Detection of Foodborne Bacterial Pathogens from Individual Filth Flies

There is unanimous consensus that insects are important vectors of foodborne pathogens. However, linking insects as vectors of the pathogen causing a ...

U.S. Food and Drug Administration

Eighty-seven culturable endophytic bacterial isolates in 19 genera were obtained from coffee plants collected in Colombia (n = 67), Hawaii (n = 17), and Mexico (n = 3). Both Gram positive and Gram negative bacteria were isolated, with a greater percentage (68%) being Gram negative. Tissues yielding bacterial endophytes included adult plant leaves, various parts of the berry (e.g., crown, pulp, peduncle and seed), and leaves, stems, and roots of seedlings. Some of the bacteria also occurred as epiphytes. The highest number of bacteria among the berry tissues sampled was isolated from the seed, and includes Bacillus , Burkholderia , Clavibacter , Curtobacterium , Escherichia , Micrococcus , Pantoea , Pseudomonas , Serratia , and Stenotrophomonas . This is the first survey of the endophytic bacteria diversity in various coffee tissues, and the first study reporting endophytic bacteria in coffee seeds. The possible role for these bacteria in the biology of the coffee plant remains unknown.

Endophytic bacteria in Coffea arabica L.

Quatre-vingt-sept cultivables souches de bact&eacute;ries endophytes dans 19 genres ont &eacute;t&eacute; extraites des plantes de caf&eacute; r&eacute;colt&eacute;es en Colombie (n = 67), Hawaii (n = 17) et le Mexique (n = 3). Fois bact&eacute;ries Gram positives et Gram n&eacute;gatifs ont &eacute;t&eacute; isol&eacute;s, avec un pourcentage plus &eacute;lev&eacute; (68 %) &eacute;tant les Gram n&eacute;gatif. Tissus produisant des endophytes bact&eacute;riens plantes adultes inclus feuilles, diff&eacute;rentes parties du grain (p. ex., Couronne, pulpe, p&eacute;doncule et graines) et feuilles, tiges et racines des semis. Certaines des bact&eacute;ries s&#39;est produite &eacute;galement comme des &eacute;piphytes. Le plus grand nombre de bact&eacute;ries parmi les tissus berry &eacute;chantillonn&eacute;s a &eacute;t&eacute; isol&eacute; de la graine et inclut les Bacillus, Burkholderia, Clavibacter, Curtobacterium, Escherichia, Micrococcus, Pantoea, Pseudomonas, Serratia et Stenotrophomonas. Il s&#39;agit de la premi&egrave;re enqu&ecirc;te sur la diversit&eacute; des bact&eacute;ries endophytes dans divers tissus de caf&eacute; et les premi&egrave;res &eacute;tude consid&eacute;r&eacute;e bact&eacute;ries endophytes dans les graines de caf&eacute;. Le r&ocirc;le &eacute;ventuel de ces bact&eacute;ries dans la biologie de la plante de caf&eacute; reste inconnu.

Bact&eacute;ries endophytes de Coffea arabica L.

Ottanta-sette isolati batterici endofiti coltivabili in 19 generi sono stati ottenuti da piante di caff&egrave; raccolte in Colombia (n = 67), Hawaii (n = 17) e il Messico (n = 3). Sia Gram-positivi e batteri Gram negativi sono stati isolati, con una percentuale maggiore (68%) essendo Gram-negativi. Tessuti producendo batteri endofiti incluso pianta adulta foglie, varie parti della bacca (ad es., corona, polpa, peduncolo e seme) e foglie, steli e le radici delle piantine. Alcuni dei batteri si &egrave; verificato anche come epifite. Il pi&ugrave; alto numero di batteri tra i tessuti di berry nel campione &egrave; stato isolato dal seme e include Bacillus, Burkholderia, Clavibacter, Curtobacterium, Escherichia, Micrococcus, Pantoea, Pseudomonas, Serratia e Stenotrophomonas. Questa &egrave; la prima indagine di diversit&agrave; endofita batteri in vari tessuti di caff&egrave; e i primi studio endofita segnalazione batteri nei semi di caff&egrave;. Il possibile ruolo di questi batteri nella biologia della pianta di caff&egrave; rimane sconosciuto.

Batteri endofite in Coffea arabica L.

