Curr Microbiol (2011) 62:1363–1367 DOI 10.1007/s00284-011-9868-6
Vancomycin-resistance Transferability from VanA Enterococci to Staphylococcus aureus Simona de Niederha¨usern • Moreno Bondi • Patrizia Messi • Ramona Iseppi • Carla Sabia Giuliano Manicardi • Immacolata Anacarso
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Received: 6 July 2010 / Accepted: 4 January 2011 / Published online: 15 January 2011 Ó Springer Science+Business Media, LLC 2011
Abstract In last decade methicillin-resistant Staphylococcus aureus with high level of vancomycin-resistance (VRSA) have been reported and generally the patients with VRSA infection were also infected with a vancomycinresistant Enterococcus (VRE). Considering that the high level of vancomycin-resistance in VRSA isolates seems to involve the horizontal transfer of Tn1546 transposon containing vanA gene from coinfecting VRE strains, the authors have studied the ‘‘in vitro’’ conjugative transfer of this resistance from VanA enterococci to S. aureus. Out of 25 matings performed combining five vancomycin-resistant enterococci as donors (three Enterococcus faecalis and two Enterococcus faecium), and five S. aureus as recipients, all clinical isolates, two have been successful using E. faecalis as donor. The transfer of vancomycin-resistance was confirmed by vanA gene amplification in both transconjugants and the resistance was expressed at lower levels (MIC 32 lg/ml) in comparison with the respective VRE donors (MIC [ 128 lg/ml). The vancomycin-resistance of trasconjugants was maintained even after subsequent overnight passages on MSA plates containing subinhibitory levels of vancomycin. This study shows that the vanA gene transfer can be achieved through techniques ‘‘in vitro’’ without the use of laboratory animals employed, in the only similar experiment previously carried out by other authors, as substrate for the trasconjugant growth. Moreover, in that previous experiment, contrary to this study, the vancomycin resistant S. aureus trasconjugants were selected on erythromycin agar and not by direct vancomycin agar selection.
S. de Niederha¨usern M. Bondi (&) P. Messi R. Iseppi C. Sabia G. Manicardi I. Anacarso Department of Biomedical Sciences, University of Modena and Reggio Emilia, Via Campi 287, 41125 Modena, Italy e-mail:
[email protected]
Introduction Staphylococcus aureus, a major cause of community and hospital-acquired infections, has developed resistance to many antimicrobial agents and often glycopeptides such as vancomycin remained one of the most effective drugs to treat the methicillin-resistant S. aureus (MRSA) infections. Unfortunately, in the last decade, MRSA strains with reduced susceptibility to vancomycin have been reported with increasing frequency and the appearance of this resistance can make treatment of S. aureus infections increasingly difficult. As defined by CLSI (2008) [3], MIC breakpoints for the vancomycin intermediate resistance and vancomycin resistance were 4–8 and C16 lg/ml, respectively. The first appearance of S. aureus isolates with MIC 8 lg/ml for vancomycin (vancomycin-intermediate S. aureus, VISA), from Japan in 1997, was followed by many more cases of VISA reported globally. The first vanA-mediated vancomycin-resistant S. aureus (VRSA) (MIC [ 128 lg/ml), was isolated in a Michigan hospital in 2002 [2]. Thereafter, eleven VRSA have been recovered in the United States and confirmed by the CDC [1]. Anyway in literature are reported other VRSA out of USA such as the strain isolated from a Kolkata hospital in June 2005 [15]. In the majority of the cases, the patients with VRSA infection were also infected with a vancomycin-resistant Enterococcus (VRE) isolated at the time of VRSA isolation [20]. The high level of vancomycin-resistance in VRSA isolates therefore seems to involve the horizontal transfer of the Tn1546 transposon containing the vanA gene, frequently harboured in a plasmid belonging to the Inc18 plasmid family [19, 20], from coinfecting VRE strains. Weigel et al. [18] and Severin et al. [16] in two different experiments demonstrated the ‘‘in vitro’’ vancomycin-resistance transfer
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from clinical VRSA to the MRSA strain COL, reinforcing concerns about potential widespread of vancomycinresistance. In previous studies [4] the authors have documented that enterococci are an important source of glycopeptide resistance genes for Listeria monocytogenes via the transfer of movable genetic elements, confirming that the Tn1546 is carried by wide host-range plasmids able to overcome the species barrier. To the knowledge, the only report in literature on artificial vancomycin-resistance transferability [10] was obtained selecting the trasconjugants on erythromycin agar, result not obtained by direct vancomycin agar selection. Aim of this work was the study of the conjugative transfer ‘‘in vitro’’ of vancomycin-resistance from Enterococcus faecalis and Enterococcus faecium VanA to S. aureus by direct vancomycin selection to understand if this event can easily occur in ideal conditions.
