Multidrug-resistant enterococci in the hospital environment: detection of novel vancomycin-resistant E. faecium clone ST910

Introduction: The role of the hospital environment as a reservoir of resistant bacteria in Tunisia has been poorly investigated; however, it could be responsible for the transmission of multidrug-resistant bacteria. The objective was to study the prevalence of Enterococcus in the environment of a Tunisian hospital and the antibiotic resistance phenotype/genotype in recovered isolates, with special reference to vancomycin resistance. Methodology: A total of 300 samples were taken (March–June, 2013) and inoculated in Slanetz-Bartley agar plates supplemented or not supplemented with 8 μg/mL of vancomycin. Antibiotic resistance genes were tested by polymerase chain reaction (PCR). The clonal relatedness of the vanA isolates was assessed using pulsed-field gel electrophoresis (PFGE) and multilocus sequence testing (MLST). Results: Enterococci were recovered in 33.3% of tested samples inoculated in SB medium. E faecium was the most prevalent species, followed by E. faecalis and E. casseliflavus. Antimicrobial resistance genes detected were as follows (number of isolates): erm(B) (71), tet(M) (18), aph(3’)-IIIa (27), ant(6)-Ia (15), cat(A) (4), and van(C2) (6). Vancomycin-resistant-enterococci (VRE) were recovered from 14 samples (4.7%), when tested in SB-VAN. The 14 VRE (one per positive sample) were identified as E. faecium and contained the van(A),erm(B), tet(M), ant(6)Ia, and aph(3’)-IIIa genes. Thirteen of the VRE strains were ascribed by PFGE and MLST to a novel clone (new ST910), and only one VRE strain was typed as ST80 included in CC17. Conclusions: The emergence and spread of new clones of VRE, especially in the hospital environment in this country, could become particularly problematic.


Introduction
Enterococci are recognized now as important causes of human nosocomial infections, especially in immunocompromised patients.These microorganisms are able to cause multiple pathologies such as urinary tract infections, bacteremia, and endocarditis, among others [1].Furthermore, the capacity of enterococci to survive outside the human body for prolonged periods of time contributes to cross-contamination through the hands of healthcare workers, leading to outbreaks [1,2].The hospital environment can be a reservoir of resistant bacteria, and the role of a possible dissemination of these bacteria, especially vancomycin-resistant enterococci (VRE) via inanimate surfaces and equipment, has been documented by several researchers [3][4][5][6].Moreover, the cross-transmission, between patients via contaminated hands and surfaces, of multidrug-resistant microorganisms increases length of stays in hospitals, healthcare costs, and mortality [7,8].
Over the past 20 years, epidemiological data collected have shown the emergence of enterococci, especially E. faecium, as pathogens responsible for nosocomial infections.The expansion of E. faecium isolates belonging to clonal complex (CC17) is remarkable [9].This epidemic clonal complex is characterized by ampicillin and vancomycin resistance, as well as by the presence of virulence determinants such as hylEfm (encoding a hyaluronidase-like protein) and a putative pathogenicity island that includes espEfm (enterococcal surface protein in E. faecium), and is responsible for severe nosocomial infections and hospital outbreaks [10,11].The resistance to vancomycin in enterococci is still uncommon in Tunisia, and there are only a few previous reports of detection of vanA-containing enterococci in hospitals in that country [6,12].In a previous study performed by our group in the Military Hospital of Tunis in 2012 [6], the presence of vanA-containing E. faecium isolates was detected in clinical samples as well as in 5% of environmental samples of this hospital.The objective of the present work was to continue the epidemiological study on environmental samples in the hospital during 2013, to determine the prevalence of Enterococcus and the phenotypes and genotypes of antibiotic resistance in recovered isolates, with special reference to vancomycin resistance, as well as to analyze the evolution of environmental VRE in terms of frequency and type of detected clones.

Samples and bacterial isolates
A total of 300 environmental samples were obtained from 17 different services of the Military Hospital of Tunis, during March-June 2013.The samples were taken with sterile cotton swabs from inanimate surfaces (beds, treatment tables, toilets, faucets, wrists, sinks) after routine cleaning (n = 250), and from the hands of patients (n = 23) and staff (n = 27).Informed consent was obtained from all individuals who participated in the study.
Samples were pre-enriched in brain-heart infusion broth for 24 hours at 37°C.Next, 0.1 mL of the suspension was seeded in plates of Slanetz-Bartley (Biolife, Milano, Italy) not supplemented (SB) and supplemented with 8 μg/mL of vancomycin (BioRad, Marnes-la-Coquette, France) (SB-VAN).The samples were then incubated for 48 hours at 37ºC for the recovery of enterococci and VRE.A colony was selected of each positive sample and was identified by species-specific polymerase chain reactions (PCRs) [13].
The presence of the insertion element IS16 (usually found in hospital-associated E. faecium isolates implicated in outbreaks) was performed by PCR in all the vanA-containing isolates [17].