Ochenta y siete aislamientos bacterianos cultivables end&oacute;fitos en 19 g&eacute;neros fueron obtenidos de plantas de caf&eacute; en Colombia (n = 67), Hawaii (n = 17) y M&eacute;xico (n = 3). Gram positivas y bacterias Gram negativas fueron aisladas, con un porcentaje mayor (68%) siendo Gram negativas. Tejidos rendimiento bacterianos end&oacute;fitos planta adulta incluyen hojas, varias partes de la baya (por ejemplo, corona, pulpa, ped&uacute;nculo y semillas) y hojas, tallos y ra&iacute;ces de las pl&aacute;ntulas. Algunas de las bacterias tambi&eacute;n ocurrieron como ep&iacute;fitas. El mayor n&uacute;mero de bacterias entre los tejidos de la baya muestreadas fue aislado de la semilla e incluye Bacillus, Burkholderia, Clavibacter, Curtobacterium, Escherichia, Micrococcus, Pantoea, Pseudomonas, Serratia y Stenotrophomonas. Este es el primer estudio de la diversidad de bacterias end&oacute;fitos en varios tejidos de caf&eacute; y las primeras bacterias end&oacute;fitos informes de estudio en semillas de caf&eacute;. Se desconoce el posible papel de estas bacterias en la biolog&iacute;a de la planta de caf&eacute;.

Bacterias end&oacute;fitos en Coffea arabica L.

Coffee berry borer (CBB) is the Worlds most devastating coffee pest causing an estimated US$500 million worth of losses annually through damage and control costs. Beauveria bassiana and Metarhizium anisopliae have been employed to control this pest but their low virulence (slow kill and large inoculums) is an important factor constraining their use. M. anisopliae (AaIT-Ma549) has been modified to express the scorpion toxin (AaIT) in insect hemolymph and this greatly increased pathogenicity against Manduca sexta and Aedes aegypti. Here, we demonstrate that AaIT-Ma549 was also dramatically more virulent against CBB, and we provide a much more comprehensive analysis of infection processes and post-mortality development than in the previous research. We evaluated several spore concentrations (10(1) through 10(7)spores/ml) of both the wild type and recombinant strain. At concentrations of 10(1), 10(2) and 10(3)spores/ml, the recombinant strain significantly increased mortality of CBB by 32.2%, 56.6% and 24.6%, respectively. The medial lethal concentration (LC(50)) was reduced 15.7-fold and the average survival time (AST) was reduced by 20.1% to 2.98+/-0.1 days with 10(7)spores/ml. This is the first occasion that an entomopathogenic fungus has been found to kill CBB in less than 3 days. However, AaIT-Ma549 produces significantly fewer spores on cadavers than the parental strain.

Increased pathogenicity against coffee berry borer, Hypothenemus hampei (Coleoptera: Curculionidae) by Metarhizium anisopliae expressing the scorpion toxin (AaIT) gene.

To improve the insecticidal efficacy of the entomopathogen Beauveria bassiana, the fungus was genetically modified with an insect-specific scorpion neurotoxin AAIT and an insect cuticle degrading protease PR1A from another insect pathogen (Metarhizium anisopliae). The wild-type and the transformants were bioassayed against the larvae of Masson's pine caterpillar Dendrolimus punctatus and the wax moth Galleria mellonella. In comparison to the wild-type strain, engineered isolates took fewer spores to kill 50% of pine caterpillars, 15-fold less for the aaIT single transformant Bb13T and eightfold less for the double transformant Bb13TPR1A, respectively. The median lethal times for Bb13T and Bb13TPR1A were reduced by 40% and 36.7%, respectively against D. punctatus and 24.4% and 20.9%, respectively against G. mellonella. Our data showed that the cotransformation of these two genes produced no synergistic effects on virulence improvement. It is evident from this study that AAIT could be degraded by the protease PR1A when they are expressed together, emphasizing that protein interactions need to be evaluated when working with multiple genes, particularly if they include proteases.

Insecticidal evaluation of Beauveria bassiana engineered to express a scorpion neurotoxin and a cuticle degrading protease.

