Enterococcus faecalis OG1RF:pMV158 survives and proliferates in the house fly digestive tract.

Citation: Doud, C. W., and L. Zurek. (2012). Enterococcus Faecalis OG1RF:PMV158 Survives and Proliferates in the House Fly Digestive Tract. Journal of Medical Entomology 49 (1): 150–55. https://doi.org/10.1603/ME11167.Enterococcus faecalis is an important nosocomial pathogen and house flies have been implicated in the dissemination of this bacterium. In this study, GFP-expressing E. faecalis OG1RF:pMV158 was used to track the fate of the bacterium in the digestive tract of the house fly, Musca domestica (L.) to assess the vector potential of this insect for E. faecalis. Colony forming unit (CFU) counts were obtained from viable fluorescing E. faecalis recovered from mouthparts and digestive tract regions (labelum, foregut, midgut, and hindgut) at 1, 4, 8, 24, 48, 72, and 96 h after the bacterial exposure. Bacterial counts were significantly highest in the midgut at 1 h and 4 h and declined during the first 24 h. In the labelum, E. faecalis concentrations were low within the first 24 h and then greatly increased. Bacterial counts and direct observations of the digestive tract under a dissecting microscope with ultra violet light revealed that E. faecalis peaked in the crop after 48 h and remained high until the end of the experiment. Concentrations of E. faecalis in the hindgut were low when compared with other parts of the digestive tract. Microscopy and CFU counts suggest that E. faecalis was digested in the midgut but proliferated in the crop. Both drinking water and feed (flaked corn) sampled at the end of the assay (96 h) were contaminated by fluorescing E. faecalis, demonstrating that the flies disseminated E. faecalis. Our data support the notion that house flies can act as a bioenhanced vector for bacteria

Temporal changes in the bacterial community of animal feces and their correlation with stable fly oviposition, larval development, and adult fitness

Stable flies are blood-feeding insects with a great negative impact on animals world wide. Larvae develop primarily in animal manure and bacteria are essential for larval development; however, the principle of this dependence is not understood. We hypothesized that as the microbial community of animal manure changes over time, it plays an important role in stable fly fitness. Two-choice bioassays were conducted using 2 week old horse manure (control) and aging horse manure (fresh to 5 week old) to evaluate the effect of manure age on stable fly oviposition. Our data showed that fresh feces did not stimulate oviposition and that the attractiveness increased as manure aged but started to decline after 3 weeks. Bioassays assessing the effect of manure age at the time of oviposition on larval development demonstrated that 1–3 week old manure supported larval development significantly better than fresh, 4, and 5 week old manure. In addition, adult fitness (body size) was significantly higher in flies from 1 and 2 week old manure comparing to that of all other treatments. Analysis of the bacterial community of aging horse manure by 454-pyrosequencing of 16S rDNA revealed a great reduction in bacterial diversity and richness from fresh to 1–5 week old manure and a major shift from strict anaerobes in fresh manure to facultative anaerobes and strict aerobes in aged manure. Overall, the microbial community of 2 and 3 week old horse manure with its dominant bacterial taxa Rhizobium, Devosia, and Brevundimonas stimulated stable fly oviposition the most and provided a suitable habitat for larval development. These bacteria represent the candidates for studies focused on better understanding of stable fly – microbial interactions

Can the Gram-negative bacterium Escherichia coli colonize the gut of Lone Star Tick (Amblyomma americanum)?

Ticks are obligate blood feeding ectoparasites and vectors of several mammalian pathogens (Williams-Newkirk et al, 2014). In addition to pathogens they also carry a bacterial community with commensal and symbiotic relationships (Bonnet et al, 2017). Using a culture-dependent approach we previously reported a high prevalence of Gram-positive bacteria in the gut of field collected lone star ticks (Amblyomma americanum). These results suggested that epithelial immunity functions to control Gram-negative bacteria in A. americanum. In this study, we used a culturing and non-culturing approach to measure the outcome of E.coli (Gram-negative) when fed to female adult lone star ticks (n=16). Results showed a significant reduction of E.coli at Days 1, 3 and 7 post bacterial feeding. qPCR of 16S rDNA confirmed reduction of bacterial rDNA when compared to water fed ticks (n=16). Our results suggest that there is a midgut epithelial immune response in place, which mainly targets Gram-negative bacteria

