Genetic Detection and Multilocus Sequence Typing of vanA -Containing Enterococcus Strains from Mullets Fish ( Liza ramada )

Introduction

Enterococci are members of the intestinal microbiota of healthy humans and animals. However, they have also emerged as important cause of nosocomial and community-acquired infections. Enterococcus is intrinsically resistant to many antimicrobial agents, and its ability to acquire resistance to other agents, such as glycopeptides, is well known. ¹⁹

The thin-lipped gray mullet, Liza ramada, is a catadromous species that passes most of its life cycle in estuarine environments. On the Portuguese coast, the adult fish perform their spawning migration toward the sea during the autumn and early winter months, although there are also reports of an upstream migration in late spring. ² As mullets have served as an important source of food in Mediterranean Europe, it is possible to address the potential risk for human populations.

Glycopeptide antibiotics, such as vancomycin and teicoplanin, are widely used in the treatment of serious infections caused by gram-positive bacteria. Eight mechanisms of acquired glycopeptide resistance have been so far described in enterococci, mediated by the vanA, vanB, vanD, vanG, vanE, vanL, vanM, and vanN, the vanA mechanism being the most frequently reported on clinical vancomycin-resistant enterococci (VRE), followed by the vanB mechanism.^4,5,12,17,34 The vanA mechanism confers high-level vancomycin and teicoplanin resistance, and it is induced by vancomycin. The vanA gene is located on a transposon, named Tn1546, together with other genes of the vanA cluster, allowing the intra- and interspecies spread of resistance. ¹² vanA confers glycopeptide resistance by the inducible synthesis of precursor ending in D-Ala-D-Lac. ⁴ Enterococcus gallinarum and Enterococcus casseliflavus display an intrinsic and chromosomally encoded mechanism of vancomycin resistance, mediated by the vanC gene that confers a nontransferable low-level glycopeptide resistance. ⁴

The virulence gene encoding for enterococcal surface protein (esp) has been detected in Enterococcus faecium as part of distinct genetic elements. As the esp gene was prevalent in E. faecium isolates associated with nosocomial outbreaks, it was concluded that this genetic element constitutes a putative pathogenicity island ³³ that has been recently shown to be part of an integrative conjugative element (ICE) and thus renamed ICEEfm1. ²⁸

Other factors possibly contributing to the pathogenesis of enterococcal infections, such as a putative glycoside hydrolase (hyl), have been reported. ¹⁰ Additionally, the presence of the esp and hyl virulence genes has been associated with epidemic clones. ¹⁶ It has been also shown that the majority of E. faecium isolates causing hospital outbreaks worldwide belong to a specific genetic subpopulation designated as high-risk clonal complex 17 (CC17). ²⁹

The purpose of this study was to determine the fecal carriage of VRE in mullets fish in Portugal, analyzing the antibiotic resistance phenotype and the mechanisms implicated, the virulence gene content, and the inclusion of some specific insertion sequences within the vanA cluster of genes in the VRE isolates recovered. Further, vanA-positive E. faecium isolates have been characterized by multilocus sequence typing (MLST) to know the circulating lineages of vanA strains in this animal ecosystem.

Materials and Methods

Results

vanA-containing enterococcal strains were detected in 4 of the 103 fecal samples studied (3.8%) and showed high-level vancomycin (MIC ≥ 128 mg/L) and teicoplanin resistance (MIC 32 mg/L). Two isolates per sample were identified to the species level and their antibiotic susceptibility profile was determined. As the isolates from each positive sample exhibited the same enterococcal species and the same antibiotic resistance profile, only one isolate per positive sample was maintained for further studies. All VRE were identified as E. faecium, and their characteristics are shown in Table 1. All strains detected in this study exhibited resistance to erythromycin, three of them to ampicillin, two to tetracycline, ciprofloxacin, and kanamycin, and one to gentamicin. The erm(B) gene, associated with erythromycin resistance, was found in all vanA strains and the tet(M) gene was demonstrated in the two strains that showed tetracycline resistance. The strains resistant to gentamicin and kanamycin harbored the aph(2′′)-aac(6′) and aph(3′)-III genes, respectively. In our study, a PCR analysis of the vanX-vanY region showed amplicons larger than predicted (1.9 kb) in the four strains and the IS1216 sequence was identified by sequencing within this region in two of them (C1249 and C1250). The size of vanS-vanH region gave the expected size for all four vanA isolates, indicating the absence of any insertion or deletion at this position. These isolates harbored the IS1216 sequence, although its specific position is unknown. Virulence genes were detected in our study. All strains harbored the hyl gene and two strains the esp gene. Two isolates were classified by MLST analysis into the ST273 and another two into the ST280, including both lineages into CC17.

