Bacterial symbiosis in Bactrocera oleae, an Achilles' heel for its pest control.

Investigations on microbial symbioses in Tephritidae have increased over the past 30 years owing to the potential use of these relationships in developing new control strategies for economically important fruit flies. Bactrocera oleae (Rossi)-the olive fruit fly-is a monophagous species strictly associated with the olive tree, and among all the tephritids, its symbionts are the most investigated. The bacterium Candidatus Erwinia dacicola is the major persistent resident endosymbiont in wild B. oleae populations. Its relationship with B. oleae has been investigated since being identified in 2005. This endosymbiont is vertically transmitted through generations from the female to the egg. It exists at every developmental stage, although it is more abundant in larvae and ovipositing females, and is necessary for both larvae and adults. Studying B. oleae-Ca. E. dacicola, or other B. oleae-microbe interactions, will allow us to develop modern biological control systems for area-wide olive protection and set an example for similar programs in other important food crops. This review summarizes the information available on tephritid-microbe interactions and investigates relationships among fruit flies, bacteria and host plants; however, its focus is on B. oleae and its strict association with Ca. E. dacicola to promote environmentally friendly control strategies for area-wide pest management.

Bacteriophages specific to Shiga toxin-producing Escherichia coli exist in goat feces and associated environments on an organic produce farm in Northern California, USA.

Shiga toxin-producing Escherichia coli (STECs) contamination of produce, as a result of contact with ruminant fecal material, has been associated with serious foodborne illness. Bacteriophages (phages) that infect STECs have primarily been reported to be of cattle origin. However, they likely exist in other environments or in animals that share habitats with cattle, such as goats. To explore the presence and diversity of phages specific to STEC O157 and the top six non-O157 STECs in goat-associated environments, environmental samples consisting of feces (goat and cattle) and soil samples were collected monthly for six months from an organic produce farm. A variety of phages belonging to the Myoviridae, Siphoviridae, and Podoviridae families were isolated from all goat fecal and half of the soil samples. The most commonly isolated phages belonged to Myoviridae and were lytic against STEC O103. The isolated phages had different host ranges, but collectively, showed lytic activity against O157 and the top six non-O157 STEC strains excluding O121. Two non-O157 STECs (O174: H21 and O-antigen-negative: H18) were isolated from soil and cattle feces, respectively. Although prior studies have reported that goats shed STEC into the environment, the findings of the current study suggest that goat feces may also contain lytic STEC-specific phages. The phages of goat origin have the capacity to infect STECs implicated in causing foodborne outbreaks, making them potential candidates for biocontrol pending additional characterization steps. Further work is needed to determine if the addition of goats to the farm environment could potentially reduce the presence of STECs.

Comparison of IMS Platforms for detecting and recovering Escherichia coli O157 and Shigella flexneri in foods.

Two automated platforms using immunomagnetic separation technology were compared for detecting and recovering Escherichia coli O157 in ground beef and sprouts and Shigella flexneri in green onions. The foods were inoculated with <20 CFU/25 g and tested at 5 and 24 h postincubation. Immunomagnetic beads were mixed with food enrichments, processed through the Pathatrix Auto or KingFisher Flex, and tested by real-time PCR (qPCR) and recovery on selective agars. At 5 h, the Pathatrix Auto detected E. coli O157 in 90% and 60% of the ground beef and sprouts samples and S. flexneri in all of the green onion samples. It also recovered E. coli O157 in all the samples but could not recover S. flexneri in any of the green onion samples. In comparison, the KingFisher Flex detected E. coli O157 in 80% and 30% of the ground beef and sprouts samples and S. flexneri in all of the green onion samples. It also recovered E. coli O157 in 90% of the ground beef samples but none of the sprouts samples and S. flexneri in 20% of the green onion samples. At 24 h, both platforms detected and recovered the target bacteria in all of the samples.

A 7-plex microbead-based immunoassay for serotyping Shiga toxin-producing Escherichia coli.

Serotyping of Shiga toxin-producing Escherichia coli (STEC) has been contingent upon the availability of antisera. Here we describe a 7-plex microbead-based immunoassay to simultaneously serotype seven STECs (i.e., belonging to serogroups O26, O45, O103, O111, O121, O145, and O157) by the Luminex xMAP® technology. This technology presents many advantages: Its multiplexed format (up to 100 analytes) saves time, reagents, and test sample, and many regulatory agencies currently utilize this platform for other assays. In this study, a total of seventy-nine STEC strains belonging to the 7 different serogroups of interest were tested. These strains had been previously serotyped and their serogroup was confirmed by PCR. Except for one strain belonging to the O111 serogroup, nearly all strains (i.e., 98.7%; 78/79) were correctly identified on the Bio-Plex 100 instrument in less than 4h. This newly developed microbead-based immunoassay could be extended to include other STEC serogroups, virulence factors, and/or bacterial species.

Isolation of Shiga toxin-producing Escherichia coli from fresh produce using STEC heart infusion washed blood agar with mitomycin-C.

