AND APPLIED ENVIRONMENTAL MICROBIOLOGY, July 2005, p. 40524056 0099-2240/05/$08.00 0 doi:10.1128/AEM.71.7.40524056.2005 Copyright 2005, American Society for Microbiology. All Rights Reserved.
Vol. 71, No. 7
Mediterranean Fruit Fly as a Potential Vector of Bacterial Pathogens
Shlomo Sela,1* David Nestel,2 Riky Pinto,1 Esther Nemny-Lavy,2 and Moshe Bar-Joseph3
Department of Food Sciences, Institute for Technology and Storage of Fresh Produce,1 and Department of Entomology2 and Department of Virology,3 Institute of Plant Protection, Agricultural Research Organization (ARO), The Volcani Center, Beth-Dagan, Israel
Received 26 October 2004/Accepted 1 February 2005
The Mediterranean fruit y (Ceratitis capitata) is a cosmopolitan pest of hundreds of species of commercial and wild fruits. It is considered a major economic pest of commercial fruits in the world. Adult Mediterranean fruit ies feed on all sorts of protein sources, including animal excreta, in order to develop eggs. After reaching sexual maturity and copulating, female ies lay eggs in fruit by puncturing the skin with their ovipositors and injecting batches of eggs into the wounds. In view of the increase in food-borne illnesses associated with consumption of fresh produce and unpasteurized fruit juices, we investigated the potential of Mediterranean fruit y to serve as a vector for transmission of human pathogens to fruits. Addition of green uorescent protein (GFP)-tagged Escherichia coli to a Mediterranean fruit y feeding solution resulted in a dose-dependent increase in the ys bacterial load. Flies exposed to fecal material enriched with GFP-tagged E. coli were similarly contaminated and were capable of transmitting E. coli to intact apples in a cage model system. Washing contaminated apples with tap water did not eliminate the E. coli. Flies inoculated with E. coli harbored the bacteria for up to 7 days following contamination. Fluorescence microscopy demonstrated that the majority of uorescent bacteria were conned along the pseudotrachea in the labelum edge of the y proboscis. Wild ies captured at various geographic locations were found to carry coliforms, and in some cases presumptive identication of E. coli was made. These ndings support the hypothesis that the common Mediterranean fruit y is a potential vector of human pathogens to fruits. Outbreaks of bacterial diseases associated with the consumption of fresh produce, such as lettuce, apple cider, unpasteurized apple juice, and alfalfa sprouts, have been reported with increasing frequency during the last decade (reviewed in references 3 and 17). Although some produce-associated outbreaks may be due to cross-contamination from meat products, others more likely reect direct contamination in the eld with residues from feces of wild or domestic animals (1, 5, 7). Direct contact with manure-contaminated soil or dust might be a source of preharvest contamination. Indirect sources of contamination could also be the trophic interactions between fruits and plant foragers like birds, mammals, and insects. The association between bacteria and fruit ies has usually been mentioned in relation to the source of attractants or in relation to symbiosis, which is important to the nutrition of the insects. Fruit ies have seldom been referred to as vectors of plant or human disease. The exceptions to this include the study of Cayol et al. (4), which showed the potential capacity of the Mediterranean fruit y (Ceratitis capitata) to transmit plant disease, and more recently, the study of Janisiewicz et al. (12), which showed the possible involvement of the vinegar y (Drosophila melanogaster) in the transmission of pathogenic bacteria to postharvest wounded apples. The ability of the Mediterranean fruit y to serve as a vector for food-borne pathogens has not been reported previously, although several features of this insect suggest its potential as a vector. The Mediterranean fruit y is a generalist cosmopolitan pest that infests more than 200 species of commercial and wild fruits (6, 10, 15). Like most ies, fruit ies must feed on protein in order to develop eggs. The protein sources for the Mediterranean fruit y include rotting fruits and fecal material (FM) (9, 14, 21). Most fruit ies locate protein food sources through attraction to ammonium-releasing substances (16). This biological phenomenon was used by Pinero et al. (18) to develop an inexpensive attractant made of human urine and chicken feces for monitoring of fruit ies by resource-poor fruit farmers. After reaching sexual maturity and copulating (in the summer, approximately 10 days after eclosion), female ies lay eggs in fruit by puncturing the skin of the fruit with their ovipositors about 1 to 2 mm deep and injecting batches of eggs into the wounds. First-instar larvae hatch from the eggs 2 to 3 days later, and two more instars feed on the fruit tissue. Fully grown third-instar larvae exit from the fruit, crawl and jump to the ground, and dig a few centimeters into the soil to pupate. After approximately 10 days (in the summer), newly emerged ies come of the ground, starting the cycle again. In a tropical setting, the Mediterranean fruit y has the potential to have more than 10 generations per year. The attraction of the Mediterranean fruit y to a variety of fecal material and its foraging on this material as a nitrogen source, in conjunction with its egg-laying activity in a variety of fruits and its extensive prevalence in numerous agricultural regions in the world, suggested that this y potentially is a vector for feces-borne pathogens. In the present study we investigated the capacity of this y to transmit Escherichia coli from contaminated fecal material to intact apples.
