Marine Pollution Bulletin 77 (2013) 325–332

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Marine Pollution Bulletin

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Impact of milk fish farming in the tropics on potentially pathogenic

vibrios
W.T. Reichardt ⇑, J.M. Reyes, M.J. Pueblos, A.O. Lluisma
Marine Science Institute, University of the Philippines, Diliman, 1101 Quezon City, Philippines

a r t i c l e i n f o a b s t r a c t

Keywords: Ratios of sucrose-negative to sucrose-positive vibrios on TCBS agar (suc/suc+) indicate the abundance of
Sucrose-negative vibrios potential human pathogenic non-cholera vibrios in coastal mariculture environments of the Lingayen
Enterovibrionaceae Gulf (Philippines. In guts of adult maricultured milkfish (Chanos chanos) of suc vibrios reached extreme
Health risk peak values ranging between 2 and 545 million per g wet weight. Suc vibrios outnumbered suc+ vibrios
Milkfish mariculture
in anoxic sediments, too, and were rarely predominant in coastal waters or in oxidized sediments.
Philippines
Suc/suc+ ratios in sediments increased toward the mariculture areas with distance from the open sea
at decreasing redox potentials. There is circumstantial evidence that suc vibrios can be dispersed from
mariculture areas to adjacent environments including coral reefs. An immediate human health risk by
pathogenic Vibrio species is discounted, since milkfish guts contained mainly members of the Enterovibrio
group. A representative isolate of these contained proteolytic and other virulence factors, but no genes
encoding toxins characteristic of clinical Vibrio species.
Ó 2013 Elsevier Ltd. All rights reserved.

1. Introduction 2013). Yet, this assumption can be biased, as currently available

Marine coastal pollution caused by intensified milk fish farming
in the Lingayen Gulf (South China Sea, Philippines) shows chemical
and biochemical effects in water and sediment (Holmer et al. 2003;
Reichardt et al. 2011; Nacorda et al. 2012). Repeated fish kills, dis-
eases of maricultured invertebrates, a cholera epidemic in 2005,
and enhanced mortality of reef-building corals raise concerns
about sustainability of intensive milk fish mariculture in relation
to environmental health (Villanueva et al. 2006; Reichardt et al.
2007). In terms of bacterial health risks, vibrios with at least a doz-
en pathogenic Vibrio species play a dominant role in coastal marine
environments used for mariculture (Tantillo et al. 2004; Das et al.
2009, Senderovitch et al., 2010). As copiotrophic heterotrophs,
marine vibrios are frequently associated with maricultured organ-
isms including fish intestines (De Paola et al., 1994, Beneduce et al.
2010; Ganesh et al. 2010; Matsunaga et al. 2011). Diversified use of
coastal waters at Cape Bolinao (Lingayen Gulf, Philippines) for
mariculture, restoration of marine wildlife, and tourism offers a
promising basis for assessing the impact of tropical mariculture
on potential health risks. Whereas numerous marine vibrios have
been classified as pathogens affecting marine animals as well as
humans (Farmer and Hickman-Brenner 2006; Austin 2010), only
a small fraction of these pathogenic species may be virulent in
their marine environment (Oberbeckmann et al. 2011; Bier et al.,
⇑ Corresponding author. Tel.: +63 2 922 3959; fax: +63 2 924 7678.
E-mail address: wtreichardt@yahoo.com (W.T. Reichardt).
information stems from non-tropical marine environments. Viru-
lent pathogenic Vibrio species may be expected more frequently
in tropical marine environments, since the expression of virulence
genes seems to increase at elevated environmental temperatures
(Mahony et al., 2010). Hence tropical coastal waters with year-
round temperatures near 30 °C would ensure most favorable
growth conditions for these pathogens.
Initial steps to examine this bacteriological health risk relied
on selective viable counts of (presumptive) vibrios on TCBS agar
(Bolinches et al., 1988). This highly selective medium allows dis-
tinction between sucrose positive and sucrose negative phenotypic
subgroups of vibrios that comprize valid Vibrio species as well as
reclassified former Vibrio species (Farmer and Hickman-Brenner
2006). Since sucrose negative phenotypes harbor the bulk of po-
tential human pathogens – ratios of sucrose negative to sucrose
positive vibrios (suc/suc+) are considered as useful health risk
indicator in sea food microbiology (Lopez-Joven et al. 2011).
Human pathogenic vibrios such as Vibrio vulnificus and Vibrio
parahaemolyticus can occur in the intestine of certain finfish (de
Paola et al. 1994; Das et al. 2009). But our information about selec-
tive forces governing both the enrichment in fish guts and dis-
persal of milkfish-borne vibrios is scarce (Matsunaga et al. 2011).
Routine bacteriological surveys of fish farming sites leave only
limited scope for diagnostic analyses at the species level. Therefore
this investigation examines suc/suc+ ratios as possible candidate
for a practicable indicator of the likely presence of non-cholera
human pathogenic vibrios that would be suited for low cost and

