Ludek Zurek

Association of Escherichia coli O157:H7 with houseflies on a cattle farm.

ABSTRACT The ecology of Escherichia coli O157:H7 is not well understood. The aims of this study were to determine the prevalence of and characterize E. coli O157:H7 associated with houseflies (HF). Musca domestica L. HF (n = 3,440) were collected from two sites on a cattle farm over a 4-month period and processed individually for E. coli O157:H7 isolation and quantification. The prevalence of E. coli O157:H7 was 2.9 and 1.4% in HF collected from feed bunks and a cattle feed storage shed, respectively. E. coli O157:H7 counts ranged from 3.0 × 101 to 1.5 × 105 CFU among the positive HF. PCR analysis of the E. coli O157:H7 isolates revealed that 90.4, 99.2, 99.2, and 100% of them (n = 125) possessed the stx1, stx2, eaeA, and fliC genes, respectively. Large populations of HF on cattle farms may play a role in the dissemination of E. coli O157:H7 among animals and to the surrounding environment.

Diversity and contribution of the intestinal bacterial community to the development of Musca domestica (Diptera: Muscidae) larvae

Abstract The bacterial diversity in the intestinal tract of Musca domestica L. was examined in larvae collected from turkey bedding and corn silage. Aerobic culturing yielded 25 bacterial species, including 11 from larvae collected from turkey bedding and 14 from larvae collected from corn silage. Providencia rettgeri (Hadley, Elkins & Caldwell) was the only species common to both environments. Two mammalian pathogens, Yersinia pseudotuberculosis (Pfeiffer) and Ochrobactrum anthropi (Holmes), were isolated from the larval intestinal tracts. The majority of isolates represented facultatively anaerobic heterotrophs capable of fermentation. The significance of these bacteria for development of house fly larvae was evaluated by bioassays on trypticase soy egg yolk agar. Pure cultures of individual bacterial species isolated from the intestinal tract of larvae from turkey bedding supported development of flies to a much greater extent than those isolated from larvae from corn silage. House fly development was best supported by a Streptococcus sanguis (White) isolate. The significance of bacteria for development of house flies is discussed.

Ecology of Antibiotic Resistance Genes: Characterization of Enterococci from Houseflies Collected in Food Settings†

ABSTRACT In this project, enterococci from the digestive tracts of 260 houseflies (Musca domestica L.) collected from five restaurants were characterized. Houseflies frequently (97% of the flies were positive) carried enterococci (mean, 3.1 × 103 CFU/fly). Using multiplex PCR, 205 of 355 randomly selected enterococcal isolates were identified and characterized. The majority of these isolates were Enterococcus faecalis (88.2%); in addition, 6.8% were E. faecium, and 4.9% were E. casseliflavus. E. faecalis isolates were phenotypically resistant to tetracycline (66.3%), erythromycin (23.8%), streptomycin (11.6%), ciprofloxacin (9.9%), and kanamycin (8.3%). Tetracycline resistance in E. faecalis was encoded by tet(M) (65.8%), tet(O) (1.7%), and tet(W) (0.8%). The majority (78.3%) of the erythromycin-resistant E. faecalis isolates carried erm(B). The conjugative transposon Tn916 and members of the Tn916/Tn1545 family were detected in 30.2% and 34.6% of the identified isolates, respectively. E. faecalis carried virulence genes, including a gelatinase gene (gelE; 70.7%), an aggregation substance gene (asa1; 33.2%), an enterococcus surface protein gene (esp; 8.8%), and a cytolysin gene (cylA; 8.8%). Phenotypic assays showed that 91.4% of the isolates with the gelE gene were gelatinolytic and that 46.7% of the isolates with the asa1 gene aggregated. All isolates with the cylA gene were hemolytic on human blood. This study showed that houseflies in food-handling and -serving facilities carry antibiotic-resistant and potentially virulent enterococci that have the capacity for horizontal transfer of antibiotic resistance genes to other bacteria.

Role of bacteria in the oviposition behaviour and larval development of stable flies.

