Local spread of Tn1546-like element among three species of vancomycin resistant enterococci in an intensive care unit

Introduction: In an intensive care unit (ICU) of the Emergency Center in the Clinical Center of Serbia, four species of vancomycin resistant enterococci (VRE) were isolated in a 17-month period mostly from blood cultures, including E. faecalis, E. faecium, E. raffinosus and E.gallinarum. Methodology: The relationship between isolates from each species was investigated by PFGE, and PCR experiments for detection of pathogenicity factor genes and van genes to determine the nature of each clone. A PCR-based method, using 10 primer pairs (p1/2-p19/20), was used to investigate the presence of the Tn1546-like structure. Results: PFGE indicated the presence of two different E. faecium clones, while the three other enterococcal species belonged to one clone each. Transposon typing revealed that isolates of E. raffinosus (4), E. gallinarum (4) and E. faecalis (3) yielded gene sequences identical to 10 primer pairs (p1/2-p19/20), suggesting the possibility of identical transposon-like structure in these species. Conclusions: The results of the study indicate probable horizontal spread of Tn1546-like structure in three species of VRE obtained from the same ICU.


Introduction
VRE displaying the VanA and VanB resistance phenotype have been reported widely as a cause of nosocomial infections in the United States and Europe [1][2][3]. The genes encoding the VanA and VanB types of vancomycin resistance are often located on mobile DNA elements and are therefore easily transmitted between members of the same species or genus or even different genera [4][5][6]. For that reason, the horizontal transfer of resistance genes among enterococci may have a more significant impact on the dissemination of vancomycin resistance than the clonal spread of resistant enterococci. The vanA gene is the most frequently encountered gene among isolates causing VRE infections in humans [6]. This gene is part of the transposable element Tn1546, cluster consisting of genes: orf1 and orf2, associated with transposition functions; vanR and vanS (regulatory genes); vanH, vanA and vanX (critical for resistance determination) and vanY and van Z (accessory genes) [7]. Previous authors developed a set of 10 primers (p1p2-p19p20), encoding overlapping fragments of prototype vanA element, to investigate the content of the element. These investigations documented genetic heterogeneity in Tn1546-related elements [8,9].
In this study, in addition to investigation of resistance elements, we therefore investigated the content of virulence factor genes. Virulence factors of interest include those that promote binding to extracellular matrix proteins, like collagen binding protein Ace and cell wall adhesins expressed in serum by E. faecalis and E. faecium, encoded by ace, EfaAfs and EfaAfm genes, respectively; adaptation and survival in different environments, such as Enterococcus surface protein Esp and aggregation substance AS, encoded by esp and asa1 genes; factors that enable secretion of toxins (e.g. cytolysin -production regulated by an operon which includes cylA and cyl B genes) and enzymes that damage and degrade host tissue (gelatinase and hyaluronidase, encoded by gelE and hyl genes). Sex pheromone genes (cpd, cob) are part of a complex system of gene transfer by conjugation controlled by intercellular signalling via peptide mating pheromones.
E. faecium and E. faecalis are well known and frequent human pathogens, which account for 12% of healthcare-associated infections [1]. Outbreaks of other enterococci, E. raffinosus and E. gallinarum [2,10,11], have been reported less frequently than the two most frequently encountered species. This study therefore also investigated the presence of Tn1546 in four species of enterococci, taking into account that they were isolated at the same ICU of the Emergency Center at the Clinical Center of Serbia (ICUEC) in a relatively short period. There is very little information from earlier studies on molecular epidemiology specific to the transposon itself, or its association with specific antimicrobial resistance or virulence traits. Our hypothesis was that the same genetic element carrying vanA resistance gene could have been shared among these different species of enterococci. Having an understanding of epidemiology is critical in designing control measures in intensive care units.

Methodology
The study was carried out between May 2005 and October 2006. It was focused on patients hospitalized at ICUEC, also called "intensive care unit for septic patients". It is intended for the patients already hospitalized in other intensive care units, either in the Clinical Center of Serbia or other healthcare institutions, whose status became septic and critical. In most of the patients abdominal operations had been performed, but some were suffering from traffic injuries. Blood cultures were incubated in semiautomatic blood culture system BACTEC 9240 (Becton Dickinson Diagnostic Instrument Systems, Sparks, USA) and subsequently cultivated on blood agar. Isolate of vancomycin resistant enterococcus from vaginal swabs, provided from colleagues from Bacteriology laboratory at Emergency Center, Department of Microbiology, was included in the study. VRE were identified to the species level by conventional tests [12].

PFGE
Genomic DNA for PFGE was prepared in agarose plugs and digested with SmaI (New England Biolabs, Beverly, USA) enzyme [14]. PFGE was performed on a CHEF-DRIII (Bio-Rad, Hercules, USA). SmaIdigested Staphylococcus aureus NCTC 8325 was a molecular mass standard on all PFGE gels. Identical fragment patterns were considered the same type while those with two or three band differences resulting from a single genetic event [15] were considered closely related and assigned as subtype. The different types or subtypes were identified by numbers and small-letter codes for E. faecium or capital-letters with number added (for subtype) for other enterococci.

