa .................. Laboratory strain
Salmonella typhimurium .................. Laboratory strain
Serratia marcescens .................. Laboratory strain
Staphylococcus aureus .................. Laboratory strain
Bacillus subtilis .................. Laboratory strain
Micrococcus luteus .................. Laboratory strain
a CVD, Center for Vaccine Development, University of Maryland.
V. vulnificus were prepared by using a variation of the
method of Linder and Oliver (16). Briefly, cells were grown
overnight at 20°C with shaking in 50 ml of MSWYE broth
(29) in a 125-ml flask. An inoculum (1% final concentration)
was then made into 1 liter of sterile artificial sea water. This
microcosm was then incubated at 5°C and periodically
sampled to determine culturability of the cells by the total
viable count method. As the number of culturable cells
reached fewer than 10/ml (average, 18 days), 10-ml samples
from the 1-liter flask were filtered in order to increase the
detection limit to 0.1 cell per ml. These filters were placed on
heart infusion agar and incubated at 37°C overnight. Direct
viable counts, by the method of Kogure et al. (15), were
performed to determine cell viability. Briefly, nonculturable
cells are added to a solution containing yeast extract as a
nutrient supplement and nalidixic acid as an antibiotic which
inhibits cross wall formation upon cell division. Hence,
viable cells will start growing but will not be able to divide.
Upon staining with acridine orange and observation under
UV light, viable cells appear greater than or equal to three
times their normal length. When less than 0.1 cell per ml was
plateable (average, 25 days), the cells were harvested and
their DNA was extracted.
DNA extraction. After trying several DNA extraction
strategies, DNA was isolated by the method of Saunders (35)
with the following modifications: lysozyme incubation was
extended to 25 min, and after addition of the chloroform-
isoamyl alcohol, the solution was allowed to stand for 10
min. The DNA was precipitated with ethanol and resus-
pended in water. Purity was calculated following determina-
tion of A26JA280 ratios, and DNA concentrations were
obtained from the A260 values.
PCR. Oligonucleotide primers 24 bp in length (V. vul-
nificus oligonucleotide 1 = 75% G + C; V. vulnificus oligonu-
cleotide 3 = 66.6% G+C) complementary to opposing
strands flanking a 340-bp sequence (Fig. 1) within the
1,416-bp hemolysin gene were synthesized by the Center for
Vaccine Development at the University of Maryland, Balti-
more. A Perkin-Elmer-Cetus GeneAmp kit (Norwalk,
Conn.) was utilized for the DNA amplifications. The follow-
ing were the final conditions employed for both culturable
and nonculturable DNA: 1.25 U of Taq polymerase, 0.6 ,uM
(each) primer, 250 p.M (each) deoxynucleoside triphosphate,
1.25x reaction buffer, and 2.8 mM Mg2+ (including Mg2+
present in the buffer) in a total reaction volume of 40 p.l.
These parameters were obtained following optimization
studies on DNA from culturable V. vulnificus cells. Reac-
APPL. ENVIRON. MICROBIOL.
Vv oligo 1
5' (C GCC GCTCAC TGG GGCAGTGGC TGG GTA 1T1 GATMG ACG MG TTC MC CCT
ATC TCTTAT TCC MC TTC AAA CCG MC TAT GAC GTT TTG TAC GM GCG CCC GTG TCT GM
ACT GGC GTAACG GATT17 GAG ATG GGC GTG MA CTC MC TAT CGT GCA CGC 1 GGTACC
GTr CTT CCT TCA GCG CTG TTT TCG GTT TAG GGC TCT GCG GGC TCG TCA ACC AGCAGT
ACT GTG AAACM CGT ATr CGC ATC GAC TGG MT CAC CCA CTG ITT GM GCG GAA CGA CAC
GTr ACA CTG CAG TCA CTG AGC MC AAC GAT CTC TGC CTG GAT GTr TAT GGT GAG MC GGT
GACAAA ACG GTr (GOG GGTGGTTCGGTTAAC GC TGG 3
Vv oligo 3
FIG. 1. Nucleotide sequence of the amplified region of the V.
vulnificus hemolysin gene, bp 726 to 1113. V. vulnificus oligonucle-
otides (Vv oligo) 1 and 3 (primers) in parentheses. The probe used
for dot blots is underlined. This purified 25-bp probe was supplied by
the University of Maryland Center for Vaccine Development and
was labeled for dot blot hybridization with 32P by using a nick
translation kit (Bethesda Research Laboratories, Gaithersburg,
Md.).
tions were performed in an Ericomp (San Diego, Calif.)
thermal cycler. For DNA extracted from culturable cells of
V. vulnificus, a total of 40 cycles with a denaturing temper-
ature of 94°C and an annealing/extension temperature of
65°C (1 min at each temperature) were run. For noncultura-
ble V. vulnificus cells and for the cultures listed in Table 1, 50
cycles, with 30 s at each temperature, were employed. In
each case, the annealing and extension step of the last cycle
was increased for all extracts to 10 min to ensure proper
extension of the bases. In all cases, 50 ,ul of mineral oil was
used to overlay the reaction mixtures to minimize evapora-
tion.