0025-326X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.marpolbul.2013.09.018
326 W.T. Reichardt et al. / Marine Pollution Bulletin 77 (2013) 325–332
routine environmental monitoring. At the same time it addresses
as yet unresolved environmental health risks associated with cer-
tain bacterial loads that accompany the dispersal of fish farming
waste in adjacent marine environments.
Bacteriological monitoring data in 2007 had suggested a tempo-
rary spillover of sucrose-negative water-borne vibrios from a fish
cage site into the waters of a nearby coral reef. Subsequent analy-
ses of intestinal contents of milk fish, water and sediment samples
in 2011/12 were to provide suc/suc+ ratios for vibrios on a larger
scale during NE monsoonal dry season. This choice minimized the
possibility of interfering selective salinity effects on Vibrio popula-
tions during the SW monsoonal wet season and covered both peak
harvest and near fallow periods for milk fish. Intestinal contents
were obtained exclusively from adult milk fish, because only these
had indicated a predominance of sucrose-negative vibrios in previ-
ous analyses (Reichardt et al. (2007).
2. Materials and methods
Mariculture zones at Cape Bolinao are located in close proxim-
ity to coral reefs and areas devoted to tourism. Milkfish farming
using 18  18 m cages in the Anda–Bolinao sections of the Ling-
ayen Gulf has been restricted to a southern (Anda) and a northern
fish farming zone (Bolinao) along the Caquiputan Strait that
diverges at Siapar Island (Fig. 1). Seasonal distribution patterns of
water borne viable bacterial counts were obtained at monthly
intervals in 2006/2007 from a fish cage site at Siapar (N16°20.90 ,
E119°57.50 (11 m water depth) and from a neighboring shallow
coral reef site at Cangaluyan Island (N21°58.90 , E119° 58.30
(1–2 m w.d.).
A survey of surface water and sediment extended over a dis-
tance of more than 10 km from the northern fish farming zone of
the Caquiputan Strait toward the mouth of the main tidal channel.
This survey was conducted on eight sampling dates at intervals of
roughly three weeks during NE monsoonal seasons in 2011/2012.
It included the peak harvest season in December followed by a sub-
sequent nearly fallow period in January. Water samples were taken
(A) at the main tidal channel exit near Lucero, N16°24.6060 , E
119°53.9160 at 18 m water depth (w.d.), (B) at a coral reef site at
Malilnep (Fig. 1,# 1), N16°26.3200 , E119°56.4580 (5 m w.d.), (C)
near inhabited shoreline at Bolinao (Fig. 1,# 9), N16°22.8400 ,
E119° 54.6260 (21 m w.d.), (D) in the fish cage area of Bolinao
(Fig. 1, #10), N16°23.1600 , E119°55.4800 (15 m w.d.), (E) at Poro
Panaien islet (Fig. 1, # 11), N16°22.2470 , E119°, 55.7750 (6 m
w.d.), and (F) near Siapar island (Fig. 1, # 12), N16°21.0710 ,
E119°57.6310 (7 m w.d.).  On each sampling date single speci-
mens of adult milk fish (Chanos chanos) were obtained from the
fish cage site of Bolinao (Fig. 1, # 10).
Complimentary sediment samples were obtained from the
same locations as the water samples, except for replacing the coral
reef site at Malilnep with fine sandy sediment from Gawa (Fig. 1,
#4), (N 16°23.5400 , E 119°54.5390 ,13 m water depth). In March
2012 additional sediment samples were obtained from a total of
17 locations extending from the southern fish cage areas of Anda
to Lucero near the open sea. These sediment sampling sites are
indicated by numbers in Fig. 1.
Triplicate water samples from 1 m depth were collected using a
Niskin sampler and were immediately filled into sterile 50 ml
Corning tubes. Triplicate sediment subsamples were obtained
using plexiglass tubes (inner diameter: 5 cm) either by scuba di-
vers or with a gravity corer. The top 2 cm layers of sediment were
subsampled using sawed-off 5 cm3 sterile syringes from which
0.5 ml aliquots were distributed into serial dilution tubes contain-
ing sterile sea water. A special sediment survey in 2012 followed a
redox potential gradient stretching from anoxic and sulfidic
sediments in the fish farming zones of Anda and Bolinao toward
oxidized sediments northwest of Santiago Island. Redox potential
readings were taken with a Pt redox electrode (WTW) in combina-
tion with an Ag/AgCl electrode serving as reference at 194 mV.
Freshly caught milkfish were aseptically dissected within 1–2 h.
Weighed contents of the distal end of the intestine were distrib-
uted into 50 ml Corning tubes containing 40 ml of sterile seawater
for serial dilutions and dispersed three times for 5 s using a high
speed ultraturrax blender, before serial dilutions were completed.
For comparison with a filter feeding organism as alternative, the
same procedure was applied to the aseptically dissected soft tissue
of freshly caught bivalve ‘‘tahong’’ (Perna viridis). Samples of this
bivalve were collected at the same time from immersed structures
of the same fish cages that provided the milkfish specimens.
From serial dilutions of water, sediment, and animal samples
targeting roughly estimated titer ranges, 0.1 ml aliquots were dis-
tributed and spread plated onto petridishes. Means of viable
counts of presumptive vibrios (mesophilic Vibrionaceae and other
closely related vibrios) were based on triplicate plate counts after
1–2 d of incubation at 37–38 °C on selective thiosulfate-citrate-bile
salts sucrose (TCBS) agar (Difco; Bolinches et al. 1988). Sucrose fer-
menting and sucrose non fermenting colonies (yellow and green in
the presence of bromothymolblue) were counted separately. To
mark the predominance of sucrose negative (sucrose non ferment-
ing) colony forming units (CFUs), ratios of sucrose-negative to su-
crose positive CFUs (suc/suc+) were recorded according to Lopez-
Joven et al. (2011). For comparison, plate counts of total copio-
trophic bacteria were obtained on ZoBell’s marine agar 2216
(HiMedia) after incubation for two weeks at 30 °C using 3–4
replicates.
Sucrose-negative isolates from TCBS agar were grown on
marine agar plates and maintained in alkaline peptone ‘‘CDC
1494’’ liquid medium (Farmer and Hickman-Brenner 2006). Pheno-
typic characterization focused mainly on tests recommended by
Noguerola and Blanch (2008) using an API 20E kit (Biomerieux,
France).
A representative isolate from milkfish intestine (V6) was chosen
for whole genome sequencing using the Roche 454 GS Junior Sys-
tem. The protocols and kits recommended by the manufacturer
were used for the preparation of the sequencing libraries. The se-
quences were then assembled using the software Newbler (ver.
2.7). The resulting contigs were searched for sequences encoding
putative virulence factors, including the known toxin genes of clin-
ically pathogenic vibrios (Chen et al., 2005). In addition, phyloge-
netic analysis of selected markers (16S rRNA, rpoA, pyrH;
Thompson et al. 2005a) obtained from the genomic data was also
carried out using MEGA5 (Tamura et al. 2011) to provide the basis
for the taxonomic classification of the isolate.
Statistics were based on GraphPad InStat 3, version 3.06 for
Windows (Graph Pad Software, Inc.).
3. Results
3.1. Bacterial load of vibrios in maricultured organisms
Intestinal contents of freshly collected adult milkfish from the
Bolinao fish farming zone showed strong anoxia with redox values
ranging between -150 and 290 mV. Viable counts of vibrios
yielded almost exclusively sucrose negative CFUs. Ratios of sucrose
non-fermenting to sucrose fermenting CFUs (suc/suc+) exceeded
by far the equilibrium ratio of one. Milk fish specimens analyzed
on seven sampling dates between January 2011 and January 2012
contained from 2.0  106 to 545  106 CFUs per g of wet weight
(median: 52.3  106) on TCBS agar (Table 1). Suc/suc+ ratios rang-
ing between 2.1 and 59, with a mean of 22.4, indicated an extreme
 W.T. Reichardt et al. / Marine Pollution Bulletin 77 (2013) 325–332 327