Vancomycin resistance and pathogenicity factor genes
Bacterial DNA in this study was extracted by use of InstaGene Matrix (Bio-Rad Laboratories, Hercules, USA), as suggested by manufacturer. Mutiplex PCR investigation of vanA and vanB genes was performed according to the method of Dutka-Malen [16]. PCR experiments for detection of pathogenicity factor genes: esp, cylA, cylB, efaAfs, efaAfm, cpd and cob were performed as described previously [17]. Briefly, PCR was performed in a GeneAmp PCR System 9600 (PerkinElmer Life and Analytical Sciences, Wellesley, USA). PCR amplifications were performed in 50-μl reaction mixtures using 5 μg of DNA, 15 mM MgCl2, 20 pmol of each primer, and 1 U of Taq DNA polymerase (Invitrogen, Carlsbad, USA). Samples were subjected to an initial cycle of denaturation (94°C for 2 minutes), annealing (at an appropriate temperature for 2 min and elongation (72°C for 2 minutes), followed by 29 cycles of denaturation (92°C for 15 seconds), annealing (at an appropriate temperature for 15 seconds), and elongation (72°C for 15 seconds). A multiplex PCR for the pathogenicity factor genes: asa1, gelE, and hyl was carried out as previously described [18]. In brief, Each 50-μl PCR mixture consisted of 5 μl of bacterial suspension; a 0.1 μM concentration (each) of primers specific for asa1,gelE, and hyl; 25 μl of Master mixture, which consisted of 2.5 U of Taq DNA polymerase, 1.5 mM MgCl2, and 200 μM deoxynucleoside triphosphates; and an additional 1.0 mM MgCl2. An initial activation step at 95°C for 15 minutes was followed by 30 cycles of denaturation (94°C for 1 minute), annealing (56°C for 1 minute), and extension (72°C for 1 minute), followed by one cycle consisting of 10 minutes at 72°C. PCR products were run on 2% agarose gels at 60V for 1.5 hours. A 100-bp DNA ladder (Invitrogen, Carlsbad, USA) was used as a molecular size marker.
Primers for detection of virulence and van genes are listed in Table 1.
Briefly, the primers (Table 2) were adjusted, and a touchdown PCR strategy was employed using Master mix. The initial denaturation step of 15 minutes at 95°C was followed by 10 cycles consisting of 30 seconds at 94°C, 30 seconds at 61°C, down to 51° C and 1 minute at 72°C, followed by 30 cycles of 30 seconds at 94°C, 30 seconds at 51°C and 1 minute at 72°C, with a final extension step of 7 minutes at 72°C. BM4147 was included as a positive control. The result was visualized by running the PCR products on a 1% agarose gel with 0.01% ethidium bromide.
The VanA element in each isolate was scored for the presence or absence of each of the 10 overlapping fragments of Tn1546 [7,8].

Results
In the study period 1156 blood cultures from ICUEC were processed. Bacterial isolates were  (Figure 1a), one pattern of E. gallinarum -clone G (Figure 1b), and two closely related patterns of E. raffinosus -clones R3 and R4 (Figure 1c), while members of E. faecium were placed in two different groups named 3b and 29, which included subgroup 29a. Multiplex PCR proved vanA gene in all VRE, whereas results for pathogenicity factor genes are displayed in Table 3. Amplification experiments showed that 4 E. gallinarum, 4 E. raffinosus and 3 E. faecalis carried a Tn1546-like element, given the amplification of all primers (p1p2-p19p20), constituents of Tn1546 (Table  4). The same table contains antimicrobial susceptibility patterns of the investigated strains. The isolates were found to be multidrug resistant, uniformly demonstrating resistance to penicillins, tetracycline and high concentrations of aminoglycosides. The antimicrobial resistance pattern was identical within each clone, with characteristic susceptibility of E. faecalis to ampicillin; clear susceptibility of E. gallinarum and E. raffinosus to ciprofloxacin and resistance of E. raffinosus to chloramphenicol. The order of appearance of the clones of enterococci containing the entire and defective Tn1546-like structure is presented on Figure 2. The study commenced in May 2005 and the first isolate containing vanA gene was E. raffinosus, obtained in July 2005. It was also the first clone to disappear, four months after. E. faecalis was detected in August 2005, a month after the appearance of the first E. raffinosus, and the last member of that clone was isolated in April 2006, eight months later. E. gallinarum persisted for seven months. Clones 29a and 3b persisted for four and five months. If Tn1546-like structure was common to three species  GGA TTT ACA ACG CTA AGC C  GCC TTT ATC AGA TGC TAC C  p3/p4  GGT TTT CGA TTA TTG GAA G  TAA AAA TAA TAG AAC GCA TCG AAT AC  p5/p6  CTT GAA AGT CAC GGA ATG  GGT TAA CAC CAG CCA TTA C  p7/    of enterococci it seems that they persisted for 4-8 months.