Detection of amplified DNA. PCR-amplified DNA (15 to 20
,ul) from V. vulnificus cells was electrophoresed on a 1.0%
agarose gel containing 0.5 ,ug of ethidium bromide per ml.
The gels were run in Tris-borate-EDTA (TBE) buffer (17) at
60 V for 2 to 3 h with PvuII-digested lambda DNA as a size
standard.
Restriction analysis. Each amplified product (20 [l) was
digested for 1 h with TaqI (Sigma, St. Louis, Mo.) in 1x
universal buffer (Stratagene, La Jolla, Calif.) in a total
volume of 25 p.l. Electrophoresis was performed in a 3%
NuSieve (FMC, Rockland, Maine) gel in TBE buffer at 60 V
for 2 to 3 h. The gel was stained for 30 min in 1 p.g of
ethidium bromide per ml.
Nucleotide sequence accession number. The nucleotide
sequence of the V. vulnificus hemolysin gene has been
assigned GenBank accession number M34670.
RESULTS AND DISCUSSION
Since current microbiological culturing techniques fail to
demonstrate bacterial cells in the nonculturable state, it is
important that methods to detect such cells be developed.
Further, the ability to detect small numbers of cells is
essential, as many bacteria are only present in natural
environments at low cell densities. Such detection is espe-
cially important for bacteria such as V. vulnificus, which in
certain individuals produces rapidly fatal infections (23).
PCR techniques seem ideally suited for this goal, as the
method allows amplification of the DNA obtained from as
few as one cell (36). It is essential, however, that the portion
of DNA to be amplified by this technique is unique to the
species of bacterium under investigation. The hemolysin
gene of V. vulnificus has been cloned (39) and sequenced
(41), and this gene has been shown to be unique to V.
vulnificus (18). Hence, using as primers for the PCR a pair of
VOL. 57, 1991 PCR TO DETECT V. VULNIFICUS CELLS 2653

FIG. 2. Restriction digests of culturable and nonculturable V.

vulnificus DNA after PCR amplification. Lane 1, lambda PvuII; lane
2, unrestricted culturable V. vulnificus DNA after PCR amplifica-
tion; lanes 3 and 4, TaqI-restricted culturable and nonculturable V.
vulnificus DNA, respectively.

24-bp oligonucleotides flanking a region of 340 bp within the

hemolysin gene, the resultant product would be expected to
be specific to this organism. The specificity of this 340-bp
region was confirmed in that, after PCR amplification, no
bands were detected upon electrophoresis of the DNA from
any strains (Table 1) except V. vulnificus (data not shown).
Restriction analysis (Fig. 2) of the PCR product of V.
vulnificus confirmed the identity of the amplified sequence.
Autoradiographs utilizing a 32P-labeled hemolysin probe also
confirmed the identity of the amplification (data not shown).
From a starting cell density of 106/ml, V. vulnificus be-
came nonplateable in an average of 25 days, at which time
between 7 and 15% could be shown to be viable by the
method of Kogure et al. (15). These figures are consistent
with previous studies on nonculturable cells of V. vulnificus
(16, 25, 38). Using PCR, we were able to amplify and detect
an initial 72 pg of DNA extracted from culturable V. vul-
nificus cells. However, using the same PCR parameters
optimized for culturable V. vulnificus cells, 4.2 ,ug of DNA
extracted from the microcosm containing cells that were
nonculturable yet viable gave no amplified product unless an
additional 40 cycles were employed. As a consequence of FIG. 3. PCR products of culturable and nonculturable V. vul-
this decreased amplification of DNA extracted from noncul- nificus DNA. Lane 1, lambda HindlIl; lane 2, culturable V. vulnifi-
cus; lane 3, nonculturable V. vulnificus; lane 4, negative control (no
turable cells, the strategy for subsequent studies on noncul- template); lane 5, lambda PvuII.
