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The Effect of Preparation of Cebiche on the Survival
of Enterotoxigenic Escherichia coli, Aeromonas hydrophila, and
Vibrio parahaemolyticus
Andrés Herrera, MD, Benjamin J. Espinosa, PhD, Gladys Nuñez, BSc, Nereyda Espinoza, BSc,
Ryan C. Maves, MD, and Gregory J. Martin, MD
United States Naval Medical Research Center Detachment, Lima, Peru
DOI: 10.1111/j.1708-8305.2010.00465.x
Background. Cebiche is a common dish in Latin America, prepared using raw fish mixed with vegetables and marinated with
lime juice. The acidity of the lime juice is commonly believed to destroy bacteria and render cebiche as safe to eat. Little data exist
concerning rates of cebiche-associated gastroenteritis outbreaks, although these may be high given the popularity of the dish.
Methods. We inoculated raw fish with Aeromonas hydrophila, Vibrio parahaemolyticus, and enterotoxigenic Escherichia coli to
determine the effect of the cebiche preparation process on bacterial viability. Raw fish were exposed to a suspension of 1.0 × 108
colony-forming units (CFUs) of each organism in a 50-mL solution, prior to the addition of cebiche ingredients. A typical Peruvian
cebiche recipe was used combining limes, onions, sweet potatoes, cilantro, and hot peppers marinated together for 30 minutes. A
homogenized mixture of the dish was then evaluated for pH and bacterial counts at 0, 10, and 30 minutes. As much as 100 μL of
inocula were streaked onto tryptic soy agar (TSA) agar plates and incubated for 24 hours.
Results. The initial average pH of the fish was 6.4 prior to adding cebiche ingredients and 5.0 immediately afterwards. The pH
at 10- and 30-minute periods was 5.4 and 5.2, respectively. Little reduction in bacterial counts was observed at either the 10- or
30-minute time periods, with counts increasing at 30 minutes.
Conclusions. The putative bactericidal role of lime juice in the preparation process is not sufficient to reduce the microbial
population present in cebiche. Pathogens may remain viable after exposure to acidic conditions. The increasing popularity of
Peruvian cuisine may also lead to cebiche-associated illness outside of Latin America.
C
ebiche is a common seafood dish in Latin America,
prepared using raw fish mixed with vegetables and
marinated together with citrus juice, commonly from
limes. It is commonly believed that the acidity of lime
juice effectively sterilizes any microbial contamination,
since it has the capacity to change both the color
and texture of the fish, making it appear slightly
‘‘cooked.’’ A previous study in Costa Rica demonstrated
significant reductions in Vibrio cholerae contamination
using a Costa Rican cebiche recipe.1 Conversely, a
1994 study in Mexico showed that Salmonella spp.
were isolated in 35/221 (15.8%) of 221 cebiche samples
analyzed.2
There is little available information in Peru about
the current rates of acute illnesses related to the
consumption of cebiche, despite the large number of
This data was presented in part as a poster at the 56th Annual
Meeting of the American Society of Tropical Medicine and
Hygiene, November 2007, Philadelphia, Pennsylvania, USA.
Corresponding Author: Ryan C. Maves, MD, 3230
Lima Place, Washington DC 20521-3230, USA. E-mail:
[email protected]
persons who consume it annually. No surveillance
studies concerning food-borne pathogens in cebiche
have been performed in Peru. This is of potential public
health importance for a number of reasons, as cebiche
is a commonly consumed national dish, eaten not only
by Peruvians but also by tourists. Hence, it may be a
common source of diarrhea among visitors as well as
local residents.
Food-borne illness is an important cause of morbidity and mortality worldwide, especially in developing
countries where food safety measures and hygiene
practices may be less emphasized or inadequate.3,4
Given the scale and complexity of the food supply, it
is difficult to ensure that all food is kept free from
potential sources of contamination. Despite recent
advances in the methods to eliminate pathogens
from food items, food-borne diseases remain a
major cause of illness worldwide. A total of 17,883
laboratory-confirmed cases of food-borne-related infections were reported during the year 2007 in the
United States according to available data obtained from
the Foodborne Diseases Active Surveillance Network
(FoodNet) of the Centers for Disease Control and
Prevention.5
Published 2010. This article is a US Government work and is in the public domain in the USA.