Fig. 1. Map of sampling area along main tidal channel of Caquiputan Strait and coral reefs at Malilnep and Cangaluyan, northern (Bolinao) fish farming zone (# 6 and 11), and
southern (Anda) fish farming zone (# 13 and 17). Numbers 1–17 indicate sediment sampling sites at different redox potential values in 2012. Sediment sites # 3, 1, 9, 10, 12,
and 14, are identical with sites A, B, C, D, E, and F, respectively. – Insert: location of Cape Bolinao on the Philippine island of Luzon.

prevalence of sucrose-negative vibrios. In contrast, samples of the
maricultured bivalve Perna viridis collected on the same sampling
dates contained only between 0.1 and 31.3  106 CfUs per g wet
weight. Both mean and median (suc/suc+) ratios of 0.8 and 0.4 ob-
tained for the bivalve indicated a prevalence of sucrose positive vib-
rios. This was significantly different from the mean (22.4) and
median (11.2) (suc/suc+) ratios obtained for the milkfish intestine
(Table 1, Mann-Whitney test, two-tailed P value = 0.0012).
When extrapolated to a ten years (2002-2011) average of 493
fish cages in the Bolinao fish farming zone, the approximate total
cage area of 159732 m2 with an average stocking density of
50 000 specimens per cage contained 7.99 109 fish. With a median
of 52.3  106 CFUs per fish (Table 1) and based on minimum intes-
tinal contents of 1 g (wet weight) per fish, the standing stock of
intestinal milkfish-borne vibrios in the fish farming zone can be
estimated at 418  1015 CFUs.