Detection of virulence genes
The presence of esp (enterococcal surface protein) and hyl (encoding a hyaluronidase-like protein) virulence genes was detected by PCR in the vanAcontaining E. faecium isolates, and the esp gene was also tested in the vancomycin-susceptible enterococci [18].

Pulsed-field gel electrophoresis (PFGE)
The clonal relatedness of the vanA-containing isolates was assessed using PFGE of the genomic DNA digested with SmaI enzyme [13].Interpretation of banding patterns was performed according to the criteria specified by Tenover et al. [19].The dendrogram of PFGE patterns of SmaI digested DNA of 14 VRE was prepared by FPQuest (BioRad, Hercules, USA) software using UPGMA algorithm.

Multilocus sequence typing (MLST)
Internal fragments of seven housekeeping genes (atpA, ddl, gdh, purK, gyd, pstS, and adk) of our vanApositive E. faecium isolates were amplified and sequenced.The sequences obtained were analyzed and compared with those included in the website database (www.pubmlst.org) in order to assign a specific sequence type (ST).STs were grouped into clonal complexes using the program eBURST (http://eburst.mlst.net)[20].The type of purK allele was analyzed in the esp-positive vancomycin-susceptible E. faecium isolates to determine if they could have the purK1 allele (CC17-associated).
Antimicrobial resistance distributed by species of enterococcal strains isolated in SB medium not supplemented with vancomycin is shown inTable 2. All enterococci, including E. faecium isolates, were susceptible to ampicillin and to a high dose of gentamicin.The antimicrobial resistance genes detected in these isolates by PCR are shown in Table 3.
The majority of the erythromycin-resistant enterococci (71 of 73 isolates, 97.3%) harbored the erm(B) gene.All the 18 tetracycline-resistant enterococci (12 E. faecium, 4 E. faecalis, and 2 E. casseliflavus) carried the tet(M) gene.High-level resistance to streptomycin and kanamycin was also detected in some of the isolates; 15 of 27 streptomycinresistant strains carried the ant(6)-Ia gene, and 27 of 28 kanamycin-resistant strains carried the aph(3')-IIIa gene.The cat(A) gene was found in four chloramphenicol-resistant E. faecium isolates.No acquired vancomycin resistance was demonstrated among the enterococci recovered in SB agar plates not supplemented with vancomycin, although 6 E. casseliflavus isolates were obtained (with intrinsic lowlevel vancomycin resistance,with the vanC2gene).
The esp gene, usually associated with nosocomial isolates, was detected by PCR in only 5 strains isolated from inanimate surfaces, all of them identified as E. faecium.These 5 esp-positive E. faecium strains had the purK17 allele, indicating that they were not ascribed to clonal complex CC17.

Prevalence and characterization of VRE strains isolated in SB-VAN agar plates
To determine the potential carriage of VRE in the 300 samples taken from the environment of the hospital, the samples were inoculated in SB-VAN agar plates (supplemented with 8 μg/mL of vancomycin).4.These VRE isolates were recovered from different origins (12 from inanimate surfaces, 1 from the hand of a patient, and 1 from the hand of a healthcare worker), and from 6 different services of the hospital (Table 4).All the VRE isolates showed the same multiresistance phenotype to erythromycin and tetracycline as well as high-level resistance to streptomycin and kanamycin, and harbored erm(B), tet(M), ant(6)-Ia, and aph(3')-IIIa genes.The VRE isolates showed resistance to ampicillin and ciprofloxacin, but they were susceptible to high dose of gentamicin.All of them carried the IS16 element.The gene encoding esp was detected by PCR screening in all VRE isolates, but none of them contained the hylEfmvirulence gene (encoding a hyaluronidase-like protein).

Analysis of clonality of vanA-containing E. faecium isolates
The clonal relatedness of isolates was assessed using PFGE and MLST.The macrorestriction analysis of genomic DNA in the 14 vanA-containing E. faecium isolates revealed two different profiles: 13 isolates showed the profile S and 1 isolate the profile A (Table 4) (Figure 1).

