Virulence Factors in Enterococci from Partridges ( Alectoris rufa ) Representing a Food Safety Problem

Introduction

E nterococci are Gram-positive bacteria and are commonly found in the gastrointestinal tract of healthy humans and animals. In addition to their commensal nature, the capacity of these bacteria to acquire virulence traits enhances their ability to break the normal commensal mode in the gastrointestinal tract and invade extraintestinal niches, resulting in disease (Richards et al., 2000). In this sense, enterococci have become recognized as leading causes of nosocomial bacteremia, urinary tract infections, and surgical wound infection (Tendolkar et al., 2003). Genes encoding for the virulence factors such as aggregation substance, surface adhesins, sex pheromones, lipoteichoic acid, extracellular superoxide, gelatinase, hyaluronidase, and cytolysin (hemolysin) were most extensively studied in enterococci (Kayaoglu and Orstavik, 2004). Some of these virulence factors such as esp, gelE, and fsr genes showed a significant association with the biofilm production by some Enterococcus faecalis strains. These structures can form on a wide variety of surfaces, and during infection, they serve as a nidus for bacteria sloughing off to colonize other sites (Mohamed et al., 2004). The partridge (Alectoris rufa) is a game bird with great economic and gastronomic relevance in the southern European countries, particularly Spain and Portugal. The partridge hunting in Portugal is allowed between December and January, organized by different corporations of hunters, and usually performed by walk-up hunting with dogs, which can be combined with other game species hunting such as rabbits.

There are reports that describe Enterococcus strains of different origins that produce different virulence factors (Engelbert et al., 2004; Kayaoglu and Orstavik, 2004; Mohamed et al., 2004; Roberts et al., 2004; Klare et al., 2005; Poeta et al., 2006), but studies in wild animal enterococcal isolates are very scarce (Poeta et al., 2008) and not documented in enterococci from partridges. The aim of the present study was to determine the incidence of gelatinase and β-hemolysis activities in a series of enterococci from partridges samples, to know the prevalence of these activities among intestinal bacteria of animal origin. The gelatinase and β-hemolysis production was correlated with the presence of fsr-gelE genes and the cyl operon, respectively. In addition, further genes encoding virulence factors were analyzed.

Materials and Methods

The presence of enterococci was studied in 43 fecal samples (one isolate per sample) recovered from partridges (A. rufa) from December 2006 to January 2007 in the north of Portugal during the partridge hunting season organized by different corporations of hunters. This hunting is supervised by the Agriculture, Rural Development, and Fishery Ministry of Portugal under the Decree-Law no. 202/2004. Samples were seeded in Slanetz-Bartley agar plates and were incubated for 48 h at 37°C. Colonies with typical enterococcal morphology (one per sample) were identified and enterococci were identified to the species level by polymerase chain reaction (PCR) (Torres et al., 2003). The gelatinase and hemolytic activity were assessed according to Poeta et al. (2006). The presence of genes encoding different virulence factors (gelE, fsr, cyl operon, cpd, hyl, agg, esp, and ace) were tested by PCR in all our enterococci, using primers and conditions previously described (Poeta et al., 2008).