Table 1
Total viable counts on TCBS agar and prevalence of sucrose-non-fermenting vibrio CFUs expressed as ratio suc/suc+ in milkfish intestine as compared with mussel (Perna viridis)
from Bolinao mariculture zone for n sampling dates between January 2011 and January 2012.

Sample (106) Total CFU/g Ratio (suc/suc+)

Range Median Mean ± SE Range Median Mean ± SE
Milkfish intestine (n = 7) 2.0–545 52.3 113 ± 79.8 2.1–59.0 11.2 22.4 ± 9.7
Mussel (n = 7) 0.1–31.3 0.9 10.6 ± 5.4 0.1–3.7 0.4 0.8 ± 0.5
328 W.T. Reichardt et al. / Marine Pollution Bulletin 77 (2013) 325–332

Table 2
Comparison of selected phenotypic characteristics of majority of 8 isolates (V6 phenotype) out of a total of 16 oxidase positive, sucrose-negative
isolates on TCBS agar from adult milkfish intestines collected in Bolinao mariculture zone from January to March 2011.

Characteristic Majority Group (V6 phenotype) Vibrio (tasmaniensisa Enterovibrionaceaeb

Growth at 38 °C
Arginine dihydrolase
Lysine decarboxylase
Ornithine decarboxylase
Indol production
Acetoin production
Gelatinase activity
Mannitol utilization
b-Galactosidase activity
+  
  v
  
  
+ + v
 + 
  
 + v
+  +

Reference taxa:
a
Thompson et al. (2003a, 2003b, 2004).
b
Thompson et al. (2004,2005b), Pascual et al. (2009).

Adult milkfish collected in January and March 2011 yielded 3.4
 106 and 52.3  106 CFUs on TCBS agar at suc/suc+ ratios of 14.4
and 2.1. A total of sixteen sucrose-negative colonies were isolated
from TCBS agar and purified for taxonomic analyses using a pheno-
typic diagnostic key (Noguerola and Blanch 2008). This rapid iden-
tification scheme classified eight of sixteen isolates as Vibrio
tasmaniensis; but their ability to grow in 8% NaCl and at 40 °C,
and their inability to utilize glucose, mannitol, and amygdalin,
and to produce acetoin (Voges-Proskauer test) and bgalactosi-
dase (ONPG test) deviated from the phenotypic description of that
species (Thompson et al., 2003a; Thompson et al., 2003b). Using
the same diagnostic key, the other less frequent phenotypes
resembled Vibrio splendidus, Vibrio gigantis, Vibrio mimicus and Vib-
rio mediterranei. Isolate V6 considered as median of the dominating
presumptive V. tasmaniensis phenotype was subjected to whole
genome sequencing. Preliminary data based on whole-genome
BLAST and multi-locus phylogenetic analysis using 16S, rpoA, and
pyrH as markers) indicate, however, that isolate V6 is most closely
related to Enterovibrio calvensis, E. norvegicus, and Grimontia (previ-
ously: Vibrio) hollisae (Enterovibrionaceae, Table 2). The molecular
markers applied were, however, not informative enough to permit
classification of the isolate to the species level.
3.2. Water analyses
Near the margin of the southern fish farming zone (Siapar
Island) and at a shallow coral reef site (Cangaluyan) situated
approximately 2 km apart from each other, monthly water samples
(1 m water depth) were analyzed from November 2006 to Decem-
ber 2007. Viable counts indicated similar trends for vibrios and total
heterotrophs (Fig. 2). Sucrose negative vibrios at the coral reef re-
mained near their detection limit (as indicated by suc/suc+ ratios
far below one), with the remarkable exception of a single peak in
September that occurred at both sampling sites. At the fish farming
site of Siapar predominance of sucrose-negative vibrios (suc/suc+
ratios > 1) began shortly after a fish kill event (documented on June
15, 2007) and lasted until October. That fish kill event coincided fur-
ther with a peak value of 8% for the calculated relative abundance of
total vibrios as percent of total copiotrophs in June 2007.
Water analyses in the northern fish farming zone (Bolinao) and
its neighboring environments were conducted during the NE mon-
soonal season from January 2011 to March 2012. Predominance of

Table 3a
Viable counts of vibrios on TCBS agar [CFU/ml] with pertaining suc-/suc+ ratios in
water samples from 1 m depth on TCBS in 2011/2012.