Discussion
In this study, VRE isolates were detected in 14 of the 300 (4.7%) environmental samples at the hospital setting studied.All VRE isolates obtained were E. faecium and contained the vancomycin resistance vanA gene.In Tunisia, the presence of VRE in hospitals is scarcely reported and there are only two previous reports of detection of vanA-containing enterococci [6,12].VRE was first isolated in this country in 2007 [12], and it still appears to be uncommon [6].Nevertheless, since VRE could be an emerging pathogen, further studies of VRE infection and colonization are important.Nowadays, acquired vanAtype vancomycin-resistant E. faecium are widely prevalent in hospitals around the world [21],and this situation could be similar in the future in Tunisia if appropriate control measures are not adopted.
According to previous studies, the E. faecium species was the most prevalent among VRE clinical isolates [22,23].The gene vanA has also been detected in E. faecalis by other authors [18,24].Thirteen of our vanA-positive isolates were ascribed by PFGE and MLST to a novel clone (new ST910) and only one strain was typed as ST80 and belonged to CC17.In a previous study performed by our group, three different PFGEtypes (A-C) and two sequence types (ST18 and ST80) were identified, a clone PFGE-A/ST80 being detected both among clinical and environmental vanA E. faecium isolates during 2012 in the same hospital [6].It is of interest that profile A detected in one of our isolates was indistinguishable from the profile A previously detected (with the same ST80/CC17) [6], indicating that this clone is still present in the environment of the hospital.All remaining vanA-positive isolates belonged to a new clone, ST910/PFGE-S.These strains were vancomycin and ampicillin resistant, had purK (1), the esp gene, and all of them contained the insertion element IS16.All these characteristics are markers of nosocomial clones belonging to CC17.However, according to e-BURST, this ST did not belong to CC17, but was a double locus variant of ST80, from which it could be derived.The appearance of this new clone in the studied hospital could be an alert of the emergence and rapid evolution and of these clinically important resistant bacteria and the necessity for active surveillance in other hospitals and regions of the country.
In several recent studies carried out in different countries around the world (including Tunisia), VRE outbreaks seem to be related to monoclonal spread [6,25,26].The homogeneity detected among our acquired vancomycin-resistant E. faecium isolates could be explained by the endemic dissemination of these VRE strains in the hospital.We found VRE in six different services of the hospital.The dissemination of vanA-containing E. faecium strains in different areas of the hospital might be due to direct contact with colonized patients or even to indirect contact via the hands of healthcare workers [4,27].The esp expression on the surface of E. faecium seems to play a role in adherence and biofilm formation [28].This gene could have a great role in the exchanging genetic mobile elements and could also be implicated in the adaptation of these bacteria to the hospital environment [29,30].Remarkably, our vanA E. faecium isolates presented a multiresistance phenotype, being resistant, in addition to glycopeptides, to erythromycin, tetracycline, ciprofloxacin, ampicillin, kanamycin, streptomycin, pristinamycin, and trimethoprimsulfamethoxazole.In the case of resistance genes erm(B) and tet(M), it is interesting to mention that they have been previously detected in the same conjugative plasmid as the van(A) gene, which indicates that coselection processes might have happened [31].Our VRE strains were still susceptible to high dose of gentamicin, similar to what was found in a previous study reported in Tunisia showing the emergence of epidemic CC17 clones of vanA E. faecium that were also sensitive to high dose of gentamicin [6].On the other hand, the detection of enterococcal species (in non-supplemented media) in one-third of samples tested (inanimate surfaces of hospital and in the intact skin hand of some patients and healthcare workers) can be explained by the fact that these microorganisms are able to survive in the environment outside the host for a long period of time and are able to resist routine cleaning [32].The species more frequently detected, E. faecium and E. faecalis, are implicated in most enterococcal infections [33,34].In fact, environmental contamination may contribute to transmission of pathogens by contact of medical equipment or patients with contaminated surfaces [35].
The wide dissemination of antibiotic resistance among our enterococci isolates could be partly due to the horizontal transfer of mobile genetic elements between strains and, on the other hand, to crosstransmission of multiresistant enterococci between patients, staff, and the environment [5].

Conclusions
The dissemination of multi-resistant bacteria, including VRE of genotype vanA, in the hospital has become particularly problematic.An active surveillance program should be carefully prepared to stop this uncontrollable evolution and emergence of these resistant bacteria.Further studies are needed and strict infection control strategies should be adopted in hospital environments to reduce potential infections by these microorganisms.

Table 1 .
Prevalence of Enterococcus strains in the hospital environment (isolated in Slanetz-Bartley medium not supplemented with antibiotics).

Table 2 .
Antimicrobial resistance of enterococci strains isolated in Slanetz-Bartley medium not supplemented with antibiotics.were recovered from 14 of these 300 samples (4.7%).The 14 VRE strains (one per positive sample) were identified as E. faecium and carried the van(A)gene, showing high-level vancomycin and teicoplanin resistance (MIC > 256 μg/mL).The characteristics of the 14 vanA-containing E. faecium isolates are shown in Table * 6 E. casseliflavus isolated in Slanetz-Bartley not supplemented with vancomycin showed a low level resistance to vancomycin (intrinsic resistance conferred by the vanC2 gene).VRE

Table 3 .
Resistance genes among enterococci isolated in Slanetz-Bartley medium not supplemented with antibiotics.