Discussion

Portugal is one of the European countries in which higher prevalence of vancomycin resistance has been reported in invasive E. faecium and E. faecalis isolates.^8,31 vanA-E. faecium isolates were mainly responsible for the high rates of infections caused by VRE in Portugal. ²⁰ The first large VRE surveillance study in this country, which included data from 10 participating hospitals, was performed in 1994 and revealed a rate of 9% of vancomycin-resistant E. faecium among clinical isolates. ²⁹ A remarkable increase was documented in subsequent years, with rates rising from 20% in 1996 to 47% in 2003. ²⁹ Dissemination of persisting E. faecium clones, belonging to the high-risk CC17, is an important fact in Portuguese VRE setting; however, polyclonality was frequently observed among VRE, corroborating that horizontal transfer of vanA transposon (Tn1546) types could play an important role in rapid and extensive spread of VRE in Portuguese hospitals.^20,29

Different studies carried out in Portugal reveal the existence of a reservoir of vanA-containing enterococci in nonhospital ecosystems, as is the case of the intestinal tract of animals such as wild buzzards, wild boars, or pets, among others (present in 1.4%–9% of tested animals).^22–24 Our actual study shows that mullets fish, in the aquatic environment, is also a reservoir of this type of resistant bacteria. The flux direction of vancomycin-resistant bacteria between the hospital and environment is not clear, but it is presumed that the wide dissemination of vanA-containing enterococci in natural ecosystems might have an impact in hospitals.

The Douro River seems to be more polluted than the Mondego River according to some analytical data.^25,26 Nevertheless, the number of animals positive for vanA-containing E. faecium isolates was similar in both rivers.

It is important to underline the detection of the ST280 and ST273 lineages belonging to the high-risk CC17 in our vanA-containing E. faecium strains, because this CC have been associated with E. faecium isolates that are well adapted to hospital environments and are frequently implicated in human infections. ¹⁵ The CC17 has, apparently, evolved by a multistep process to raise a good adaptation to a novel ecologic niche, the hospital environment. This CC17 represents the first globally dispersed nosocomially adapted clonal lineage, which is frequently associated with ampicillin and quinolone resistance as well as to the presence of esp gene. ³²

In a recent study performed in Portugal, ¹¹ CC17 was identified in 24 VRE and vancomycin-sensible isolates from hospitals, healthy volunteers, swine, piggeries, and the environment in different regions from 1997 to 2007. In this report, the CC17 meroclone consisted of a high diversity of STs (ST16, ST18, ST78, ST80, ST125, ST132, ST280, ST368, ST369, ST390, ST393, ST430, and ST431), particularly enriched by ST18. However, unlike to our study, the ST273 was not detected and ST280 was only found in an outbreak strain disseminated in two hospitals between 2002 and 2003.

Few studies have been reported in which virulence factors are demonstrated in enterococci of food and animal origin, ⁹ and even fewer reports have been focused on strains of wild animals. ²³ Two strains that showed ampicillin resistance also harbored the hyl and esp genes encoding virulence factors. The incidence of vancomycin-resistant E. faecium isolates in clinical setting was attributed mainly to the occurrence and spread of epidemic-virulent ampicillin/vancomycin-resistant, vanA- and vanB-positive E. faecium clones, most of which contained the esp gene and some of them the hyl gene. ¹⁴

All our strains were erythromycin resistant. Some authors refer that the persistence of VRE after the banning of glycopeptides in animal production may be explained by the genetic link between erm(B) and vanA genes and their possible coselection by macrolide use. ¹ Another study showed that currently a wide dissemination of E. faecium clones resistant to vancomycin, macrolides, and tetracycline may be taking place in different European countries. ²¹

The detection of CC17 among nonclinical sources may indicate a hospital contribution of community strains with different genetic contents further than contamination from the hospital setting and might clarify its high prevalence and global spread. In the future, surveillance studies should be carried out to supervise the prevalence of antimicrobial resistance and virulence in enterococci of different origins to detect the emergence and dissemination of new or already-known antimicrobial resistance genes and virulence factors in different ecosystems.