Sampling site Lucero Malilnep Bolinao Fish Poro Siapar

cage panaien
No. of 8 8 8 8 8 8
sampling
dates
TCBS CFU
Mean 150 912 1513 332 579 198
(SE) (45) (797) (1405) (154) (363) (78)
Median 154 192 180 167 210 114
Range 32– 67–6127 123– 43– 17–2870 17–
395 10 710 1237 540
Suc/suc+
Mean 0.63 0.21 0.72 1.72 1.04 0.86
Fig. 2. Annual cycle (2006/2007) of mean viable counts (n = 3) of vibrios on TCBS (SE) (0.22) (0.07) (0.20) (1.21) (0.56) (0.17)
agar and of total copiotrophic heterotrophs on ZoBell’s marine agar in surface Median 0.66 0.29 0.69 0.43 0.88 0.81
waters of mariculture zone (Siapar) and at nearby coral reef (Cangaluyan). Suc/ Range 0.02– 0.01– 0.15– 0.15– 0.13– 0.39–
suc+ ratios of vibrio counts are shown as open triangles. Asterisk indicates widely 1.67 0.57 1.70 9.57 4.33 1.54
spread fish kill event.
 W.T. Reichardt et al. / Marine Pollution Bulletin 77 (2013) 325–332 329

Table 3b sucrose-negative vibrios prevailed in the fish farming area and its
Viable counts of vibrios on TCBS agar [CFU cm-3]  103 with pertaining suc-/suc+ immediate vicinity at Bolinao, Poro Panaien and near Siapar
ratios in surface sediment samples in 2011/2012. Significant differences (P = 0.0004,
Dunns multiple comparisons test) between certain sites are indicated in brackets.
(Table 3b). At greater distance from the fish farming zone, how-
ever, oxidized sediments showed an overwhelming predominance
Sampling site A. B. C. D. Fish E. Poro D. of sucrose positive vibrios nearest to the open sea (Lucero sedi-
Lucero Gawa Bolinao Cage Panaien Siapar
ment). This predominance (suc/suc+ < 1) was weaker in sediment
No. of 7 6 8 8 4 4 at Gawa, a sampling site closer toward the fish farming area that
sampling
dates
still showed redox potential readings in the positive range. Signif-
TCBS CFU icant differences (P = 0.0004) were noted for suc/suc+ ratios be-
Mean 42.1 94.3 16.8 23.2 11.5 14.7 tween oxidized sandy sediments at Lucero and strongly reduced
(SE) 9.4 83.2 3.5 10.4 6.3 7.6 sediments from the fish farming area (fish cage and locations at
Median 35.6 9.4 13.8 13.4 10.8 12.1
Bolinao and Siapar – (Table 3b, Dunn’s multiple comparisons test).
Range 16.2– 2.3– 13.2– 0.5– 1.2–23.3 2.5–
261.7 471.2 433.4 353.4 32.2 When sediment analyses were further extended into the south-
ern fish farming area of Anda in 2012, the resulting topographic
Suc/suc+ (C)(D)(F) (A) (A) (A)
Mean 0.81 0.31 1.27 1.46 1.38 1.84 gradient revealed an even stronger predominance of sucrose nega-
(SE) (0.03) (0.10) (0.41) (0.33) (0.82) (0.48) tive vibrios with increasing distance from a reference site close to
Median 0.04 0.45 1.2 1.3 0.85 2.10 the open sea; for suc/suc+ ratios were significantly correlated
Range 0.03– 0.03– 0.07– 0.5– 0.40–3.50 0.70– with the distance of sampling sites from the open sea (r = 0.89,
0.20 0.50 8.20 3.40 2.50
two tailed P < 0.0001, Spearman Rank correlation, Fig. 3a). Further-
more, suc/suc+ ratios were also negatively correlated with the
redox potential values of the sediment sampled (r = 0.56, two-
tailed P = 0.0184, Spearman Rank correlation). This correlation
sucrose negative vibrios (in 11 of a total of 48 samplings) was
was significant, but only moderately as compared with the strong
mainly confined to fish cage sites and their vicinity (Bolinao, Poro
distance-dependent decline of suc/suc+ ratios toward the open
Panaien, Siapar), but not completely absent from Lucero waters at
sea. In the northern fish farming area at Bolinao, frequently sulfidic
the exit of the main tidal channel. Due to high variances between
sediments contained, despite their extremely low redox potential
sampling dates no significant differences between water sampling
values, less sucrose negative vibrios as compared with sediments
sites outside (Lucero, Malilnep) and inside fish farming areas
from locations near Anda at a greater distance from the open sea
(remaining sites) could be established (Table 3a, non-parametric
(Fig. 3b).
ANOVA, Kruskal-Wallis test).