Abstract.  Stable flies, Stomoxys calcitrans (L.), are the most important pests of cattle in the United States. However, adequate management strategies for stable flies, especially for pastured cattle, are lacking. Microbial/symbiont‐based approaches offer novel venues for management of insect pests and/or vector‐borne human and animal pathogens. Unfortunately, the fundamental knowledge of stable fly–microbial associations and their effect on stable fly biology is lacking. In this study, stable flies laid greater numbers of eggs on a substrate with an active microbial community (> 95% of total eggs oviposited) than on a sterilized substrate. In addition, stable fly larvae could not develop in a sterilized natural or artificial substrate/medium. Bacteria were isolated and identified from a natural stable fly oviposition/developmental habitat and their individual effect on stable fly oviposition response and larval development was evaluated in laboratory bioassays. Of nine bacterial strains evaluated in the oviposition bioassays, Citrobacter freundii stimulated oviposition to the greatest extent. C. freundii also sustained stable fly development, but to a lesser degree than Serratia fanticola. Serratia marcescens and Aeromonas spp. neither stimulated oviposition nor supported stable fly development. These results demonstrate a stable fly bacterial symbiosis; stable fly larval development depends on a live microbial community in the natural habitat, and stable fly females are capable of selecting an oviposition site based on the microbially derived stimuli that indicate the suitability of the substrate for larval development. This study shows a promising starting point for exploiting stable fly–bacterial associations for development of novel approaches for stable fly management.

Role of bacteria in mediating the oviposition responses of Aedes albopictus (Diptera: Culicidae).

Abstract The responses of Aedes albopictus to sources of oviposition attractants and stimulants were evaluated with a behavioral bioassay in which females attracted to odorants emanating from water were trapped on screens coated with an adhesive. Gravid mosquitoes were attracted to volatiles from larval-rearing water and soil-contaminated cotton towels. Bacteria were isolated from these substrates and from an organic infusion made with oak leaves. Through fatty acid-methyl ester analyses, six bacterial isolates from larval-rearing water, two isolates from soil-contaminated cotton towels, and three isolates from oak leaf infusion were identified to species. The response of gravid mosquitoes to these isolates was also evaluated in behavioral bioassays. Water containing Psychrobacter immobilis (from larval-rearing water), Sphingobacterium multivorum (from soil-contaminated cotton towels), and an undetermined Bacillus species (from oak leaf infusion) elicited significantly higher oviposition than control water without bacteria. Only volatiles collected from larval rearing water elicited significant electroantennogram responses in females.

Insects in confined swine operations carry a large antibiotic resistant and potentially virulent enterococcal community

BackgroundExtensive use of antibiotics as growth promoters in the livestock industry constitutes strong selection pressure for evolution and selection of antibiotic resistant bacterial strains. Unfortunately, the microbial ecology and spread of these bacteria in the agricultural, urban, and suburban environments are poorly understood. Insects such as house flies (Musca domestica) and German cockroaches (Blattella germanica) can move freely between animal waste and food and may play a significant role in the dissemination of antibiotic resistant bacteria within and between animal production farms and from farms to residential settings.ResultsEnterococci from the digestive tract of house flies (n = 162), and feces of German cockroaches (n = 83) and pigs (n = 119), collected from two commercial swine farms were isolated, quantified, identified, and screened for antibiotic resistance and virulence. The majority of samples (93.7%) were positive for enterococci with concentrations 4.2 ± 0.7 × 104 CFU/house fly, 5.5 ± 1.1 × 106 CFU/g of cockroach feces, and 3.2 ± 0.8 × 105 CFU/g of pig feces. Among all the identified isolates (n = 639) Enterococcus faecalis was the most common (55.5%), followed by E. hirae (24.9%), E. faecium (12.8%), and E. casseliflavus (6.7%). E. faecalis was most prevalent in house flies and cockroaches, and E. hirae was most common in pig feces. Our data showed that multi-drug (mainly tetracycline and erythromycin) resistant enterococci were common from all three sources and frequently carried antibiotic resistance genes including tet(M) and erm(B) and Tn916/1545 transposon family. E. faecalis frequently harbored virulence factors gelE, esp, and asa1. PFGE analysis of selected E. faecalis and E. faecium isolates demonstrated that cockroaches and house flies shared some of the same enterococcal clones that were detected in the swine manure indicating that insects acquired enterococci from swine manure.ConclusionsThis study shows that house flies and German cockroaches in the confined swine production environment likely serve as vectors and/or reservoirs of antibiotic resistant and potentially virulent enterococci and consequently may play an important role in animal and public health.

