Comparison of the incidence of virulence determinants and antibiotic resistance between Enterococcus faecium strains of dairy, animal and clinical origin

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Abstract

Enterococci are part of the dominant microbiota of several dairy products. They are also present in the gut of humans and animals. Their presence in traditional raw milk cheeses is probably due to faecal contamination of milk during milking. Due to their importance as a cause of nosocomial infections, enterococci are acquiring increased significance. Such infections are becoming more and more difficult to treat as resistance to antibiotics increases. The aim of this investigation was to compare the potential virulence of Enterococcus faecium isolated from different ecological habitats and to establish if strains isolated from dairy products should really be considered as potential pathogens. In the present work, the antibiotic resistance pattern of 40 E. faecium strains isolated from dairy products, 26 E. faecium isolated from ewes' faeces and 28 clinical isolates of the same species was studied, and checks were made to see if known virulence determinants were present. Resistance to 12 different antibiotics commonly used in the treatment of human infections was tested using the broth microdilution method as described by the NCCLS. In addition, polymerase chain reaction (PCR) tests were carried out to see if genes for vancomycin resistance were present. The presence of the aggregation substance (AS) gene, the surface protein gene esp, the accessory colonisation factor ace, the Enterococcus faecalis endocarditis antigen efaA and the gelatinase gelE gene, which are involved in the virulence of enterococci, were also tested by PCR. The results of this study clearly indicate that E. faecium strains isolated from both cheese and sheep faeces are less pathogenic than those isolated from clinical samples. A similar pattern of resistance to antibiotics was observed in both dairy and animal strains. It was also found that there was difference in the kind of virulence determinants present in dairy and clinical isolates, while no virulence traits were found in sheep faeces strains. The results of this study suggest that E. faecium from traditional Sardinian raw milk cheeses should not be considered to be the main source of untreatable nosocomial enterococcal infections in humans in the island of Sardinia.

Introduction

Enterococci comprise a bacterial group widely diffused in several habitats. They are present in the microbial association of a variety of fermented foods such as cheese, fermented sausages and vegetables Ledda et al., 1994, Leclerc et al., 1996, Cogan et al., 1997, Mannu et al., 1999, Mannu et al., 2002, Müller et al., 2001. In cheese, particularly in traditional raw milk cheeses, they are involved in the primary fermentation activity, but they also form part of the secondary non-starter microbiota involved in the ripening process and in aroma development Ledda et al., 1994, Cogan et al., 1997, Mannu et al., 2002. These beneficial effects have been attributed to proteolytic and lipolytic activities Wessels et al., 1990, Centeno et al., 1999, Durlu-Ozkaya et al., 2001, Sarantinopoulos et al., 2002. Some enterococcal strains have also been used as human probiotics because they can survive and compete in the gastrointestinal tract (Franz et al., 1999). The use of enterococcal strains to produce bacteriocin in cheese and meat has also been reported Giraffa, 1995, Núñez et al., 1997. They are not, however, considered “generally recognised as safe” (GRAS) and their presence is an indicator of faecal contamination. Indeed, they normally colonise the intestinal tract of humans and animals, although they are also known to be opportunistic pathogens responsible for a wide variety of human infections such as endocarditis, urinary and genital tract infections, meningitis and septicemia (Murray, 1990). These microorganisms are the third most common cause of hospital-acquired bacteraemia (Garnier et al., 2000). Enterococci have also been found to be increasingly resistant to multiple anti-microbial drugs in the last few years, limiting the number of therapeutic options available to the physicians Handwerger et al., 1993, Montecalvo et al., 1994, Satake et al., 1997. Vancomycin resistance in particular, especially when it emerges in strains highly resistant to ampicillin, is a clinically relevant characteristic, which has been steadily rising over the past decade. A significant increase in the percentage of clinical Enterococcus strains displaying high-level vancomycin resistance has been observed, and very often, the resistance to glycopeptides is associated with high levels of resistance to aminoglycosides (Endtz et al., 1999). In addition, vancomycin resistance can be disseminated through both clonal expansion of resistant enterococci and horizontal transmission of resistance genes (Stobberingh et al., 1999). This is increasingly a cause of concern for infectious disease specialists. Several types of both acquired and intrinsic glycopeptide resistance, with either low or high levels of resistance, have been found in enterococci Leclercq et al., 1992, Arthur et al., 1996, Périchon et al., 1997, Fines et al., 1999. The virulence of enterococci associated with infections can be still further enhanced by the presence of additional virulence characteristics. Several virulence molecules have been described in enterococci, in particular associated with infection-derived Enterococcus faecalis strains Libertin et al., 1992, Lowe et al., 1995, Nallapareddy et al., 2000a, Süßmuth et al., 2000, Qin et al., 2001, Shankar et al., 2001, Toledo-Arana et al., 2001. Many of these may interact with host cells and colonise the mucosal surfaces. Such interactions and colonisation are considered trigger events in the pathogenesis of many infections. In addition, haemolysin production can increase the severity of enterococcal infections. The aggregation substance (AS) is one of the postulated virulence factors. It is a surface protein that is encoded by sex pheromone plasmids and promotes donor and recipient cell aggregation, leading to conjugative transfer of plasmids Süßmuth et al., 2000, Waters and Dunny, 2001. Other surface proteins, such as Ace, E. faecalis antigen A (EfaA) and enterococcal surface protein (Esp) have been described. Their precise role in the pathogenesis is not always completely clear, but they are presumed to be involved in mechanisms by which the enterococcal cells adhere to biotic and abiotic surfaces and in biofilm formation Lowe et al., 1995, Nallapareddy et al., 2000b, Shankar et al., 2001, Toledo-Arana et al., 2001. Another property of enterococci that has been considered a possible virulence factor is gelatinase gelE, a metalloproteinase. It is mainly found in endocarditis isolates and it is presumed to contribute to virulence (Singh et al., 1998).