Results and Discussion

The gelatinase activity of the 43 enterococci of partridges origin as well as the genotype for the presence of gelE and fsr genes are shown in Table 1. Most of E. faecalis isolates (10 of 15 isolates) showed gelatinase activity, and all of them harbored the gelE and fsr genes. Most of the reports refer to the frequent detection of gelE and its regulator fsr genes in E. faecalis isolates (Gilmore et al., 2002; Roberts et al., 2004). Similarly, in a previous study carried out by our group, the E. faecalis isolates of poultry origin that exhibited gelatinase activity showed the fsr + -gelE+ genotype (Poeta et al., 2006). The fsr genes have been shown to be important for the regulation and expression of the gelE gene and for enterococcal infection in animal models (Singh et al., 1998; Qin et al., 2000; Engelbert et al., 2004). The gelatinase activity was not detected in the other enterococcal species. The prevalence of gelatinase activity found in our E. faecalis isolates is similar to that reported for isolates recovered from feces of healthy community-based volunteers (66.6%) (Roberts et al., 2004). Cytolysin is a toxin that causes a β-hemolytic reaction on certain blood erythrocytes, and its expression requires the presence of the whole cyl operon (Gilmore et al., 2002). Table 1 shows the β-hemolytic activity of the enterococcal isolates and the presence of genes of the cyl operon. One of our enterococci (E. faecalis) showed β-hemolysis, whereas no β-hemolysis was detected in the remaining isolates. Further, seven different genotypes of genes of the cyl operon were detected in our enterococci isolates. Only the β-hemolytic–positive enterococcal isolate harbored the five tested genes of the cyl operon (cylLL, cylLS, cylA, cylB, and cylM). The remaining 42 enterococcal isolates showed six different combinations of cyl operon genes, and all of them lacked at least one of the five tested genes. Although the cyl genes had been more associated with E. faecalis, our isolates of different species (E. faecalis, E. faecium, E. casseliflavus, E. hirae, and E. durans) harbored at least one of the cyl operon genes. In a previous study in poultry, different enterococcal species also harbored genes of the cyl operon (Poeta et al., 2006). PCR analysis allowed us to identify additional virulence factors in our enterococci isolates (Table 1). It is of interest to emphasize the significant prevalence and the wide variety of genes encoding virulence factors detected among the E. faecalis isolates compared with those found in other enterococcal species. The cpd gene (encoding a sex pheromone determinant) was detected in 9 of the 15 E. faecalis isolates and in the E. casseliflavus isolate. The agg gene (encoding the aggregation substance) was found in 5 of the 15 E. faecalis isolates and the ace gene (encoding an accessory colonization factor) was found in four isolates. In a previous study carried out by our group, the agg gene was not found in enterococci isolated from wild boars (Poeta et al., 2008). The hyl gene (encoding hyalorunidase activity) was identified in one E. faecium. The association of hyl gene with specific clonal complexes of ampicillin/vancomycin-resistant E. faecium strains was observed, which have emerged in the last years in several hospitals (Klare et al., 2005). The esp gene, encoding an extracellular surface protein associated with the ability to form biofilms on abiotic surfaces (Toledo-Arana et al., 2001; Kristich et al., 2004; Mohamed et al., 2004), was not detected in our enterococci isolates. The esp gene is more frequently found in clinical isolates (Shankar et al., 1999; Mohamed et al., 2004) and this fact could be the reason for the absence of detection among the commensal isolates analyzed in our study. In clinical setting, the incidence of vancomycin-resistant E. faecium isolation was attributed mainly to the occurrence and spread of epidemic, virulent, ampicillin/vancomycin-resistant, vanA- and vanB-positive E. faecium clones, most of which exhibited the virulence factor enterococcal surface protein (esp) (Klare et al. 2005). Enterococci have clearly appeared as a medically important organism, causing outbreaks of many nosocomial infections. Formerly considered a not dangerous bacterium, it has emerged as a virulent pathogen accounting for more hospital-borne diseases. In general, enterococci isolated from medically indwelling devices produced high amount of virulence factors (Klare et al. 2005) when compared with isolates recovered from poultry (Poeta et al., 2006) or wild animals (Poeta et al., 2008).

In the present study, we show that enterococci of the intestinal tract of partridges can constitute a reservoir of genes encoding virulence factors. The partridges are an important food source of many animals, and for this reason, enterococci with virulence determinants can be transferred to other wild animals. Further, the partridges are often used in human diet. During their cooking preparation, when pulling out the guts, there is a strong probability of transmission of enterococci from feces to the meat. Nevertheless, depending of the thermal process, the enterococci could survive during the cooking process and may enter into the human food chain, making them a potential transmitter of genes to humans. Further, the bacterium can cause food intoxication through production of biogenic amines and can be a reservoir for opportunistic infections and virulence traits, representing a public health problem. From a food safety perspective, additional studies, for example, in the meats, become essential in this research area with respect to their contribution to microbial risk assessment.