The ability to detect and isolate Shiga toxin-producing Escherichia coli (STEC) remains a major challenge for food microbiologists. Although methods based on nucleic acids and antibodies have improved detection of STECs in foods, isolation of these bacteria remains arduous. STEC isolation is necessary for matching food, environmental, and clinical isolates during outbreak investigations and for distinguishing between pathogenic and nonpathogenic organisms. STEC heart infusion washed blood agar with mitomycin-C (SHIBAM) is a modification of washed sheep blood agar prepared by adding mitomycin-C and optimizing both the washed blood and base agar to better isolate STECs. Most STEC isolates produce a zone of hemolysis on SHIBAM plates and are easily distinguishable from background microbiota. Here, we present data supporting the use of SHIBAM to isolate STECs from fresh produce. SHIBAM was tested for accuracy in identifying STECs (365 of 410 STEC strains were hemolytic, and 63 of 73 E. coli strains that did not produce Shiga toxin were not hemolytic) and for recovery from artificially inoculated fresh produce (11 of 24 romaine lettuce samples and 6 of 24 tomato samples). STEC recovery with SHIBAM agar was greatly improved when compared with recovery on Levine's eosin-methylene blue agar as a reference method.

Rapid O serogroup identification of the ten most clinically relevant STECs by Luminex microbead-based suspension array.

Identification and serotyping of Shiga toxin-producing Escherichia coli during foodborne outbreaks can aid in matching clinical, food, and environmental isolates when trying to identify the source of illness and ultimately food contamination. Herein we describe a Luminex microbead-based suspension array to identify the O serogroup of the ten most clinically relevant STECs: O26, O45, O91, O103, O111, O113, O121, O128, O145, and O157. The use of PCR followed by Luminex xMAP® technology enables the detection of multiple analytes in a single multiplex reaction with high throughput capabilities. One hundred and fourteen STEC isolates were correctly identified with no false positives among forty-six other organisms using this assay. Assay performance was tested in multiple laboratories using a panel of eleven different STEC serogroups on the Bio-Plex 200 and MAGPIX instruments. The STEC microbead-based suspension array can be performed in a 96-well plate format for high throughput screening in less than 4h. Furthermore, it is expandable, allowing for the addition of O serogroups should the need arise.

O serogroup specific real time PCR assays for the detection and identification of nine clinically relevant non-O157 STECs.

TaqMan™ real time PCR assays were designed for each of the non-O157 STEC O serogroups most commonly associated with human illness: O26, O45, O91, O103, O111, O113, O121, O128, and O145. The nine RT-PCR assays can be run as single assays when a known pathogen is of concern, or multiplexed in three reactions, to quickly screen for the most clinically relevant O serogroups. All assays included an internal amplification control constructed from the green fluorescent protein gene as an indicator of PCR inhibition. Of 103 strains tested, the inclusive tests accurately identified the O serogroup for 101 strains. The exclusive tests for each assay yielded no false positives for over 120 Escherichia coli strains and 23 non-E. coli bacteria tested. Furthermore, the RT-PCR assays were tested by inoculating four food matrices, milk, apple juice, lettuce, and ground beef, at ≤30 CFU/25 g or mL. Following a 24h selective enrichment, the RT-PCR assays detected STECs in all foods except for one ground beef sample inoculated with O111, and all apple juice samples inoculated with O113. The assays could also detect each O serogroup in human stool specimens inoculated with STECs at 1000 CFU/0.5 g of stool following 24 h enrichment.

Technological advances to enhance agricultural pest management.

Biotechnology offers new solutions to existing and future pest problems in agriculture including, for the first time, possible tools to use against insect transmitted pathogens causing plant diseases. Here, we describe the strategy first described as Autocidal Biological Control applied for the development of conditional lethal pink bollworm strains. When these strains are mass-reared, the lethal gene expression is suppressed by a tetracycline repressor element, which is activated by the presence of chlorotetracycline, a normal component of the mass-rearing diet. Once removed from the tetracycline diet, the lethal genes are passed on to offspring when ordinary lab-reared pink bollworms mate with special lethal strains. Lethality is dominant (one copy sufficient for lethality), expressed in the egg stage and affects all eggs (100% lethal expression). The initial investment by the California Cotton Pest Control Board is an outstanding example of research partnerships between agriculture industry, the USDA and land grant universities.

Effect of probiotic adult diets on fitness components of sterile male Mediterranean fruit flies (Diptera: Tephritidae) under laboratory and field cage conditions.