In the present work, the antibiotic resistance patterns of Enterococcus faecium strains isolated from ewe's raw milk cheeses, from ovine faeces and from clinical samples were defined and the presence of known virulence determinants was determined. Although the molecular taxonomy of E. faecium does not distinguish between a pathogen strain and a safe food use strain, much of the available literature suggests that antibiotic resistance and virulence determinants may have been acquired in the hospital environment and may be particularly frequent in clinical isolates. The aim of this work was, therefore, to evaluate if the diffusion of antibiotic resistances and of virulence determinants in strains of E. faecium were at the same levels in dairy, animal and clinical environments. If such pathogenic traits in E. faecium, which are part of the dominant microbiota of ewe's milk cheese, could be excluded, this would be of great importance. When one considers that the high levels of enterococci in milk, particularly in ewe's milk, is probably due to faecal contamination from the animal, then investigating E. faecium isolated from ovine faeces is also important for the same reason.

Section snippets

Bacterial strain isolation and growth conditions

A total number of 94 strains were used in this study. All the strains were isolated in Sardinia between 1998 and 2001. Forty of them were isolated from different traditional ewe's raw milk cheeses produced in the island of Sardinia. Twenty-six of them were isolated from faeces of Sarda breed sheep, and 28 were isolated from different clinical samples from patients at Sassari Hospital (Sardinia, Italy). The list of the strains and their origin are reported in Table 1. Cheese sampling and

Taxonomic identification of the isolates

The preliminary phenotypic characterisation of the isolates suggested that they might belong to the Enterococcus genus. PCR with genus-specific primers confirmed this result, and all the isolates gave the expected amplification product of 112 bp on 2% agarose gel.

The PCR with E. faecium species-specific primers showed that the isolates belonged to this species.

Antibiotic susceptibility testing

The antibiotic susceptibility testing of the three groups of tested strains is summarised in Fig. 1. The MIC data obtained in vitro with

Conclusions

The results obtained in this study clearly indicate that E. faecium strains isolated from both cheese and sheep faeces have lower potential pathogenicity than those isolated from clinical samples. A similar antibiotic resistance pattern was observed in both dairy and animal strains. The presence of virulence determinants also was found to be different between dairy and clinical isolates, while no virulence trait was found in sheep faeces strains. These results suggest that the consumption of

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