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 x 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.

FIG. 1.

Antibiotic resistance screening of identified isolates by phenotype. T, tetracycline; VA, vancomycin; AM, ampicillin; CIP, ciprofloxacin; K, kanamycin; E, erythromycin; S, streptomycin; C, chloramphenicol.

FIG. 2.

Distribution of tet(M), tet(O), tet(W), and erm(B) genes in E. faecalis and E. faecium isolates phenotypically resistant to tetracycline (n = 120 and n = 4, respectively) and erythromycin (n = 46 and n = 2, respectively) and distribution of Tn916 and Tn916/Tn1545 family transposons in all E. faecalis (n = 181) and E. faecium (n = 14) isolates.

FIG. 3.

Restriction fragment length polymorphism of tet(M), determined using RsaI and MspI. Lane L, 100-bp molecular marker; lane PCa, tet(M) from E. faecalis OG1RF(pCF10); lanes 1a to 7a, E. faecalis isolates digested with RsaI; lanes 1b to 6b, E. faecalis isolates digested with MspI.

FIG. 4.

Prevalence of virulence genes gelE, asa1, cylA, and esp in identified isolates. (A) Data from multiplex PCRs. (B) Data from phenotypic tests (except esp data).