Isolation of Multidrug-Resistant Helicobacter pylori from Wild Houseflies Musca domestica with a New Perspective for the Treatment.

Background: High frequency of Helicobacter pylori infection and the unknown mode of transmission prompted us to investigate H. pylori-wild housefly relationship. H. pylori causes chronic gastritis, peptic ulcers, and stomach cancer. H. pylori persists in the gut of the experimentally infected houseflies. The existence of H. pylori strains isolated from wild houseflies, on the other hand, has never been documented. Materials and Methods: In this study, 902 wild houseflies from different sites were identified as Musca domestica, then 60 flies were screened by traditional microbiological techniques and H. pylori-specific 16S rRNA gene. The antibiotic resistance (ART) was investigated phenotypically. Wild housefly gut bacterial isolates were further evaluated genotypically to have 23S rRNA gene mutation related to clarithromycin resistance. To find efficient therapeutic alternatives, the potency of three plant extracts (garlic, ginger, and lemon) and the wasp, Vespa orientalis venom was evaluated against H. pylori. The cytotoxic effect of the crude wasp venom, the most potent extract, against Vero and Colon cancer (Caco2) cell lines was investigated using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Results: All isolates from houseflies were positive. The isolated bacteria have variable resistance to frequently used antibiotics in all isolates. Minimum inhibitory concentration values of 15.625 mg/mL for both ginger and lemon extracts, 7.8125 mg/mL for garlic extract, and 0.0313 mg/mL for wasp venom were recorded. Wasp venom has the most potent antibacterial activity compared with the four antibiotics that are currently used in therapies against H. pylori. Conclusion: We conclude that wild houseflies can play a role in disseminating H. pylori. The housefly gut may be a suitable environment for the horizontal transfer of ART genes among its associated microbiome and H. pylori. Wasp venom proved its potential activity as a new and effective anti-H. pylori drug for both therapeutic and preventative usage.

Preparation, characterization, and insecticidal activity evaluation of three different formulations of Beauveria bassiana against Musca domestica.

Three formulations; bait, encapsulation, and emulsion of Beauveria bassiana were prepared and evaluated for their insecticidal activity in simulated field settings. Tea waste-based bait formulation of B. bassiana showed 100% mortality (within 72 h) in lab assay against adult houseflies. In field assay using traps, 65% relative entrapment and 100 % mortality (within 60 h) of entrapped flies was observed. Although the bait formulation was low cost and easy to prepare and transport, its storage ability was limited. Hence, more advanced formulations in form of encapsulation and emulsion was attempted. Encapsulated B. bassiana conidia (using skimmed milk powder, polyvinyl pyrrolidone K-90 and glucose as additives) showed 100% conidial germination and retained 78% conidial viability, even after storage for 12 months at 30 °C. Encapsulated product showed 54.8% (freshly prepared) and 30.6 % (after 12-months storage) mortality of housefly larvae in a simulated field condition. Emulsion formulation was prepared by using Tween 20 as surfactant with seven vegetable oils: soybean, rapeseed, sunflower, olive, castor, til, and linseed. Emulsion with linseed oil showing maximum conidial germination (94%) was evaluated for shelf life and pathogenecity against housefly larvae. Shelf life analysis of emulsion revealed 28% conidial germination and 19.9% housefly larval mortality after 12 months of storage as opposed to 94% conidial germination and 51.7% of larval mortality with fresh product. Significant increase in shelf × targeted application of formulation is expected to increase its mass applicability for housefly control. Also, the variability among products presents diverse opportunities for commercialization.

Antimicrobial activity of alcohols from Musca domestica.

Information on the stimulatory and inhibitory effects of cuticular alcohols on growth and virulence of insecticidal fungi is unavailable. Therefore, we set out to describe the content of cuticular and internal alcohols in the body of housefly larvae, pupae, males and females. The total cuticular alcohols in larvae, males and females of Musca domestica were detected in comparable amounts (4.59, 3.95 and 4.03 µg g(-1) insect body, respectively), but occurred in smaller quantities in pupae (2.16 µg g(-1)). The major free alcohol in M. domestica larvae was C(12:0) (70.4%). Internal alcohols of M. domestica larvae were not found. Among cuticular pupae alcohols, C(12:0) (31.0%) was the most abundant. In the internal lipids of pupae, only five alcohols were identified in trace amounts. The most abundant alcohol in males was C(24:0) (57.5%). The percentage content of cuticular C(24:0) in males and females (57.5 and 36.5%, respectively) was significantly higher than that of cuticular lipids in larvae and pupae (0.9 and 5.6%, respectively). Only two alcohols were present in the internal lipids of males in trace amounts (C(18:0) and C(20:0)). The most abundant cuticular alcohols in females were C(24:0) (36.5%) and C(12:0) (26.8%); only two alcohols (C(18:0) and C(20:0)) were detected in comparable amounts in internal lipids (3.61±0.32 and 5.01±0.42 µg g(-1), respectively). For isolated alcohols, antimicrobial activity against 10 reference strains of bacteria and fungi was determined. Individual alcohols showed approximately equal activity against fungal strains. C(14:0) was effective against gram-positive bacteria, whereas gram-negative bacteria were resistant to all tested alcohols. Mixtures of alcohols found in cuticular lipids of larvae, pupae, males and females of M. domestica generally presented higher antimicrobial activity than individual alcohols. In contrast, crude extracts containing both cuticular and internal lipids showed no antifungal activity against the entomopathogenic fungus Conidiobolus coronatus, which efficiently kills adult house flies.

Quantitative contamination and transfer of Escherichia coli from foods by houseflies, Musca domestica L. (Diptera: Muscidae).

The housefly, Musca domestica L. (Diptera: Muscidae), is recognized as an important factor in the dissemination of various infectious diseases such as cholera, shigellosis, and salmonellosis. They can also serve as a cross-contamination vector for other foodborne pathogens. However, the potential for bacterial transfer by houseflies has been demonstrated in a qualitative rather than quantitative manner. In this study, the numbers of bacteria a housefly can carry on its body and transfer to a clean surface after exposure to a sugar-milk aqueous solution, steak, and potato salad contaminated with a fluorescent gene Escherichia coli (8 log10 CFU/ml) were determined. In the first series of experiments to quantify bacterial numbers on the flies, about 40-60 flies were transferred into a sterile cage, exposed to the food for 30 min, the flies immobilized and the attached E. coli on each fly enumerated. Detectable E. coli (>1.7 log10 CFU/fly) were found on 43% (29/67), 53% (23/43), and 62% (32/52) of the flies in the cages with sugar/milk, steak, and potato salad, respectively. For the positive flies, the geometric mean carriage (log10 CFU/fly) was 2.93+/-1.24 for sugar-milk, 3.77+/-1.28 for steak, and 2.25+/-0.64 for the potato salad. In the second series of experiments, the transfer of bacteria by individual flies from contaminated food to the inner surface of a sterile jar per each landing was determined. E. coli transferred from the sugar-milk was 3.5+/-0.7 log10 CFU/fly-landing, 3.9+/-0.7 for steak and 2.61+/-1.16 for the potato salad. From the initial contamination levels of bacteria and the number of transferred bacteria, it can be calculated that flies contaminate clean surfaces with approximately 0.1 mg of food per landing.