The Effect of Preparation of Cebiche on the Survival of Enterotoxigenic Escherichia coli, Aeromonas hydrophila, and Vibrio parahaemolyticus

Authors


  • 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.

Ryan C. Maves, MD, 3230 Lima Place, Washington DC 20521-3230, USA. E-mail: Ryan.maves@med.navy.mil

Abstract

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.

Cebiche 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 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

Although the food items implicated in food-borne 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, non-typhoidal 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 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

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).

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.

Table 1.  Pre-inoculation bacterial counts
SpeciesInfectious dose (number of organisms)Inoculum (number of colony-forming units)
  1. Quantity of bacteria present in each inoculum prior to the cebiche preparation. Typical infectious doses of each organism are presented for comparison.

Aeromonas hydrophila (ATCC 7966)Unknown171.11 × 108
Enterotoxigenic Escherichia coli (H10407)>1.0 × 106 50.91 × 108
Vibrio parahemolyticus (Outbreak isolate, Lima, 1994)>1.0 × 106 3,51.09 × 108

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, 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).

Table 2.  Bacterial counts
SpeciesMean viable count (CFUs)/100 µL
Initial10 min30 min
  1. Mean bacterial load detectable following exposure of cebiche to lemon juice at baseline and after 10 and 30 minutes of marination.

Aeromonas hydrophila0.93 × 1070.39 × 1070.47 × 107
ETEC0.85 × 1070.45 × 1070.64 × 107
Vibrio parahaemolyticus0.45 × 1070.31 × 1070.39 × 107

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 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.

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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