3.3. Sediment analyses

4. Discussion

During NE monsoonal season sediment and water analyses

were conducted on the same sampling dates. In sediment samples
Fig. 3. Suc/suc+ ratios of vibrios based on means of triplicate subsamples in
sediment on 14 March 2012 at (A) incremental distances from reference site near
open sea on; and (B) measured redox potential.
Coastal marine environments that serve as resource base for
mariculture can cause human health hazards by favoring toxigenic
Vibrio species (Rivera et al. 1989; Barbieri et al. 1999; Beneduce
et al. 2010). As these human pathogenic vibrios are mesophiles
with growth temperature optima around 37 C, maximum human
health risks ought to be expected in permanently warm tropical
environments. Yet, currently published health hazards arising from
non-cholera mesophilic vibrios have raised particular awareness of
global warming in non-tropical climates (Vezzulli et al. 2012). This
provokes the question whether human health hazards caused by
vibrios go usually unreported in the tropics, or whether they are
considered here as less alarming. This could be either due to miti-
gating environmental factors or because of better immunization of
local human populations in combination with lowered awareness.
Growth and virulence of mesophilic vibrios can rise with increas-
ing temperature even beyond 30 °C (Farmer and Hickman-Brenner
2006; Mahony et al., 2010). On the other hand, a temperature of
30 °C has already been considered as an upper threshold for detect-
ing maximum abundances of classic clinical pathogens (Tantillo
et al. 2004).
Intestines of marine finfish can harbor 105 to 106 CFU/g of Vibrio
vulnificus (De Paola et al. 1994; Fukushima and Seki 2004). Also
toxigenic Vibrio parahaemolyticus and even Vibrio cholerae are
known to reside in finfish guts (Das et al. 2009, Senderovitch
et al., 2010). Vibrio vulnificus (biotype 2) serovars can infect both
fish and humans (Fouz et al. 2010). With maximum viable counts
approaching half a billion CFU/g (Table 1) intestines of adult milk-
fish at Bolinao contain a remarkably high load of presumptive
vibrios representing almost exclusively sucrose negative vibrios.
The result that filter feeding organisms (bivalves) from the same
sampling location as the milkfish show an opposite prevalence of
sucrose- positive vibrios, suggests the existence of different modes
of selective enrichment in milk fish intestine and Perna viridis
330 W.T. Reichardt et al. / Marine Pollution Bulletin 77 (2013) 325–332
mussels. Strongly anoxic enrichment conditions that prevail in
milk fish intestines seem to favor different physiological groups
of vibrios than the entrails of filter feeding mussels with their per-
manent exposure to oxygenated water from the same fish cage.
The subgroup of sucrose negative vibrios comprizes toxigenic
pathogens such as Vibrio vulnificus, Vibrio parahaemolyticus, Vibrio
(now: Grimontia) hollisae, Vibrio damselae (now:Photobacterium
damselae) and Vibrio mimicus (Farmer and Hickman-Brenner
2006). This subgroup has become a practical target in depuration
techniques for shellfish in sea food microbiology where elevated
ratios of sucrose negative to sucrose positive CFUs on TCBS agar
serve as health risk indicators of toxigenic vibrios (Lopez-Joven
et al. 2011). Its members are also associated with vibrioses in mari-
cultured animals including finfish (Paillard 2004; Das et al. 2009;
Fouz et al., 2010; Austin 2010). Phenotype based numerical taxon-
omy has identified a mesophilic cluster of sucrose negative coastal
Vibrio species with elevated growth temperature optima (>38 °C)
and moderate NaCl requirement (Simidu and Tsukamoto 1985).
While most human pathogenic non-cholera Vibrio spp. are sucrose
negative (Farmer and Hickman-Brenner 2006), confirmation of
pathogenicity of environmental isolates may not always be
achieved by conventional phenotypic diagnostics and can even
go beyond proper taxonomic classification.
Classification as pathogenic Vibrio species using both classic
phenotyping as well as genotyping needs to be supplemented by
the identification of toxin genes and additional virulence factors
as these are characteristic of clinical pathogenic strains. Current
taxonomic revisions of vibrios do not only suffer from mismatches
between classic phenotypes and 16S r DNA-based genotypes, but
also from genotypic differences that require multilocus genetic
analyses (Thompson et al. 2005a). Apparent mismatches between
rapid phenotypic identification schemes based on dichotomous
keys (Noguerola and Blanch, 2008) and genotyping seem also to
apply to our isolates of sucrose negative vibrios from milkfish
intestine.
Phenotype-based keys have led to members of the Vibrio splen-
didus group dominated by Vibrio tasmaniensis. But this classifica-
tion does not match with whole genome analysis of isolate V6
(representing half of the total) that leads to Enterovibrionaceae.
Since there is considerable sequence divergence between Entero-
vibrionaceae and the Vibrio splendidus group including Vibrio tas-
maniensis (Thompson et al. 2004), isolate V6 has been classified
as a member of the Enterovibrio group (Enterovibrionaceae). Fur-
thermore the key-based phenotypic identification proved to be in