Hydrocarbon synthesis by enzymatically dissociated oenocytes of the abdominal integument of the German Cockroach, Blattella germanica

In insects, hydrocarbons waterproof the cuticle, protect the insect from the external environment, and serve as semiochemicals or their metabolic precursors. In the German cockroach, Blattella germanica, hydrocarbons are synthesized by the abdominal integument, but the precise site of biosynthesis is not known. We developed a method for separation of oenocytes from other cells in the abdominal integument using enzymatic dissociation followed by Percoll gradient centrifugation. Radiolabeled propionate was then used to monitor de novo synthesis of hydrocarbons by dissociated cells. Oenocyte-enriched cell suspensions of abdominal sternites synthesized hydrocarbons, whereas suspensions enriched with epidermal cells did not. Our results show conclusively that hydrocarbons are produced by oenocytes not only in insects whose oenocytes are localized within the hemocoel, but also in those insects whose oenocytes are within the abdominal integument. Furthermore, these data support a hemolymph pathway for transport and delivery of hydrocarbons to both external and internal tissues, including the epicuticle, fat body, and ovaries.

Insects Represent a Link between Food Animal Farms and the Urban Environment for Antibiotic Resistance Traits

ABSTRACT Antibiotic-resistant bacterial infections result in higher patient mortality rates, prolonged hospitalizations, and increased health care costs. Extensive use of antibiotics as growth promoters in the animal industry represents great pressure for evolution and selection of antibiotic-resistant bacteria on farms. Despite growing evidence showing that antibiotic use and bacterial resistance in food animals correlate with resistance in human pathogens, the proof for direct transmission of antibiotic resistance is difficult to provide. In this review, we make a case that insects commonly associated with food animals likely represent a direct and important link between animal farms and urban communities for antibiotic resistance traits. Houseflies and cockroaches have been shown to carry multidrug-resistant clonal lineages of bacteria identical to those found in animal manure. Furthermore, several studies have demonstrated proliferation of bacteria and horizontal transfer of resistance genes in the insect digestive tract as well as transmission of resistant bacteria by insects to new substrates. We propose that insect management should be an integral part of pre- and postharvest food safety strategies to minimize spread of zoonotic pathogens and antibiotic resistance traits from animal farms. Furthermore, the insect link between the agricultural and urban environment presents an additional argument for adopting prudent use of antibiotics in the food animal industry.

Vector competence of Musca domestica (Diptera: Muscidae) for Yersinia pseudotuberculosis.

Abstract The vector potential of adult house flies, Musca domestica L., for Yersinia pseudotuberculosis (Pfeiffer), a pathogen of domestic animals and humans, was investigated. Adult flies were allowed to feed on trypticase soy broth (TSB) containing Y. pseudotuberculosis for 6 h and then transferred to sterile containers with sterile TSB as a source of water and nutrients. At 6-h intervals, all flies were transferred to sterile containers with sterile TSB and 10 randomly selected flies were examined for the pathogen. Yersinia pseudotuberculosis did not establish a permanent population in the house fly colony; however, viable cells were detected from the digestive tract of flies for up to 36 h after the initial exposure, and flies contaminated their environment (sterile TSB) for up to 30 h after the exposure. These results demonstrated that house flies can carry Y. pseudotuberculosis for a considerable period and therefore must be considered as a potential mechanical vector of pseudotuberculosis infection.

Occurrence of ciprofloxacin-, trimethoprim-sulfamethoxazole-, and vancomycin-resistant bacteria in a municipal wastewater treatment plant.

The occurrence of antibiotic-resistant bacteria was evaluated in aqueous samples obtained from a municipal wastewater treatment plant. Samples collected from the influent, clarifier effluent, and disinfected effluent were assayed for fecal coliforms, E. coli, and enterococci exhibiting resistance to ciprofloxacin, trimethoprim-sulfamethoxazole, and vancomycin. Membrane filtration of samples was followed by plating on growth media containing various concentrations of antibiotic. Bacterial colonies on plates with antibiotic exposures greater than the clinical minimum inhibitory concentrations were counted and considered resistant. The numbers of drug-resistant organisms in influent ranged from nondetectable to 7 x 10(5) colony-forming units (CFU)/100 mL for fecal coliforms, nondetectable to 5 x 10(4) CFU/100 mL for E. coli, and nondetectable to 6 x 10(5) CFU/100 mL for enterococci. Fecal coliforms, E. coli, and enterococci with reduced susceptibility to antibiotics were also detected in influent and clarifier effluent; however, the disinfected effluent did not contain resistant bacteria. Species-level identification of enterococci revealed that resistant enterococci were predominantly E. faecalis.