Materials and Methods Bacterial Strains Three Enterococcus faecalis (EMB04 from urethral swab, EMM09 and EMB10 from urine cultures) and two Enterococcus faecium (EMB07 from surgical wound and EMB12 from urine culture), all containing a plasmid harbouring the vanA gene cluster and already employed in previous studies [4, 14], were used as donors in the mating experiments. The recipients were five Staphylococcus aureus (STM17 and STM384 from blood cultures, STM359, STM594 and STM653 from skin infections) resulted plasmid free by analysis of extrachromosomal DNA (data not show). Selection of S. aureus Rifampicin-Resistant Mutants S. aureus strains rifampicin-resistant mutants were selected so as to use rifampicin as selective agent for VanA transconjugants in the mating experiments. A single colony of each S. aureus was streaked on gradient plates containing rifampicin (1–128 lg/ml), and colonies growing on the maximum drug concentration were isolated and confirmed by plating on the highest rifampicin concentration.
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Laboratories, Detroit, MI). After an incubation of 6 h at 37°C, mating was carried on spreading 2 ml of parental strains on a Tryptic Soy agar (TSA, Difco) plate surface (90 mm diameter). The plates were incubated in slight movement on a platform shaker for 5 hours at 37°C and after a further 24 h incubation at the same temperature, the overlying lawn was removed, placed in 5 ml of fresh TSB and incubated for 12 h at 37°C. Transconjugants were selected spreading 100 ll of this TSB culture on a Mannitol Salt agar (MSA, Difco) plate supplemented with the selective agents, rifampicin 64 lg/ml and vancomycin 16 lg/ml (Sigma Chemical Co., St. Louis, MO, USA) and incubated for 24 h at 37°C. The Rifr Vanr colonies of the putative S. aureus transconjugants were isolated and identified by API Staph system (bioMerieux, Marcy l’Etoile, France). Minimum Inhibitory Concentration (MIC) The minimum inhibitory concentration for the putative transconjugants and the respective parental strains against vancomycin, rifampicin, erythromycin, oxacillin (all from Sigma Chemical Co.) was evaluated, by agar dilution method, according to the NCCLS (2003) [9]. To test the stability of vancomycin-resistance, the trasconjugants were passaged daily, for 14 days, on MSA containing 16 lg/ml vancomycin and then the MIC was determined. Plasmid DNA Analysis and vanA Gene Amplification To compare the plasmid profile of parental strains and putative transconjugants, small-scale plasmid isolation was performed [11]. DNA plasmid was analysed in 0.7% agarose gel electrophoresis at 3.5 V/cm for 8 h in a Tris-acetate buffer. Purified plasmids of Escherichia coli V517 [7], were used as size reference plasmids for molecular weight determinations. Total DNA of tranconjugants, donors and recipients, was subjected to amplification assay employing the oligonucleotide primers for the vanA gene A1 (50 -GGGAAAA CGACAATTGC-30 ) and A2 (50 -GTACAATGCGGCCG TTA-30 ) and for the ddlE.faecalis gene E1 (50 -ATCAA GTACAGTTAGTCT-30 ) and E2 (50 -ACGATTCAAAGCT AACTG-30 ) [5].
Results Mating Experiments Mating Experiments Twenty-five distinct mating experiments were performed by combining the five VRE (Vanr Rifs) as donors, and the five S. aureus rifampicin-resistant mutants (Vans Rifr) as recipients. For each conjugation, 1 ml overnight cultures of both donors and recipients (ratio of one donor to one recipient) were mixed in 5 ml fresh Tryptic Soy broth (TSB, Difco
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Out of 25 matings performed, vancomycin-resistance was successfully transferred in two conjugations subsequently confirmed. In particular, the transfer occurred from E. faecalis EMM09 to S. aureus STM359 and from E. faecalis EMB04 to S. aureus STM17. In both cases the
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plasmid of donor harbouring the vanA gene not belongs to the Inc18 plasmid family (data not show). Other authors also [19] reported that Inc18-like vanA plasmids are not the only plasmids capable of transferring vancomycin resistance from Enterococcus spp. to S. aureus. In MSA plates supplemented with selective agents, multiple colonies, representing clonal twins derived the same original transconjugation event, have been observed. No transconjugants were obtained employing the other VRE, and in particular E. faecium strains, as donor. Minimum Inhibitory Concentration (MIC) Both E. faecalis donors (EMM09 and EMB04) showed a high-level resistance towards vancomycin (MIC [ 128 lg/ ml) and were resistant to erythromycin (MIC [ 128 lg/ml), while were susceptible to oxacillin and rifampicin. The S. aureus recipients (STM359 and STM17), rifampicinresistant mutants, have high MIC values for rifampicin ([128 lg/ml) and lower for erythromycin than enterococci (MIC 1 and 4 lg/ml for STM359 and STM17, respectively). In addition S. aureus STM17 was resistant to oxacillin (MIC [ 128 lg/ml). When the vancomycin resistance of both S. aureus transconjugants (STM359-EMM09 and STM17-EMB04) was evaluated, the resistance was expressed at lower levels (MIC 32 lg/ml) in comparison with the respective VRE donors (MIC [ 128 lg/ml). For the other antibiotics, transconjugants showed the same MIC of the parental S. aureus. The two transconjugants continued to grow even after fourteen overnight passages on MSA plates containing subinhibitory levels of vancomycin. This outcome indicated a stable transfer of vancomycin-resistance from clinical E. faecalis donors EMM09 and EMB04 to S. aureus STM359 and STM17. Plasmid DNA Analysis and vanA Gene Amplification By plasmid DNA analysis it was observed in S. aureus STM359-EMM09 transconjugant the presence of a 53 kb band corresponding to the higher molecular weight plasmid of E. faecalis EMM09 donor (Fig. 1). No plasmids were recovered in S. aureus STM17-EMB04 transconjugant, however, the transfer of the vanA gene was confirmed by PCR amplification in both S. aureus recombinants (Fig. 2a). The absence of the amplification of the ddlE.faecalis fragment using the S. aureus transconjugants DNA as template, ruled out a contamination of enterococci donors (Fig. 2b).