disagreement with the complete phenotypic description of Vibrio
tasmaniensis (2004). In contrast to Vibrio tasmaniensis the pheno-
typic majority of milk fish isolates led by isolate V6 does neither
produce acetoin nor use mannitol, but possesses b-galactosidase
activity and can grow above 35 °C (Table 2). The latter preference
for elevated growth temperatures is absent from both Vibrio tas-
maniensis and Enterovibrionaceae. With several phenotypic key
characteristics reported as variable for the novel family of Entero-
vibrionaceae, this is the final taxonomic classification we are cur-
rently able to reach for a representative of the majority group of
isolates from milkfish intestine.
Universal virulence markers such as haemolysin genes are often
absent from environmental isolates of pathogenic Vibrio species as
confirmed for Vibrio parahaemolyticus in temperate or cold marine
waters (Oberbeckmann et al. 2011). Expression of such virulence
genes seems to increase with elevated temperatures and salinities
(Mahoney et al. 2010). Hence such virulence factors of pathogenic
Vibrio spp. could be assumed to occur at higher frequencies in
coastal marine environments of the tropics. Yet no known toxin
genes related to clinically pathogenic vibrios were detected in
the analyzed isolate. However, other classes of virulence factors
were found in the genome of isolate V6. These included metallo-
proteinases, iron sequestration and transport components, and
possibly antibiotic resistance proteins (JR, unpubl.). These have
been previously documented to contribute to the pathogenicity
of non-toxigenic Vibrio vulnificus (Jones and Oliver 2009), Vibrio
parahaemolyticus (Goshima et al. 1978) and Vibrio mimicus (Hasan
et al. 2010). Among Enterovibrionaceae Grimontia (previously: Vib-
rio) hollisae has been reported to be causative of gastroenteritis and
sepsis in humans as well (Edouard et al. 2009; Hinestrosa et al.
2007). It is also noteworthy that certain bacterial functions en-
coded in ‘‘genomic islands’’ can be related, though not restricted,
to pathogenicity. As these genomic islands may be transferred en
bloc into recipient genomes, horizontal gene transfer in the rather
selective microenvironments of milk fish guts can be most effective
(Thompson et al. 2004). Equally low redox potential values in both
the intestine of adult milkfish and in the sediments underneath
fish cages (<-200 mV) would eventually provide potent sites for
subsequent enrichment and dispersal of members of the Enterovib-
rio group. More in-depth analyses will be required to fully assess
potential, as yet unknown health risks associated with this group.
Predominance of sucrose-negative vibrios in surface layers of
sediment in the vicinity of fish cages (Table 3, Fig. 3) matched with
the extreme prevalence of this phenotypic subgroup in the
intestine of adult milkfish (Table 1). Selective enrichment of
sucrose-negative vibrios in anaerobic milkfish guts (as opposed
to enrichment of sucrose-positive vibrios in ‘‘better aerated’’
bivalves) would plausibly explain that prevalence in sediments of
the fish farming area. Fecal pellets leaving the fish cages via sedi-
mentation are deposited in an anoxic environment with redox
characteristics quite similar to those prevailing in milk fish guts.
Hence presumably selective anoxic conditions that favor enrich-
ment could be carried over from intestinal to sea floor environ-
ments. This most likely explanation has yet to be confirmed by
specific taxonomic analyses in sediments.
Prevalence of sucrose-negative vibrios in fish farming areas
proved significant for sediments only (Table 3b). As a result of ex-
treme variances conferred upon water analyses under a tidal re-
gime and depending on changing weather conditions, benthic
records prove more stable and better buffered against short-term
fluctuations and interferences by drifting ‘‘vectors’’ of vibrios such
as sea wrack in the water column. Abundance of sucrose-negative
vibrios in sediment correlates with redox potential values (Fig. 3b)
and even more strongly so with distance from the open sea
(Fig. 3a). Extremely high densities in the southern fish farming
zone of Anda suggest an involvement of further factors other than
low redox potentials that would favor selective enrichment. Previ-
ous water analyses in the southern part of Caquiputan Strait indi-
cate a drastic decrease of salinities during SW monsoonal (wet)
season in 2004 when high densities of sucrose-negative vibrios
coincided with minor fish kills (Reichardt et al. 2007). It is possible
that temporarily enhanced freshwater inflow creating brackish
water conditions in this area constitutes another selective advan-
tage for sucrose-negative coastal vibrios such as ‘‘cluster S 2’’ type
mesophiles (Simidu and Tsukamoto 1985) that may be considered
as main target of viable counts on TCBS agar at 37–38 °C.
Usually observed absence of sucrose-negative vibrios from
water samples outside fish farming areas could be either due to
severely limited resuspension from sediment surfaces, or to ad-
verse conditions for growth in the presence of oxygen. The latter
appears more likely; for sucrose-negative vibrios are almost absent