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

<p>Abstract</p> <p>Background</p> <p>Extensive 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 (<it>Musca domestica</it>) and German cockroaches (<it>Blattella germanica</it>) 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.</p> <p>Results</p> <p>Enterococci from the digestive tract of house flies (<it>n </it>= 162), and feces of German cockroaches (<it>n </it>= 83) and pigs (<it>n </it>= 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 × 10⁴CFU/house fly, 5.5 ± 1.1 × 10⁶CFU/g of cockroach feces, and 3.2 ± 0.8 × 10⁵CFU/g of pig feces. Among all the identified isolates (n = 639) <it>Enterococcus faecalis </it>was the most common (55.5%), followed by <it>E. hirae </it>(24.9%), <it>E. faecium </it>(12.8%), and <it>E. casseliflavus </it>(6.7%). <it>E. faecalis </it>was most prevalent in house flies and cockroaches, and <it>E. hirae </it>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 <it>tet</it>(M) and <it>erm</it>(B) and Tn<it>916</it>/<it>1545 </it>transposon family. <it>E. faecalis </it>frequently harbored virulence factors <it>gelE, esp, </it>and <it>asa1</it>. PFGE analysis of selected <it>E. faecalis </it>and <it>E. faecium </it>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.</p> <p>Conclusions</p> <p>This 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.</p

Antibacterial Activities of Nepetalactones Against Public Health-Related Pathogens

The antimicrobial activities of (Z,E)- and (E,Z)-nepetalactones, 2 major compositional compounds from the essential oil of catnip (Nepeta cataria), were first discovered from fly larval development media studies with over 98% inhibition of bacterial growth. Further investigation demonstrated inhibition of the growth of various bacterial species of public health significance. Catnip oil showed antibacterial activity against 5 Gram-positive and 9 Gram-negative bacteria. The antimicrobial activity varied among the original essential oil from the plant and its major compositional compounds as a blended mixture or an individual compound. Growth inhibition was observed against 5 Neisseria species, with particularly strong inhibition against Neisseria sicca (with MICs ranging from 0.5 to 5 mg/mL) that provided comparable or increased levels of growth control produced by 2 antibiotics (Ceftiofur and Cephalothin). The development of plant-based antibacterial agents to prevent or delay the emergence of antimicrobial resistance in bacteria is discussed

Mortality in kittens is associated with a shift in ileum mucosa-associated enteroccoci from E. hirae to biofilm-forming E. faecalis and adherent E. coli

Approximately ~15% of foster kittens die before 8-wks of age with most of these kittens demonstrating clinical signs or post-mortem evidence of enteritis. While a specific cause of enteritis is not determined in most cases; these kittens are often empirically administered probiotics that contain enterococci. The enterococci are members of the commensal intestinal microbiota but can also function as opportunistic pathogens. Given the complicated role of enterococci in health and disease, it would be valuable to better understand what constitutes a “healthy” enterococcal community in these kittens and how this microbiota is impacted by severe illness. In this study, we characterize the ileum mucosa-associated enterococcal community of 50 apparently healthy and 50 terminally ill foster kittens. In healthy kittens, E. hirae was the most common species of ileum mucosa-associated enterococci and was often observed to adhere extensively to the small intestinal epithelium. These E. hirae isolates generally lacked virulence traits. In contrast, non-E. hirae enterococci, notably E. faecalis, were more commonly isolated from the ileum mucosa of kittens with terminal illness. Isolates of E. faecalis had numerous virulence traits and multiple antimicrobial resistance. Moreover, attachment of E. coli to the intestinal epithelium was significantly associated with terminal illness and was not observed in any kitten with adherent E. hirae. These findings identify a significant difference in species of enterococci cultured from the ileum mucosa of kittens with terminal illness compared to healthy kittens. In contrast to prior case studies that associate enteroadherent E. hirae with diarrhea in young animals, these controlled studies identified E. hirae as more often isolated from healthy kittens and adherence of E. hirae as more common and extensive in healthy compared to sick kittens

Dogs Leaving the ICU Carry a Very Large Multi-Drug Resistant Enterococcal Population with Capacity for Biofilm Formation and Horizontal Gene Transfer