Journal of Travel Medicine 2010; Volume 17 (Issue 6): 395–399
396
Although the food items implicated in foodborne illnesses are usually not definitively determined,
seafood accounts for up to 10% of food-borne-related
outbreaks in most countries.3,6 However, this rate
varies depending on each country’s per capita seafood
consumption, food preparation processes, and handling
practices. In 2004, Peru’s total fisheries production was
9.6 million tons, making it the second largest producer
of fish worldwide, after China. With a per capita
consumption of 21.4 kg/year in the year 2001, Peru
was the third largest per capita consumer of seafood in
South America, after Guyana and French Guiana.7,8
Given the often-inadequate food safety measures
in developing countries, seafood can represent an
important source of food-borne pathogens. According
to some studies carried out in the United States
and Spain, bacteria belonging to the Vibrionaceae
family (ie, Vibrio and Aeromonas spp.), are frequently
isolated from uncooked fish.3,4,6,9 Other nonindigenous
pathogenic bacteria (present due to contamination with
human or animal feces), such as Escherichia coli, nontyphoidal Salmonella, Shigella spp., Listeria monocytogenes,
Plesiomonas spp., and Clostridium botulinum are also
implicated in many seafood-related outbreaks.3,6,9,10
Since the ingestion of raw or undercooked food is a
risk factor commonly associated with infection, the rate
of seafood-related outbreaks tends to be higher in those
countries where seafood is consumed raw or only slightly
cooked. In comparison to developed countries, where
shellfish accounts for almost all cases of seafood-related
outbreaks, the ingestion of uncooked fish is responsible
for most of the acute diarrheal episodes related to the
intake of seafood in developing countries.3
In Peru, consumer preferences are usually for fresh
fish, which is often eaten raw. One of the most
popular meals in Peru is cebiche, a dish made of
fresh raw fish that is prepared with lime juice and
seasoned with onions, yellow pepper, sweet potatoes,
corn, garlic, and cilantro. As cebiche is not exposed to
a typical cooking process, many pathogens that would
otherwise be inactivated by heat may remain viable after
preparation. It is believed that the acid of limes used
in the preparation of cebiche eliminates any microbial
contamination. Exposure to the acidic lime juice changes
the color and texture of the fish, making it appear slightly
‘‘cooked.’’ It is widely thought that exposing bacteria
to acid stress conditions is enough to inactivate or kill
them.
Bacterial requirements for survival and growth
include an external pH value that is between 4 and 8.
Some pathogens, such as E. coli and Salmonella, have
mechanisms that allow them to grow under low
pH conditions. These mechanisms include acid shock
protein synthesis, the development of maximal acid
tolerance through the induction of pH homeostasis,
and protein repair systems.11,12
In this project, we seek to correlate the
supposed bactericidal effect of lime juice with the
survival and growth rates of three different pathogenic
J Travel Med 2010; 17: 395–399
Herrera et al.
bacteria frequently associated with food-borne disease,
inoculated onto fish samples and exposed to the cebiche
preparation process.
Materials and Methods
Preparation of Inocula
Three different bacterial strains were selected:
Aeromonas hydrophila (ATCC 7966), enterotoxigenic
Escherichia coli (ETEC, H10407 LT/ST O78:H11),
and Vibrio parahaemolyticus (IMA635, derived from
a 1994 outbreak in Lima, Peru). An inoculum of
each strain was prepared by culturing each separately
in duplicate on tryptic soy agar (TSA) plates (lot
6228427, BDDIFCO) and incubating overnight at
37◦ C. Strains were harvested and suspended separately
in phosphate-buffered saline (PBS; pH 7.2). Serial
dilutions were obtained to give a final concentration
of 1 × 108 CFU/mL. Bacterial growth was determined
by measuring the absorbance at 600 nm (with optical
densities of each 1/100 dilution between 0.106 and
0.111) and by plate count on TSA. The infectious
dose for each pathogenic strain was considered before
obtaining the final inocula to confirm that each
450 g portion would contain sufficient bacteria to be
potentially infectious if consumed.
Sample Preparation
Prior to the addition of cebiche ingredients, we
inoculated each fish filet sample (450 g) with a
50 mL bacterial suspension13 containing approximately
1 × 108 CFU/mL of each organism. The bacterial
suspension remained in contact with the surface of
the fish for 10 minutes at room temperature.14 Ten
grams of portions were then collected and blended
for 2 minutes in an electric blender with 90 mL PBS.