Metarhizium anisopliae is a model system for studying insect fungal pathogenesis. The role of cAMP signal transduction in virulence was studied by disrupting the class I PKA catalytic subunit gene (MaPKA1). The PKA mutant (DeltaMaPKA1) showed reduced growth and greatly reduced virulence. PKA was dispensable for differentiation of infection structures (appressoria), but differentiation was delayed and the appressoria were defective because of reduced turgor pressure. DeltaMaPKA1 germinated at similar rates as the wild type in glucose and glycerol, but germination was delayed on alanine. Conidial adhesion and appressorium formation by DeltaMaPKA1 against a plastic surface was fully inhibited with glucose as sole nutrient source. Adhesion to plastic was not inhibited with glycerol or alanine, but appressorium formation was delayed. DeltaMaPKA1 showed reduced tolerance to the oxidative agent diamide, but not to H(2)O(2) and methyl-viologen. Comparative transcriptome analysis of DeltaMaPKA1 and the wild type strain showed that PKA is responsible for up-regulating approximately one-third of the genes induced by insect cuticle, including subsets of those responsible for differentiation of appressoria and penetration pegs, cuticle degradation, nutrient acquisition, pH regulation, lipid synthesis, cell cycle control and the cytoskeleton. PKA was not however required for expression of toxin-producing genes. We conclude therefore that MaPKA1 is required for sensing host-related stimuli and transduction of these signals to regulate many infection processes.

Protein kinase A regulates production of virulence determinants by the entomopathogenic fungus, Metarhizium anisopliae.

Many strains of Metarhizium anisopliae have broad host ranges, but others are specialists and adapted to particular hosts. Patterns of gene duplication, divergence, and deletion in three generalist and three specialist strains were investigated by heterologous hybridization of genomic DNA to genes from the generalist strain Ma2575. As expected, major life processes are highly conserved, presumably due to purifying selection. However, up to 7% of Ma2575 genes were highly divergent or absent in specialist strains. Many of these sequences are conserved in other fungal species, suggesting that there has been rapid evolution and loss in specialist Metarhizium genomes. Some poorly hybridizing genes in specialists were functionally coordinated, indicative of reductive evolution. These included several involved in toxin biosynthesis and sugar metabolism in root exudates, suggesting that specialists are losing genes required to live in alternative hosts or as saprophytes. Several components of mobile genetic elements were also highly divergent or lost in specialists. Exceptionally, the genome of the specialist cricket pathogen Ma443 contained extra insertion elements that might play a role in generating evolutionary novelty. This study throws light on the abundance of orphans in genomes, as 15% of orphan sequences were found to be rapidly evolving in the Ma2575 lineage.

Comparative genomics using microarrays reveals divergence and loss of virulence-associated genes in host-specific strains of the insect pathogen Metarhizium anisopliae.

Metarhizium anisopliae and Beauveria bassiana are ubiquitous insect pathogens and possible plant symbionts, as some strains are endophytic or colonize the rhizosphere. We evaluated 11 strains of M. anisopliae and B. bassiana, and two soil saprophytes (the non-rhizospheric Aspergillus niger and the rhizosphere-competent Trichoderma harzianum) for their ability to germinate in bean root exudates (REs). Our results showed that some generalist strains of M. anisopliae were as good at germinating in RE as T. harzianum, although germination rates of the specialized acridid pathogen Metarhizium acridum and the B. bassiana strains were significantly lower. At RE concentrations of <1 mg ml(-1), M. anisopliae strain ARSEF 2575 showed higher germination rates than T. harzianum. Microarrays showed that strain 2575 upregulated 29 genes over a 12 h period in RE. A similar number of genes (21) were downregulated. Upregulated genes were involved in carbohydrate metabolism, lipid metabolism, cofactors and vitamins, energy metabolism, proteolysis, extracellular matrix/cell wall proteins, transport proteins, DNA synthesis, the sexual cycle and stress response. However, 41.3% of the upregulated genes were hypothetical or orphan sequences, indicating that many previously uncharacterized genes have functions related to saprophytic survival. Genes upregulated in response to RE included the subtilisin Pr1A, which is also involved in pathogenicity to insects. However, the upregulated Mad2 adhesin specifically mediates adhesion to plant surfaces, demonstrating that M. anisopliae has genes for rhizosphere competence that are induced by RE.

The rhizosphere-competent entomopathogen Metarhizium anisopliae expresses a specific subset of genes in plant root exudate.