* Corresponding author. Mailing address: Department of Food Sciences, ARO, The Volcani Center, Beth-Dagan 50250, Israel. Phone: [phone]. Fax: [phone]. E-mail: [e-mail]. 4052
MATERIALS AND METHODS Bacteria and bacteriological determination. E. coli strain TG1 (Amersham Biosciences, United Kingdom), which expresses green uorescence protein
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TRANSMISSION OF E. COLI BY MEDITERRANEAN FRUIT FLY TABLE 1. Presence of coliforms and E. coli in wild Mediterranean fruit iesa
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Sampling location
Ecosystem type
No. of ies in sample
No. of coliforms (CFU/y)
No. of E. coli (CFU/y)b
Beth-Dagan 1 Beth-Dagan 2 Kfar-Saba Rehovot
Mixed Mixed Home Home
citrus orchard citrus orchard gardens with bearing fruit trees in rural areas gardens with bearing fruit trees in urban areas
16 23 3 4
1.3 4.9
104 104 c
1.1
104 c
a Mediterranean fruit y samples were obtained from different geographic locations in central Israel and different ecosystems. Flies samples were collected with McPhail traps loaded with Biolure. The traps were exposed for 2 weeks during March and April 2004. b Presumptive identication as E. coli. c Bacteria were detected following enrichment.
(GFP) on a high-copy-number plasmid (a gift from I. Benhar, Tel-Aviv University) was used in this study. When this organism is grown on a Trypticase soy agar plate, uorescence is easily observed under long-wavelength UV light. For each experiment, bacteria were grown in tryptic soy broth supplemented with 100 g/ml ampicillin for 20 h at 37C and washed twice with double-distilled water (DDW). A number of CFU were added to the feeding solution, as described below. Contamination of ies by GFP-tagged E. coli was determined as follows. Twenty ies were put into a 15-ml tube containing 2 ml phosphate-buffered saline, pH 7.4, and vortexed (Vortex-Genie 2) three times at maximal power for 30 s. One hundred microliters of the solution and 10-fold serial dilutions were spread plated on Trypticase soy agar containing ampicillin. The plates were incubated for 24 h at 37C, and the number of ampicillin-resistant GFP-expressing bacteria was recorded. Preparation of ies. Mediterranean fruit y pupae (strain Sade) were obtained from the laboratory colony of the Institute of Biological Control (Israel). Approximately 100 puparia 2 days before adult eclosion were placed for emergence in a petri dish in a Perspex insect cage (40 by 30 by 30 cm) and supplied with water and with a 20% sucrose solution. All feeding materials consisted of 10 ml of solution soaked into cotton wool in a petri dish to prevent drowning of the feeding ies. Maturation of insects and experiments were conducted in a temperature-controlled room at 27C 2C. Contamination of Mediterranean fruit ies with E. coli present in the feeding solution. Adult ies (ca. 2 days old) that were previously maintained on a 20% sucrose solution were starved for 8 h and then exposed to a 20% sucrose solution containing a predetermined concentration of bacteria (6, 7, 8, or 9 log10 cells per ml). In some experiments, the feeding solution was replaced with FM enriched with GFP-tagged E. coli. The FM solution was prepared by vigorously mixing 4 g of fresh human feces with 36 ml of DDW containing 2 109 CFU GFP-tagged E. coli per ml. All feeding solutions (sucrose and FM) were introduced into the cages by using soaked cotton wool, and the ies (100 ies per cage) were allowed free access to the solutions for 20 h. After this, 10 males and 10 females were randomly sampled from each concentration and cage (one cage per concentration), pooled by sex, and subjected to bacteriological determination, as described above. For bacterial survival experiments, following exposure of the ies to E. coli, the contaminated solution was removed from the cage, and regular (noncontaminated) food was reintroduced. Flies were sampled, as described above, at time zero (control, before exposure) and 1, 2, 3, 6, and 7 days after the beginning of exposure, and the number of GFP-tagged E. coli was determined, as described above. The experiment was conducted with three replicate cages. Contamination of apples by Mediterranean fruit ies. The experimental design of the model system followed essentially the design of the transmission experiments conducted by Janisiewicz et al. (12). Adult ies were fed for 3 days after eclosion on protein-hydrolyzed yeast and sucrose (1:3) cake to stimulate egg development. The cake was prepared by mixing the ingredients and allowing the formation of a