The aim of the current study was to investigate the effect of probiotic adult diets, i.e., adult diets containing viable symbiotic intestinal bacteria, on the pheromone-calling activity, mating success, life expectancy, and survival of mass-reared male Mediterranean fruit flies, Ceratitis capitata (Wiedemann), as an avenue for improving the field performance of sterile males in release programs to eradicate, suppress, or prevent spread of wild populations. The effect of inoculation of two standard adult diets (sugar-yeast granulate [SY] and sugar agar [s]) and two experimental formulations (yeast-reduced granulate [Sy] and yeast-enhanced sugar agar [sy]) with Enterobacter agglomerans and Klebsiella pneumoniae (typically occurring in the gut of wild flies) on the different fitness components was assessed in the laboratory and on field-caged host trees. We found that, in the laboratory, males reared on the probiotic yeast-enhanced agar, sy, had a significant mating advantage over competitors fed the standard s agar (probiotic and control) or noninoculated sy agar; no effect of probiotic enrichment (or lowering the yeast content) was found with the granular diets. Mating test results obtained in the field were inconsistent with laboratory data in that no differences in the numbers of matings were observed between males reared on any of the probiotic and control agar diets (or the SY granulate), whereas males feeding on the probiotic modified granulate, Sy, scored significantly more matings than their control competitors. The pheromone-calling activity of males maintained on the granular diets was not affected by probiotic enrichment on any of the seven observation days. Agar-fed males, however, "called" more frequently on days 6 and 7 (but not on days 1-5) when their diet contained the probiotic load. Laboratory survival of granulate-fed males was found to be significantly prolonged with probiotic inoculation and lowering the yeast content of the standard SY granulate (but not with probiotic inoculation of sy). Similarly, males reared on the probiotic and control modified agars (sy) survived significantly longer than those feeding on the standard s agars (inoculated and control). Again, the results obtained in the field were inconsistent, because no differences between treated and control males were found for any of the diets. The findings are discussed in the light of other published studies on adult nutrition and behavioral ecology in C. capitata.

Volatiles production and attractiveness to the Mexican fruit fly of Enterobacter agglomerans isolated from apple maggot and Mexican fruit flies.

We investigated two strains of uricase (+) Enterobacter agglomerans, one isolated from the apple maggot fly (AMF) and one from the Mexican fruit fly (MFF), for 1) attractiveness to MFF, and 2) production of attractive chemicals. Regarding chemicals demonstrated attractive to the MFF, the MFF bacterial strain produced more 2,5-dimethylpyrazine, 2-phenylethanol, and indole than the AMF strain, whereas the AMF, but not the MFF strain, produced 3-hydroxybutanone. Cell types that predominated in plated subcultures varied from batch to batch resulting in variation in volatiles production, especially by the AMF strain where indole was sometimes a major component of the odor and at other times not detectable. Despite the greater production of attractive chemicals by the MFF strain, the AMF strain was consistently more attractive and the MFF strain was not different from uninoculated control plates. Statistical analyses indicated negative correlations of attractiveness with production of indole, 2,5-dimethylpyrazine, and 2-phenylethanol, and positive correlation with 3-hydroxybutanone. Results support previous findings with the Mexican fruit fly that showed combinations of attractive chemicals sometimes are not attractive.

Purine metabolizing capability of Enterobacter agglomerans affects volatiles production and attractiveness to Mexican fruit fly.

We investigated two strains of Enterobacter agglomerans that differ in their ability to metabolize uric acid for (1) attractiveness to sugar-fed Mexican fruit flies, and (2) production of volatile chemicals that may be responsible for the attractiveness. The two strains were cultured on a medium that contained uric acid as the primary nitrogen source to simulate bird feces, a natural substrate for this bacterium. Active cultures of both strains were more attractive than uninoculated uric acid medium to both sexes of sugar-fed flies in wind-tunnel bioassays. The uricase(+) strain was more attractive than the uricase(-) strain to males and to females <9 days old, but not to older females. Volatiles found by solid-phase microextraction in greater amounts in headspace above active cultures of both strains than above uninoculated medium were ammonia, dimethyldisulfide, 3-methylbutanol, 2-phenylethanol, 2,5-dimethylpyrazine, and trimethylpyrazine. The uricase(+) strain produced more ammonia, dimethyldisulfide, and trimethylpyrazine than the uricase(-) strain. An additional chemical, 3-hydroxybutanone, appears to be produced exclusively by the uricase(+) strain. The uricase(-) strain produced more 2-phenylethanol than the uricase(+) strain. Differences in volatiles are consistent with the generally greater attractiveness of the uricase(+) strain compared with the uricase(-) strain as ammonia, 3-hydroxybutanone, and trimethylpyrazine have been demonstrated attractive to sugar-fed Mexican fruit flies.

Transformed bacterial symbionts re-introduced to and detected in host gut.

Strains of Enterobacter agglomerans and Klebsiella pneumoniae isolated from Rhagoletis completa Cresson were engineered to express transgenic fluorescent proteins (ECFP, DsRed). These bacteria were introduced into flies by feeding the flies a sucrose solution in which the bacteria were suspended. The transgenic and heterologous marker protein was expressed and visible in the bacteria after they were ingested by WHF and while they were in the fly gut. We describe the plasmids used to transform these bacteria and demonstrate expression of heterologous proteins from the transforming plasmids and discuss the implications for future pest control strategies.