The enterococcal community from feces of seven dogs treated with antibiotics for 2–9 days in the veterinary intensive care unit (ICU) was characterized. Both, culture-based approach and culture-independent 16S rDNA amplicon 454 pyrosequencing, revealed an abnormally large enterococcal community: 1.4±0.8×108 CFU gram−1 of feces and 48.9±11.5% of the total 16,228 sequences, respectively. The diversity of the overall microbial community was very low which likely reflects a high selective antibiotic pressure. The enterococcal diversity based on 210 isolates was also low as represented by Enterococcus faecium (54.6%) and Enterococcus faecalis (45.4%). E. faecium was frequently resistant to enrofloxacin (97.3%), ampicillin (96.5%), tetracycline (84.1%), doxycycline (60.2%), erythromycin (53.1%), gentamicin (48.7%), streptomycin (42.5%), and nitrofurantoin (26.5%). In E. faecalis, resistance was common to tetracycline (59.6%), erythromycin (56.4%), doxycycline (53.2%), and enrofloxacin (31.9%). No resistance was detected to vancomycin, tigecycline, linezolid, and quinupristin/dalfopristin in either species. Many isolates carried virulence traits including gelatinase, aggregation substance, cytolysin, and enterococcal surface protein. All E. faecalis strains were biofilm formers in vitro and this phenotype correlated with the presence of gelE and/or esp. In vitro intra-species conjugation assays demonstrated that E. faecium were capable of transferring tetracycline, doxycycline, streptomycin, gentamicin, and erythromycin resistance traits to human clinical strains. Multi-locus variable number tandem repeat analysis (MLVA) and pulsed-field gel electrophoresis (PFGE) of E. faecium strains showed very low genotypic diversity. Interestingly, three E. faecium clones were shared among four dogs suggesting their nosocomial origin. Furthermore, multi-locus sequence typing (MLST) of nine representative MLVA types revealed that six sequence types (STs) originating from five dogs were identical or closely related to STs of human clinical isolates and isolates from hospital outbreaks. It is recommended to restrict close physical contact between pets released from the ICU and their owners to avoid potential health risks

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

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 × 10(3) 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

Association of Escherichia coli O157:H7 with Houseflies on a Cattle Farm

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 × 10(1) to 1.5 × 10(5) 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

Influx of Enterococci and Associated Antibiotic Resistance and Virulence Genes from Ready-To-Eat Food to the Human Digestive Tract▿

The influx of enterococcal antibiotic resistance (AR) and virulence genes from ready-to-eat food (RTEF) to the human digestive tract was assessed. Three RTEFs (chicken salad, chicken burger, and carrot cake) were sampled from five fast-food restaurants five times in summer (SU) and winter (WI). The prevalence of enterococci was significantly higher in SU (92.0% of salad samples and 64.0% of burger samples) than in WI (64.0% of salad samples and 24.0% of burger samples). The overall concentrations of enterococci during the two seasons were similar (∼103 CFU/g); the most prevalent were Enterococcus casseliflavus (41.5% of isolates) and Enterococcus hirae (41.5%) in WI and Enterococcus faecium (36.8%), E. casseliflavus (27.6%), and Enterococcus faecalis (22.4%) in SU. Resistance in WI was detected primarily to tetracycline (50.8%), ciprofloxacin (13.8%), and erythromycin (4.6%). SU isolates were resistant mainly to tetracycline (22.8%), erythromycin (22.1%), and kanamycin (13.0%). The most common tet gene was tet(M) (35.4% of WI isolates and 11.9% of SU isolates). The prevalence of virulence genes (gelE, asa1, cylA, and esp) and marker genes for clinical isolates (EF_0573, EF_0592, EF_0605, EF_1420, EF_2144, and pathogenicity island EF_0050) was low (≤12.3%). Genotyping of E. faecalis and E. faecium using pulsed-field gel electrophoresis revealed that the food contamination likely originated from various sources and that it was not clonal. Our conservative estimate (single AR gene copy per cell) for the influx of tet genes alone to the human digestive tract is 3.8 × 105 per meal (chicken salad). This AR gene influx is frequent because RTEFs are commonly consumed and that may play a role in the acquisition of AR determinants in the human digestive tract