compact mass through hygroscopic absorption of ambient humidity. The ies then were starved for 20 h to stimulate hunger and exposed either to FM alone or to FM enriched with GFP-tagged bacteria. After 1 h, one previously washed apple (cv. Starking; weight, 110 to 190 g) was introduced into each cage for 15 h. After this, the fruits were carefully removed from the cages to avoid cross-contamination and put individually into sterile stomacher bags. The bags were shaken in 250 ml DDW in an orbital shaker at 180 rpm for 20 min, and samples were taken for bacterial enumeration, as described above. The presence of at least 1 GFP-expressing CFU indicated apple contamination. Contaminated fruits typically harbored 10 to 103 E. coli cells per g. To verify that no mechanical contamination occurred during handling of the FM solution and apples, control experiments were performed with apples and E. coli-enriched FM but without ies. Each experiment was conducted with three replicate cages and
repeated three times on different dates. Thus, the entire set of experiments consisted of nine exposed apples and nine control apples. To check the effect of the common home-style fruit decontamination treatment on bacterial persistence, a similar set of experiments was performed. However, in these experiments apples removed from cages were hand washed under running tap water for 30 s before they were subjected to bacteriologic analysis. Field survey. Wild Mediterranean fruit ies were collected in three geographic locations in central Israel during March and April 2004 using McPhail traps loaded with Biolure (trimethylamine-putrecine-ammonium acetate; Suterra, Bend, Oreg.). The locations included Beth-Dagan (10 km east of Tel-Aviv), Rehovot (20 km southeast of Tel-Aviv), and Kfar-Saba (20 km northeast of Tel-Aviv) and represented two types of ecosystems (Table 1). The traps were left in the eld for up to 2 weeks, and the ies were aseptically removed from the traps and brought to the laboratory. To estimate the level of coliforms and E. coli, all ies collected in a single trap were homogenized in a tube containing Luria broth, and samples were spread plated, as described above, on both violet red-bile agar and Chromcult TBX (tryptone bile x-glucuronide) agar (Merck). The plates were incubated at 37C for 24 to 48 h, and pink to purple colonies (diameter, 0.5 mm) on violet red-bile agar plates were considered to be coliforms, while blue-green colonies grown on Chromcult agar were presumptively identied as E. coli. Luria broth tubes containing ies (after vortexing) were further incubated at 30C for 18 h to recover possible injured bacteria. Samples that were negative for coliforms or E. coli in the direct plating test were retested for the presence of these bacteria following the enrichment step. Statistical analysis. The relationship between the bacterial loads in feed and bacterial contamination in ies was investigated using linear regression analysis (22). The relationship between the E. coli contamination of apples when they were exposed to ies and feces and the E. coli contamination of apples when they were exposed to feces but no ies (control) was inferred by analysis of frequencies from a test of independence (after Yates correction) (22). Similarly, a test of independence (after Yates correction) was used to infer the relationship between the bacterial contamination of apples after they were washed in the presence of ies and the bacterial contamination of apples after they were washed in the absence of ies. The effect of food substrate (sucrose or FM) on the ability of male and female Mediterranean fruit ies to acquire bacteria was analyzed with a two-way analysis of variance (22) after log10 transformation
RESULTS Contamination of Mediterranean fruit with ies E. coli. The results show that GFP-tagged E. coli cells are readily transmitted to Mediterranean fruit ies. The average log10 number of bacteria per y increased linearly with the log10 amount of bacteria in feed (for males, F 17.7 and P 0.05; for females, F 547.1 and P 0.05) (Fig. 1). The contamination of females (b 1.18, t 4.21, P 0.05) was slightly greater than that of males (b 1.05, t 23.4, P 0.05). After 24 h, the average number of bacteria per y when the ies were exposed to FM was similar (F 0.001, P 0.9) to the average number of bacteria per y observed when the ies were exposed to the contaminated sucrose solution (Fig. 2). There were no statistical differences between males and females (F 3.17, P
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SELA ET AL.
