Distribution of vancomycin resistant enterococci and their resistance patterns determined by surveillance

In order to identify the status of vancomycin resistant enterococi (VRE) carrier in our hospital, periodical rectal swab cultures were obtained from the patients hospitalized in the Pediatrics Unit, Neurosurgery Intensive Care Unit and Reanimation Unit. VRE strains isolated were examined for type identification, antibiotic sensitivity, High Level Gentamicin Resistance (HLGR), High Level Streptomycin Resistance (HLSR), beta-lactamase production and genotypic resistance patterns. Rectal swab cultures were obtained from 250 patients and 38 of these (15%) were VRE positive. 28 (73.6%) of the enterococci were Enterococcus faecium, 6 (15.8%) were Enterococcus casseliflavus, 3 (7.9%) were Enterococcus gallinarum and 1 (2.7%) was Enterococcus faecalis. 24 strains were identified to have Van A resistance pattern. None of the strains have beta-lactamase. HLGR was identified at a rate of 92% and HLSR at a rate of 95%. In addition to glycopeptide resistance, VRE strains had high levels of Ampicillin, Penicillin, Erythromycin, Rifampicin, Chloramphenicol and Nitrofurantoin resistance. Quinolon resistance was found to be at moderate level (34%, 45%), while Tetracycline (29%), Fosfomycine (6%), Dalfopristinquinupristin (3%) and Linezolid (0%) were found to be the most effective antimicrobials.


INTRODUCTION
Vancomycin resistance in Enterococci was first described in 1988 and then resistant strains became widespread worldwide (Uttley et al., 1998).Asymptomatic VRE colonization can easily lead to infections.VRE infections are important causes of mortality and morbidity and their treatment is expensive (Milestone et al., 2010).Early identification of VRE colonization is important for the control of VRE infections.The most important VRE reservoirs are those patients who carry VRE in their gastrointestinal systems (Robert et al., 2005).If surveillance cultures are not performed on patients having high risk, asymptomatic carriership can easily be missed.Standard culture methods and molecular techniques like PCR are important for the identification of VRE colonization and prevention of outbreaks.
The first VRE strain in our hospital was isolated in December 2004 from the urine of a patient hospitalized in our Pediatrics Unit.Considering the possibility of an outbreak, Infection Control Committee was alarmed.By putting into place an active surveillance program, rectal  swab cultures were obtained from patients under high risk.As anticipated, these patients had a considerably high rate of rectal carriership (15%) in one year surveillance cultures.For the VRE strains isolated, type level identification, antibiotic sensitivity, high level amino-glycoside resistance (HLAR), high level streptomycin resistance (HLSR), beta-lactamase production and genotypic resistance patterns were investigated.

MATERIALS AND METHODS
During the 8-month period from December 2004 to July 2005, VRE carriers' status was investigated by obtaining rectal swab cultures from the patients in the Pediatrics Unit, Neurosurgery Intensive Care Unit, Reanimation Unit and Hematology Units within the first 72 h of their hospitalization.This was followed by a weekly swab during the course of their hospitalization.Rectal swab samples were obtained with sterile swabs.The materials were transferred onto VRE agar medium (OXOID) containing 6 μ/ml Vancomycin and 1 μg/ml Meropenem; type level identification was performed with classical methods and Rapid ID 32 STREP kit (Biomerieux) on the gray-black colonies that were formed after 24-48 h.The identification of antibiotic sensitivities and HLAR was based on CLSI recommendations (CLSI, 2005) and was performed with agar dilution method and ATB Enterocci 5 (Biomerieux) kit.Antibiogram sensitivities to Fosfomycine and Linezolid were studied with disc diffusion method.The confirmation of the strains identified as Vancomycin resistant was performed by E test (AB Biodisk Sweden) with the identification of MIC values as well as identification of resistance genes by Multiplex PCR (Table 1).
A single bacterial colony was taken overnight and grown on a blood agar plate which was suspended in a 100 μl of a PCR mixture containing distillated water (65.5 μl), 10 mM PCR Buffer (10x), 50 mM MgCl2, 0.2 mM dNTP ( dATP, dCTP, dGTP,dTTP), 0.5 μm primer and 25 U Taq DNA polymerase.Thermocycler conditions were as follows: an initial cycle of 94°C for 5 min lysis and denaturation followed by 30 cycles; at 94°C for 30 s denaturation, at 58 C for 30 seconds primer binding, at 72°C for 30 s elongation of primer; at the end of 30 cycles at 72°C for 10 min: all tubes were put in the thermocycler and at the end of 30 cycles, PCR products were analyzed by electrophoresis on a 1.5 % agarose gel with etidium bromide and on a UV transilluminator (Figure 1).Betalactamase activity was analyzed with Nitrocefin discs (OXOID).Twenty four (24) strains could be studied for resistance genes with PCR which resulted in the identification of Van A type resistance pattern.All isolates exhibited resistance to vancomycin, teicoplanin, ampicillin, penicillin, erythromycin and rifampicin, which were all E. faecium.Clonal relationship among the isolates was not investigated.But all the strains were E. faecium, Van A pattern and had same antibiotic resistance.None of the investigated strains had beta-lactamase positivity.