To determine the initial bacterial count in the fish,
100 μL aliquots of diluted homogenate were streaked
in duplicate onto TSA, MacConkey agar (ETEC,
A hydrophila), and TCBS agar (V parahaemolyticus) plates
and incubated overnight at 37◦ C.
Before and after the addition of lime juice but prior
to the addition of the remaining cebiche ingredients,
baseline pH levels of the samples were determined by
obtaining 10 g from the sample and blending it with
40 mL of distilled water.
Preparation Process of Cebiche
A typical Peruvian cebiche recipe was used combining
450 g of toyo (Mustelus whitney, Mustelus lunulatusi), a
common fish found in all warm and temperate coastal
seas with cilantro, garlic, hot peppers, sweet potatoes,
and corn (all ingredients were obtained from a retail
market) marinated together with lime juice for 10 and
30 minutes (which are typical marination times for
Peruvian cebiche).15
ETEC, Vibrio, and Aeromonas in cebiche
Bacterial Load and pH Determination
After the 10- and 30-minute marination periods, all
ingredients were homogenized in a blender. A 10 g
aliquot was transferred to a blender jar containing
90 mL of PBS and blended for 2 minutes, resulting
in a 1 : 10 dilution. Serial dilutions of the original
homogenate were prepared to 1 : 1,000, 1 : 10,000,
and 1 : 100,000 concentrations in PBS. Hundred
microliters of aliquots of each dilution were transferred
using a pipette into separate and duplicate TSA,
MacConkey agar (ETEC, A hydrophila), and TCBS
agar (V parahaemolyticus) plates. Plates were incubated
overnight at 37◦ C.
The pH was then measured using an electronic pH
meter as described previously.16 Final pH measurements
were performed immediately after adding lime juice
to the sample and after a 10-minute (cebiche sample
#1) and a 30-minute (cebiche sample #2) period,
respectively.
Results
The baseline pH of the fish was 6.5 prior to the addition
of food ingredients. Specimen pH was approximately
5.0 for all three groups following the addition of lime
juice (Figure 1). Initial bacterial load was determined
as described above, with concentration of bacteria
well above described infectious doses (when known)
(Table 1).
Following cebiche preparation, homogenized specimens were tested at 0, 10, and 30 minutes for pH and
placed into culture media as above. At the 10-minute
period, pH for all the specimens was 5.36 to 5.45, before
declining to 5.22 to 5.24 at the 30-minute period. These
pH levels reached after the preparation process were not
sufficient to inhibit the bacterial population present in
the sample (Figure 1).
Bacterial loads for all three species evaluated declined
between the 0- and 10-minute points, with Aeromonas
declining more sharply than ETEC or Vibrio. However,
397
Table 1
Pre-inoculation bacterial counts
Species
Aeromonas hydrophila
(ATCC 7966)
Enterotoxigenic Escherichia
coli (H10407)
Vibrio parahemolyticus
(Outbreak isolate, Lima,
1994)
Infectious dose
(number of
organisms)
Inoculum (number
of colony-forming
units)
Unknown17
1.11 × 108
>1.0 × 106 5
0.91 × 108
>1.0 × 106 3,5
1.09 × 108
Quantity of bacteria present in each inoculum prior to the cebiche preparation.
Typical infectious doses of each organism are presented for comparison.
Table 2
Bacterial counts
Mean viable count (CFUs)/100 μL
Species
Aeromonas hydrophila
ETEC
Vibrio parahaemolyticus
Initial
10 min
30 min
0.93 × 107
0.85 × 107
0.45 × 107
0.39 × 107
0.45 × 107
0.31 × 107
0.47 × 107
0.64 × 107
0.39 × 107
Mean bacterial load detectable following exposure of cebiche to lemon juice at
baseline and after 10 and 30 minutes of marination.
after this initial decline at the 10-minute point, bacterial
growth appeared to have resumed and, in the case of
ETEC, was approaching baseline levels by 30 minutes
(Table 2).