turable cells was to decrease the duration of the denaturing
and annealing and extension steps to 30 s and to increase the
number of cycles. As a result, a clearly observable DNA
band could be seen after a total of 50 cycles after starting nonculturable cells. Whether such cells contain less DNA
with only 31 ng of extracted DNA. While we cannot exclude per cell than culturable cells is not known. In the case of
the possibility that our method amplifies DNA from dead marine bacteria undergoing nutrient deprivation, as was the
cells present in the microcosm, this seems unlikely, as DNA case in the present study, Nystrom et al. (21) reported an
would be expected to be rapidly degraded upon cell death. increase in DNA content in cells of the marine Vibrio sp.
Figure 3 shows the PCR detection of both culturable and S14. In contrast, Amy et al. (1) and Moyer and Morita (19)
nonculturable DNA but does not represent the minimal reported an overall decrease in DNA content of the psychro-
detectable limits. philic marine Vibrio sp. ANT-300, during carbon starvation,
The reason(s) for the decreased amplification of noncul- to as little as 5 to 10% of the original amount. Similarly,
turable cell DNA is not at present understood. To date, there Hood et al. (8) reported a decrease in DNA in starving cells
is no information regarding the DNA content of viable but of V. cholerae. It is also possible that alternative techniques
2654 BRAUNS ET AL.
will need to be employed for optimal extraction of DNA
from starving or nonculturable cells. Nystrom and Kjelle-
berg (22) have shown significant changes in the cell wall
peptidoglycan of marine Vibrio sp. S14 on nutrient depriva-
tion. These changes resulted in enhanced resistance to
sonication, presumably because of thickened cell walls. We
have also observed (24) significantly increased resistance to
sonication following carbon deprivation of V. vulnificus. It is
also possible that some DNA rearrangements occur as cells
enter the nonculturable state. Sadouk et al. (33) reported that
strains of Alcaligenes eutrophus, in response to heavy metal
stress, responded by "reshuffling" small sequences of DNA
between a resident plasmid and its chromosomal DNA.
Loss or rearrangement of DNA within the cytotoxin-
hemolysin gene (possibly because of cold or starvation
stress), increased resistance to cell lysis and DNA extrac-
tion, and the presence of some factor in lysed nonculturable
cells are all conditions which could result in decreased PCR
amplification of DNA obtained from nonculturable cells, as
was observed in the present study. Loss or rearrangement of
the cytotoxin-hemolysin DNA or some factor present in
nonculturable cells seems more plausible than increased
resistance to cell lysis and DNA extraction, since a signifi-
cant yield of DNA from nonculturable cells was obtained. In
fact, more nonculturable cell DNA than culturable cell DNA
was employed for the amplification reactions.
Hill et al. (7) have recently shown the specificity of
another region of the cytotoxin-hemolysin gene to V. vul-
nificus by using PCR in oyster homogenates. However,
these authors examined only culturable cells of V. vulnificus.
Bej et al. (2) have used PCR to detect dying cells of
Legionella pneumophila which could not be cultured follow-
ing exposure to hypochlorite. However, such nonculturable
cells are quite different from those found in the environment,
as they were artificially induced by a toxic chemical as
opposed to the gradual temperature changes which appear to
initiate the nonculturable response in V. vulnificus in the
natural environment (38). The present study has demon-
strated, for the first time, the amplification of DNA from
viable but nonculturable cells induced by a natural environ-
mental parameter. Such cells are capable of resuscitation
following a reversal of the inducing factor (20), whereas
hypochlorite treatment ultimately kills the cells. PCR seems
well suited for the detection of such cells, which otherwise
are undetectable by standard microbiological techniques. It
still needs to be determined whether the DNA of noncultur-
able cells is in some way altered, leading to decreased
amplification. We are currently applying PCR methodology
for the detection of culturable and nonculturable cells natu-
rally present in oysters and other environmental sources.
ACKNOWLEDGMENTS
We thank J. Glenn Morris, Jr., and Anita Wright of the University
of Maryland Center for Vaccine Development for providing the
primers and probe used in this study. We also thank Linda Simpson
for her excellent advice in preparing the manuscript, Trudi Groubert
and Susan Murphy for providing the oyster samples, and Sandy
Zane for preparation of the photographs.
This work was supported by grant 9013-ARG-0806 from the North
Carolina Biotechnology Center as well as grants from the Florida
(R/LR-Q-15) and North Carolina (R/MER-20) Sea Grant programs.
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