from permanently oxic sediments at Lucero where sucrose-posi-
tive vibrios abound (Table 3b). Despite adverse conditions for
growth in the water column, sucrose-negative vibrios are likely
to be transported by tidal currents. This would at least explain
the incidence of a single peak of water-borne sucrose-negative vib-
rios near a fish farming area at Siapar that had been mirrored at a
nearby coral reef (Fig. 2). This apparent culmination of suc/suc+
 W.T. Reichardt et al. / Marine Pollution Bulletin 77 (2013) 325–332
ratios had been preceded by a fish kill event at Siapar that occurred
at peaking relative abundance of vibrios (among heterotrophic
bacteria counted on marine agar).
Determining the predominance of mesophilic sucrose-negative
vibrios in mariculture environments is a practical, but rather rough
assessment of a phenotypic group of vibrios that is to contain the
main human pathogenic Vibrio species except Vibrio cholerae. This
study has indicated that none of the established pathogenic Vibrio
species, but members of the newly described family of Enterovib-
rionaceae make up for the bulk bacterial constituents associated
with milk fish in tropical mariculture. This result diminishes the
specificity of suc/suc+ ratios as a hypothesized indicator of human
pathogens. Enterovibrionaceae comprize both Grimontia hollisae
(Vibrio hollisae), an ‘‘old’’ re-classified human pathogen, and appar-
ently non-pathogenic inhabitants of the intestine of sea fish larvae
(Thompson et al. 2002). The absence of classic virulence genes
from a representative isolate (V6) substantially reduces the likeli-
hood of human health risks in tropical milk fish mariculture.
Future risk assessment might have to pay particular attention to
the frequency at which pathogenic traits can be acquired via hori-
zontal gene transfer in fish intestine.
Extremely high loads of sucrose negative vibrios in the anoxic
intestine of adult milk fish and predominance of this group in
the strongly anoxic sediments of fish farming areas would suggest
that the bulk of sucrose negative vibrios encountered has
originated from fecal pellets of milk fish and are most likely Ente-
rovibrionaceae. This assumption has yet to be confirmed, as their
dispersal with resuspended sediment into neighboring marine
environments including coral reefs can only be inferred from cir-
cumstantial evidence.
Particularly high prevalence of sucrose negative vibrios in
mariculture areas located at greatest distance from the open sea
(Anda) suggests the presence of further selective enrichment fac-
tors beyond low redox potentials. Previous investigations indicate
that milk fish mariculture at Anda has frequently been affected
simultaneously by lowered salinities, extremely low frequencies
of fingerling survival and high abundances of water-borne su-
crose-negative vibrios during SW monsoonal wet seasons. At
the same time sucrose-negative vibrios prevailed only in the
intestine of adult milk fish, not, however, in fingerlings (Reichardt
et al. 2007, WR unpubl.). While milkfish fingerlings or other cul-
tivated animals including holothurians and giant clams continue
to be affected by diseases in the Lingayen Gulf, the pathogenic
potential of sucrose –negative vibrios toward marine animals
has yet to be elucidated. Certain as yet unclassified sucrose-neg-
ative vibrios that cause ulcerative white spot disease in Porites
spp. corals at Bolinao may exemplify a latent pathogenic potential
of sucrose negative vibrios of the study area toward marine ani-
mals (Arboleda and Reichardt, 2010), that deserves further taxo-
nomic examination.
5. Conclusion
In coastal marine environments affected by milk fish maricul-
ture viable counts of sucrose-negative vibrios as indicators of
non-cholera pathogens predominate nearly exclusively under an-
oxic conditions. These favorable conditions are provided in the
intestines of adult milkfish and in the strongly reduced sediments
of fish farming areas. It can be assumed that the enormous load of
sucrose-negative vibrios in milk fish intestines is largely dispersed
via sedimentation of fecal pellets onto sediments that turn rapidly
anoxic under permanent sedimentation of fish farming waste. Hu-
man health risks arising from this accumulation of sucrose-nega-
tive vibrios prove lower than to be expected, since the
predominant bacterial constituents are not pathogenic Vibrio
331
species, but apparently non-toxigenic Enterovibrionaceae. On the
other hand, microbial habitats in fish guts remain a high risk envi-
ronment for genetic exchanges of pathogenic traits. Further inves-
tigations will also have to focus on the most probable involvement
of sucrose-negative vibrios from fish farming areas on diseases of
milk fish fingerlings and maricultured invertebrates as well as on
coral diseases on reefs in the vicinity of fish farming zones.
Acknowledgments
Access to municipal fish farming records by the Mayor of Boli-
nao is gratefully acknowledged.
This investigation was supported by funds from WB-COE and
from DAAD (Germany) to WTR, by a Philippine government grant
(CHED) to the Marine Science Institute, and by a UP-OVPAA
Emerging S&T grant to AOL.
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