Discussion The main concern about the vanA cluster is its capability of transfer from vancomycin-resistant enterococci to more
Fig. 1 Plasmid profiles of E. faecalis EMM09 and S. aureus STM359 (parental strains) and of S. aureus STM359-EMM09 (transconjugant). The acquired vancomycin-resistance in S. aureus T359-EMM09 transconjugant was revealed by the conjugative plasmid band of 53 kb. Lane 1 molecular size marker E. coli V517. Lane 2 S. aureus STM359 (recipient). Lane 3 E. faecalis EMM09 (donor). Lane 4 S. aureus STM359-EMM09 (transconjugant)
pathogenic organisms. As the nosocomial VRSA until now confirmed by CDC were generally recovered in association with vancomycin-resistant enterococci, an horizontal transfer of the Tn1546 containing vanA and correlated genes from VRE to S. aureus is probably involved. In 1992 vanA gene was co-transferred by Noble experiments [10] from VRE to S. aureus selecting transconjugants on agar containing rifampicin plus erythromycin, but the authors have not achieved the same result using vancomycin as selective agent. Moreover Noble and co-authors have not obtained encouraging results by filter mating and the success of their recombination was achieved using a non-traditional method that completely differs from the study. They claimed to have obtained their results by a transfer ‘‘in vivo’’ placing the staphylococcus and enterococcus parental strains on the skin of a hairless mouse obese. This study shows that the vanA gene transfer from enterococci to staphylococci, although remaining an event that can never be taken for granted, can be achieved through techniques ‘‘in vitro’’ without the use of laboratory animals, unlike previously reported [10]. Indeed, the conjugation experiments have confirmed only in two of 25 attempts the capability of the vancomycin-resistance transfer by ‘‘in vitro’’ mating. The employ of our easier procedure could be advantageous to future researchers
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Fig. 2 a, b PCR amplification of vanA a and ddlE.faecalis. b genes. a Lane 1 100 bp DNA ladder. Lane 2 S. aureus STM359-EMM09 transconjugant. Lane 3 E. faecalis EMM09 (positive control). Lane 4 S. aureus STM359 (negative control). Lane 5 S. aureus STM17EMB04 transconjugant. Lane 6 E faecalis EMB04 (positive control). Lane 7: S. aureus STM17 (negative control). b Lane 1 100 bp DNA
ladder. Lane 2 S. aureus STM359 (negative control). Lane 3 E. faecalis EMM09 (positive control). Lane 4 E. faecalis EMB04 (positive control). Lane 5 S. aureus STM17 (negative control). Lane 6 S. aureus STM17-EMB04 transconjugant. Lane 7 S. aureus STM359EMM09 transconjugant
increasing the methodological efficiency for studying the basic mechanisms of inter-species vancomycin-resistance transfer. In the S. aureus STM17-EMB04 transconjugant the electrophoresis analysis not revealed plasmid bands. This result could be do to two sequential genetic events, the transfer of the conjugative plasmid followed by excision and insertion of the transposon Tn1456 into S. aureus chromosomal DNA. Although the presence of the vanA gene was confirmed by PCR assay in both the transconjugants, the vancomycinresistance was expressed at lower levels in comparison with the VRE donors. As already reported by other authors for VRSA nosocomial isolates [12], a genetic instability or a long delay for induction of resistance by vancomycin could be responsible for the lower levels of resistance observed in the S. aureus STM17-EMB04 and STM359EMM09 transconjugants. Although only two S. aureus transconjugants were studied, considering the lower levels of vancomycinresistance expressed, these results suggest that, towards conjugation with VRE, in nosocomial infections could emerge VRSA that can pass by unnoticed as in clinical laboratories using automated methods detection of both VISA and VRSA has proved difficult [6, 8, 13]. Indeed the changes that produce the increased MICs are not readily revealed by the analyses and interpretation algorithms that are employed in many automated systems [17]. The lack of identification could delay the application of suitable hygiene practices to avoid spread of these strains.
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