APPL. ENVIRON. MICROBIOL.
5
Females Males
log CFU/ fly
3 2 1 0 3
4
log CFU/ fly
5 7 9 11
3 2 1
Bacterial concentration in feed (log10/ml)
FIG. 1. Acquisition of GFP-tagged E. coli by Mediterranean fruit ies exposed to contaminated feed. Flies were exposed for 24 h to a 20% sucrose solution supplemented with different numbers of tagged E. coli cells. Male and female ies were collected separately, and the number of Ampr, GFP-expressing bacteria in 20 ies was determined.
0
1
2
3
6
7
Days
FIG. 3. Survival of E. coli on contaminated Mediterranean fruit ies. Flies were exposed to a sucrose solution supplemented with 4.6 9 10 CFU/ml of E. coli for 20 h. After this, a sample of 10 to 20 ies was collected each day from the cage and subjected to microbiological analysis. Day 1 refers to the 24 h after the beginning of exposure. No E. coli was detected on preexposure ies.
0.1); however, there was a tendency of females to acquire larger numbers of bacteria in the two substrates. Survival of E. coli on Mediterranean fruit ies. Flies continued to harbor living E. coli for at least 7 days following the beginning of exposure (Fig. 3). No E. coli cells were detected in samples taken at time zero (before exposure). Transmission of E. coli to intact apples by contaminated ies. The presence of FM and Mediterranean fruit ies in a cage resulted in a high contamination rate. Six of nine apples were found to be contaminated with the specic GFP-tagged E. coli strain. Washing the fruits with tap water did not prevent apples from maintaining the bacterial contamination; four of nine apples were found to harbor E. coli after they were washed with tap water. In control y-free cages containing apples and FM enriched with E. coli, no contamination of the fruits was detected, supporting the hypothesis regarding the role of Mediterranean
100000
No. of E. coli CFU per fly
Males Females
10000
1000
fruit ies in transmitting the bacteria (e.g., contamination was signicantly dependent on the presence of ies in the cage; 2 4.18 and P 0.05 in experiments in which the fruit was not washed and 2 2.89 and P 0.09 in experiments in which the fruit was washed with tap water). Wild Mediterranean fruit ies carry coliforms and E. coli. Three of four samples of wild ies, collected during the end of March and the beginning of April, were found to harbor coliforms, either by direct plating or following enrichment (Table 1). The number of coliforms ranged from 1.3 104 to 4.9 104 cells. One of the samples, which contained 23 ies, also harbored a signicant number of presumptive E. coli cells (1.1 104 CFU/y). In another sample composed of four ies, presumptive E. coli was detected only after enrichment. Microscopic examination of contaminated Mediterranean fruit ies. Flies which were exposed to 2 109 GFP-expressing bacteria in the sucrose feed solution for 24 h were dissected and observed under a uorescence microscope (Leica model DMLB) equipped with a charge-coupled device camera (Leica model DC200). Fluorescent bacteria were detected in the ies labelum, specically along the pseudotrachea (Fig. 4), but not in other organs, including the ovipositor, other mouthparts, and tarsomeres. DISCUSSION Feces have been suspected as sources of pathogens on contaminated fruits, vegetables, or minimally processed produce that have subsequently been associated with or conrmed as causes of human disease outbreaks (2). Insects are a recognized contributing epidemiological factor in the spread of food-borne pathogens, especially E. coli, Listeria, Salmonella, and Shigella. Houseies, for example, were shown to be potential vectors of E. coli O157:H7 (11, 13). The vinegar y (D. melanogaster) was also implicated in the transmission of pathogenic E. coli to wounded apple tissues under laboratory conditions (12). Yet the involvement of the Mediterranean fruit y in transmission of human pathogens to fruits has not been investigated previously. Potentially, there are a number of reasons why this pest and other fruit ies should be of concern as
100
10
1
Control
+bacteria
Control
+bacteria
Sucrose (20%)
Feces
FIG. 2. Mediterranean fruit ies acquire E. coli from contaminated fecal material. Flies were exposed to a 20% sucrose solution or fecal material enriched with 2 109 CFU/ml GFP-labeled E. coli for 20 h. The numbers of labeled bacteria in males and females were determined. Flies could efciently acquire E. coli from both the sucrose solution and fecal material in similar numbers. Bacteriological determinations were performed with batches of 10 males and 10 females per replicate (three replicates per treatment).