Discussion
Recent years have seen a substantial increase in
international trade, travel, and migration. As a result
of these increased connections around the world,
there has also been a greater risk of cross-border
transmission of infectious diseases.18 With the impact
of industrialization on food production and trade,
food-borne diseases represent a leading public health
Figure 1 The pH of cebiche dishes following bacterial inoculation. The pH of cebiche in lemon juice following bacterial
inoculation by organism, measured at baseline and at 10- and 30-minute periods.
J Travel Med 2010; 17: 395–399
398
problem in the modern era. Access to international
travel has also contributed to the increased rate of
food-borne-related outbreaks. In the United States, an
estimated 10% to 19% of food-borne illnesses involve
seafood consumption.3 Norovirus, L monocytogenes,
Campylobacter spp., Aeromonas spp., V parahaemolyticus,
and scombrotoxin have been implicated as the
most commonly isolated agents in seafood-related
illnesses.3,10,19,20
When present in fish, indigenous bacteria are found
at low levels and are easily inactivated or killed when
fish is properly cooked. Seafood-associated pathogens
have been involved in a number of epidemics (eg, the
1991 cholera epidemic in Peru and the 1994 epidemic
of Vibrio vulnificus infection in Denmark).9,18 Most
of these outbreaks occur in countries where seafood
is consumed raw or slightly cooked (such as Japan
and Peru). These microorganisms have evolved stress
responses that permit their survival and persistence in
food products, despite several methods to reduce or
eliminate them from fresh fish.12
In our study, we subjected contaminated raw fish to
the cebiche preparation process. It is a commonly held
belief that the acidic conditions provided by lime juice
are sufficient to eliminate bacterial pathogens present
in fish. However, exposing bacteria to a pH of 5.0 is
insufficient to kill or even reduce bacterial counts in the
fish samples we tested.
For growth and survival, bacteria require pH values
that are between 4 and 8. Even though acidification
is usually effective in controlling bacterial growth,
organisms have evolved several mechanisms directed
toward survival in conditions of low pH.12 In this
project, the acidified conditions caused by lime juice
were insufficient to kill the pathogenic bacteria tested
and should not be relied upon to adequately sterilize
potentially contaminated fish.
Maintenance of seafood quality is central to ensuring
the safety of seafood. There is presently no way to
ensure that all food is kept free from potential sources
of contamination. Good manufacturing practices,
involving the harvest of fish from approved areas
(sewage-free harvest beds), type and size of fish caught,
methods of capture and processing immediately after
capture, can all decrease the rate of contamination
of fishery products.9,21 Good handling practices
guidelines are available for seafood restaurants, and they
recommend the use of several refrigerating, freezing,
defrosting, and storage measures to reduce the microbial
spoilage of products and to improve food safety.
This experiment has limitations that may restrict
its applicability to travelers who consume cebiche. We
tested only a focused number of enteric pathogens and
did not evaluate other common causes of infectious
diarrhea, such as Campylobacter, Salmonella, and Shigella
species. Additionally, we used high inocula in our testing
that were in excess of the described infectious doses of
the bacteria tested. We cannot state what the effect of
cebiche preparation would be on lower bacterial doses.
J Travel Med 2010; 17: 395–399
Herrera et al.
In summary, conventional methods of cebiche
preparation are not adequate to inactivate common
pathogenic bacteria. International travelers should
exercise caution when consuming uncooked seafood.
Persons at particular risk (including young children,
the elderly, immunocompromised persons, and pregnant women) should be encouraged to eat fully cooked
seafood and to avoid buying fish or shellfish from street
vendors.20,21
Acknowledgment
This work was supported by work unit number 847705
82000 25GB B0016.
IRB statement: The study protocol was approved
by the Naval Medical Research Center Institutional
Review Board (PJT.NMRCD.2007.006) in compliance
with all applicable Federal regulations governing the
protection of human subjects.
Disclaimer
The views expressed in this article are those of the
authors and do not necessarily reflect the official policy
or position of the Department of the Navy, Department
of Defense, nor the US Government. Dr Martin,
Dr Espinosa, and Dr Maves are US military service
members. This work was prepared as part of their official
duties. Title 17 United States Code (USC)/Section 105
provides that ‘‘Copyright protection under this title
is not available for any work of the United States
Government.’’ Title 17 USC Section 101 defines a
US Government work as a work prepared by a military
service member or employee of the US Government as
part of that person’s official duties.
Declaration of Interests
The authors state that they have no conflicts of interest
to declare.
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