This paper describes the main distinguishing characteristics of female and male pupae and adults of cocoa pod borer, Conopomorpha cramerella (Snellen) (Lepidoptera: Gracillariidae). Two pairs of tubercles present on the sterna of segments IX and X of the female pupae are useful in differentiating female from male pupae. The female genital opening is located anterior to the first pair of tubercles and forms a plateau in which the center has a light brown longitudinal depression that indicates the female genital opening. The male genital opening is a conspicuous, brown, longitudinal slit located between the two pairs of tubercles. The sex of the adult moth can be determined by examining the ventrocaudal segments of the abdomen. The last segment of the female abdomen is white, compressed laterally and at the tip, and the hairy anal papillae can be seen. In the male, the ventrocaudal end of the abdomen is black and robust. This information will be useful for laboratory and field diagnosis and while working on sex ratios of this important pest of cocoa.

Sexual dimorphism of pupae and adults of the cocoa pod borer, Conopomorpha cramerella.

Genetically modified Metarhizium spp represent a major new arsenal for combating insect pests and insect-borne diseases. However, for these tools to be used safely and effectively, we need a much better understanding of their evolutionary potential and invasion ecology. In order to model natural as well as anthropogenic dispersal scenarios, we investigated evolutionary processes in a green fluorescent protein tagged strain of Metarhizium robertsii following transfer from a semitropical to a temperate soil community. Adaptive changes occurred over four years despite recurrent genetic bottlenecks and lack of recombination with locally well adapted strains. By coupling microarray-based functional analysis with DNA hybridizations we determined that expression of cell wall and stress response genes evolved at an accelerated rate in multiple replicates, whereas virulence determinants, transposons, and chromosome structure were unaltered. The mutable genes were enriched for TATA boxes possibly because they are larger mutational targets. In further field trials, we showed that the new mutations increased the fitness of M. robertsii in the new range by enhancing saprophytic associations, and these benefits were maintained in subsequent years. Consistent with selection being habitat rather than host specific, populations of an avirulent mutant cycled with seasons similarly to the wild type, whereas a mutant unable to adhere to plant roots showed a linear decrease in population. Our results provide a mechanistic basis for understanding postrelease adaptations, show that agents can be selected that lack gene flow and virulence evolution, and describe a means of genetically containing transgenic strains by disrupting the Mad2 gene.

Local adaptation of an introduced transgenic insect fungal pathogen due to new beneficial mutations.

Although flies are important vectors of food-borne pathogens, there is little information to accurately assess the food-related health risk of the presence of individual flies, especially in urban areas. This study quantifies the prevalence and the relative risk of food-borne pathogens associated with the body surfaces and guts of individual wild flies. One hundred flies were collected from the dumpsters of 10 randomly selected urban restaurants. Flies were identified using taxonomic keys before being individually dissected. Cronobacter spp., Salmonella spp., and Listeria monocytogenes were detected using the PCR-based BAX system Q7. Positive samples were confirmed by culture on specific media and through PCR amplification and sequencing or ribotyping. Among collected flies were the housefly, Musca domestica (47%), the blowflies, Lucilia cuprina (33%) and Lucilia sericata (14%), and others (6%). Cronobacter species were detected in 14% of flies, including C. sakazakii, C. turicensis, and C. universalis, leading to the proposal of flies as a natural reservoir of this food-borne pathogen. Six percent of flies carried Salmonella enterica, including the serovars Poona, Hadar, Schwarzengrund, Senftenberg, and Brackenridge. L. monocytogenes was detected in 3% of flies. Overall, the prevalence of food-borne pathogens was three times greater in the guts than on the body surfaces of the flies. The relative risk of flies carrying any of the three pathogens was associated with the type of pathogen, the body part of the fly, and the ambient temperature. These data enhance the ability to predict the microbiological risk associated with the presence of individual flies in food and food facilities.

Prevalence and relative risk of Cronobacter spp., Salmonella spp., and Listeria monocytogenes associated with the body surfaces and guts of individual filth flies.

Monica Pava-Ripoll

Center for Food Safety and Applied Nutrition

U.S. Food and Drug Administration

Detection of Foodborne Bacterial Pathogens from Individual Filth Flies

Monica Pava-Ripoll

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Center for Food Safety and Applied Nutrition

U.S. Food and Drug Administration

Detection of Foodborne Bacterial Pathogens from Individual Filth Flies

Center for Food Safety and Applied Nutrition