DISCUSSION
First, VRE strains were reported in the United Kingdom in 1988 and this was immediately followed by reports from France and USA; but the spread was very fast (Uttley et al., 1989).The first VRE strain in our country was reported in 1998 from Akdeniz University and this was followed by other case reports (Kocagöz et al., 1999).In these cases, most of the strains studied for phenotype were reported as E. faecium with Van A resistance pattern (Başustaoğlu et al., 2000;Çetinkaya et al., 2004;Gündeş et al., 2002;Yiş et al., 2011;Ergani et al., 2008).In the year 2000, in a multicenter study done in Europe on 4208 enterococcus isolates, it was determined that prevalence of Van A and Van B phenotype for Turkey was about 1-2% (Schouten et al., 2000).In the studies reported abroad, the isolated strains also had Van A resistance pattern (Song et al., 2009).Of the 38 strains isolated in our hospital, resistance genes were investigated in 24.And similar to the findings of the previous studies, all of the samples were found to be E.faecium with Van A resistance pattern.
Different studies report different rates of VRE colonization and infections.Harris et al. (2004) covered 42 surgical intensive care units in their study; of the 1362 cases they studied, they reported VRE colonization in 136 (10%).In the studies reported from the USA, rectal VRE colonization rates in the intensive care units are reported as 6-20% (van den Braak et al., 2000;Song et al., 2009;Kim et al., 2012).In the studies done in Pediatric ICUs, rectal carriership rates are reported as 2-5% (Milestone et al., 2010;Gray et al., 2000).In our study, we had 15% rectal colonization rate, which is similar to high colonization rates of VRE.
Of the enterococci strains, E. faecalis is the most commonly identified strain as the cause of infections.E. faecium, on the other hand, is more resistant to antimicrobials compared to E. faecalis and can spread easily clonally, so it is accepted as a more important pathogen (Arias et al., 2008).In recently performed studies, E. faecium is being isolated more.E. faecalis to E.faecium rate was 3/1 in 2002 while it was reported as 1.2/1 in 2006 (Lester et al., 2008).In our study, for enterococci colonization, E. faecium had a higher rate than E. faecalis.Of the 38 VRE strains isolated, 28 (73%) were defined as E. faecium.Song et al. (2009) identified 33 E. faecium and 1 E. faecalis in their study.
The recommended treatment for VRE infections is the combination of an aminoglycoside with Penicillin, Ampicillin or Amoxicillin.However, Vancomycin resistant strains are usually resistant to other antibiotics as well (Harris et al., 2004).The widespread existence of HLAR is a significant problem in the treatment of enterococcal infections.HLAR eliminates the synergistic effects of the beta-lactame+aminoglycoside combination used for treatment and results in treatment failures (Agarwal et al., 2009).In our study, all the VRE strains were found to be resistant to Penicillin, Ampicillin, Erythromycin and Rifampicin.Furthermore, HLGR was identified as 92% and HLSR as 95%.Resistance to Chloramphenicol was 76%; to Nitrofurantoin, 75%; to Ciprofloxacin, 45%; and to Levofloxacin, 34%.High level aminoglycoside resistance is 28-44 % in the South American hospitals (Panesso et al., 2010).Based on EARRS data, it was 15.4 -50 % in Europe.26 European countries reported 6950 isolates of which 2484 had high level resistant to aminoglycosides ( EARS-Net, 2009).High level aminoglycoside resistance is 30-60% in the USA and is reported to be most frequent in E. faecium (Taşova, 2009;EASAC reports, 2007).In a study by Çetinkaya et al. (2004) on 49 strains, all the VRE strains that were isolated within the scope of the surveillance program at Hacettepe University Hospital were identified to have Teicoplanin and Ampicillin resistance, as well as HLGR and HLSR.Nitrofurantoin resistance was identified as 95.7%, Rifampicin resistance as 78.7%, Tetracycline resistance as 55.3% and Chloramphenicol resistance as 17.7%.The rate of Tetracycline resistance was higher than that we identified.Similar to our study, beta-lactamase positivity was not identified.Decrease of PBP or production of beta-lactamase by enterococci is responsible for the resistance to betalactame antibiotics.That is why beta-lactamase activity should be analyzed in enterococci (Robert et al., 2005).In a study by Gordon et al. (1992) on 705 strains, betalactamase positivity of enterococci was reported as 1.6%.In the study by Agarwal et al. (2009), only one out of 86 enterococci was identified to have beta-lactamase positivity.
All the strains in our study were found to be sensitive to Linezolid.In a study in Canada, from January 2010 to June 2012, of 2829 enterococcal isolates tested, 12 E. faecium were found to be resistant to linezolid (Patel et. al., 2013).According to LEADER Surveillance Program 2011, activity of linezolid on enterococci is 99.7% (Flamm et al., 2013).In our study, resistance rate to Fosfomycine was identified as 6%.However, the use of Fosfomycine is only recommended for E. faecalis strains (Robert et al., 2005).Dalfopristin-Quinupristin is also effective for E. faecium strains.In a study in the USA performed by E test, 87% of the strains were found to be sensitive (Lamb et al., 1999).In our study, E. faecium constituted 73.6% of the VRE strains and the sensitivity rate was found to be high.

Table 1 .
Amplification of Van A, Van B, Van C genes by PCR: Primer series for Multiplex PCR method.

Table 2 .
Resistance rates to antibiotics.All the isolated VRE strains were found to be resistant to Teicoplanin, Ampicillin, Penicillin, Erythromycin and Rifampicin.Antibiotic resistance rates are shown in Table2.35 isolate (92%) had HLGR and 36 (95%) isolates had HLSR.Of the 38 colonized patients, none developed infections.