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FIG. 4. Visualization of E. coli in the Mediterranean fruit y by uorescence microscopy. Flies were fed a 20% sucrose solution supplemented with 109 CFU/ml GFP-expressing E. coli. (A) Micrograph of the labelum under UV light, with bacteria clearly present in the pseudotrachea of the labelum (arrows). (B) Fine structure of the labelum with associated uorescent bacteria at a higher magnication. Bars, 100 m.
that the Mediterranean fruit y might also act as a vector for transmitting bacteria to intact apples. This model system, which facilitates bacterium-y-apple interactions, simulates the reported acquisition of microorganisms by fruit ies feeding on fecal material in nature (14). Microscopic analysis suggested that the main organ involved in bacterial uptake is the ys mouthparts. The mouthparts may also be the main vehicle for the contamination of fruits, since fruit ies forage for fruit juices on the surface of fruits (8). An alternative mechanism of fruit contamination may be through the introduction of pathogenic bacteria underneath the intact fruit skin by the mechanical activity of the female ovipositor during egg laying. Fruit y eggs derived from ies feeding on a GFP-tagged E. coli contaminated sucrose solution were associated with uorescent bacteria (Sela and Nestel, unpublished data). The nding that washing E. coli-contaminated apples under owing tap water did not remove bacteria further supports this route of transmission. It is noteworthy that investigation of the association between E. coli O157:H7 and houseies showed that a large number of the bacteria adhered to the surface of the housey mouthparts and actively proliferated in the minute spaces of the labelum. The ingested bacteria were excreted continuously for at least 3 days after feeding (13). It has not been established yet whether E. coli can also grow within the Mediterranean fruit y or whether the y merely serves as a mechanical vector. However, survival studies showed that Mediterranean fruit ies continued to harbor viable GFP-tagged E. coli for at least 7 days following contamination. The results of a restricted eld survey demonstrated that wild ies do carry coliforms and that some even harbor presumptive E. coli during a period (spring) that the Mediterranean fruit y population is still low. These ndings highlight the potential of the y to carry human pathogens and to serve as a vector for transmission of food-borne diseases. Our ndings strengthen the need for further investigations to evaluate the actual epidemiological potential of the Mediterranean fruit y to disseminate human pathogens. Control measures to reduce fruit y populations should include cultural control, as well as strict sanitation measures both within and surrounding orchards. These measures are expected to eliminate the risk of disseminating bacterial pathogens to commercially grown fruits.
ACKNOWLEDGMENTS
potential vectors. The Mediterranean fruit y, like most fruit ies, forages for food on nitrogenous sources, such as fecal material (14, 19). This natural food source often contains pathogens. For example, Robacker et al. (20) isolated a pathogenic bacterium (Staphylococcus aureus) from the mouthparts of a female Mexican fruit y (Anastrepha ludens). Moreover, different strains of E. coli (including strains typical of warmblooded animal feces) have been isolated from the apple maggot (Rhagoletis pomonella) (14). The last nding reinforces the potential risk for transmission of human diseases by fruit ies foraging for proteinaceous material and supports the hypothesis that other fruit-foraging insect species have the potential to serve as vectors in the contamination of fresh produce. While the vinegar y (D. melanogaster) was shown to transmit E. coli O157:H7 to wounds on apple fruit (12), the present study, in which a similar experimental system was used, showed
We are grateful to Yoav Gazit (The Institute for Biological Control, Beth-Dagan, Israel) for supplying the ies and for microscopic analyses. We also thank Zvi Mendel (Department of Entomology, The Volcani Center) for helpful discussions and three anonymous reviewers whose comments greatly improved the manuscript. This work was partially supported by an intramural grant from The Volcani Center to S. Sela.
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