Discussion
Glycopeptide resistant enterococci are important causative agents in bacteremias. According to Brown [19], most clinical isolates of Enterococcus spp., whether glycopeptide susceptible or resistant, represent colonization rather than infection, which is typically endogenous. Since the isolates from patients in this study were from blood cultures in critically ill patients in intensive care units, we considered the infections to be clinically significant [20].
Investigation of patients' blood cultures revealed four species, clonally spread, of enterococci carrying vanA gene. Although the most frequent causative agent in human infections is E. faecalis, outbreaks with different types of enterococci carrying vanA genotype have been previously reported [10,21]. One isolate of E. raffinosus, part of R4 clone, was obtained from vaginal swab, suggesting spread of that clone to other body parts, or that the other body parts were the source of vancomycin resistant E. raffinosus.
As vanA gene is the most frequently located on Tn1546, as a part of a plasmid, we investigated the presence of genes encoding ten fragments of Tn1546 in all members of enterococci from the outbreak (Table 3). Indeed, a total set of genes was present in all E. faecalis, E. raffinosus and E. gallinarum. Clonal relatedness of enterococci from the study was congruent with identical antimicrobial resistance within the clone. The entireTn1546-like element was likely identical or closely related to the original Tn1546 transposon detected by Woodford [8] and transmitted probably en bloc with other genes encoding high level resistance to aminoglycosides, erythromycin, and tetracycline in three species of enterococci. It remains unknown in which strain the Tn1546-like element appeared originally, since Tn1546 has a wide range of hosts, encompassing Enterococcus, Staphylococcus aureus, Staphylococcus epidermidis, Bacillus circulans, Oerskovia turbata, Arcanobacterium haemolyticum, Paenibacillus, Rhodococcus [7,22,23]. Generally, intra-species transfer of enterococcal vanA plasmids is far more frequent than transfer across species or genus barriers and transconjugants could also serve as additional donors, being capable of transferring resistance plasmids to further recipients [6]. Vancomycin resistance genes are sometimes physically linked with virulence determinants on the same plasmid [24]. Investigation of virulence determinants was previously shown to be important for E. faecalis and E. faecium; our study detected none of them in E. raffinosus and E. gallinarum. E. faecium contained only EfaAfm gene inherent to that species and hyl gene in clone 29. Instead, in the C clone of E. faecalis the presence of asa1 gene has been detected, encoding aggregation substance (AS), associated with vanA-containing pheromone-responsive plasmids in E. faecalis [25]. The clone contained pheromone genes (cpd, cob) as well. Previous mating experiments on transfer of vanA genes across species barriers revealed higher rates from E. faecalis donors to E. faecium recipients than vice versa [5,6], especially in presence of AS genes to facilitate conjugative exchange [5]. Pheromone induction results in increased transcription of numerous structural genes required for conjugation and consecutive transfer of resistance genes. According to this, it seems more likely that the source of vanA gene in our study was E. faecalis than E. raffinosus. E. faecalis clone persisted in the time when E. gallinarum clone emerged, which leaves the possibility for transfer of resistance genes to E. gallinarum as well.
In the study of Dahl [4] exconjugants containing a plasmid harbouring the vanA transposon Tn1546 persisted stably in vivo in the absence of glycopeptides for more than 20 days. Transposon stability would have been enhanced under the glycopeptide pressure, exerted to the isolates from our study, considering that the patients were in intensive care unit, receiving the high dozes of glycopeptide and carbapenem antibiotics. A period of 4-8 months when clones of enterococci from our study persisted seems to be the period of transposon stability.
Although results suggest horizontal spread of Tn1546-like element, which is usually located on the plasmid, only the complete sequencing of the plasmid genome would confirm the existence of entire Tn1546. It is necessary to determine and analyze the whole sequence of the plasmid to reveal the dynamics of the genes. This study was unable to make a direct impact on infection control measures to prevent the transmission of the strains or modify the antibiotic usage in that ICU. Finally, this study was performed in an eighteen month period and historical isolates were not tested. Further study is required to understand if there is ongoing transmission and how long the horizontal spread of the Tn1546-like element has been occurring.

Conclusion
In this study a case of polyclonal outbreak of at least three species of enterococci, E. faecalis, E. raffinosus and E. gallinarum, was described, in which horizontal transmission of entire Tn1546-like elementwasprobable. The event occurred in a 17-month period, after which the investigation was ceased. This event is an example of dissemination of single or related resistance determinants among related, but clearly different organisms in a single Intensive Care Unit.