Vancomycin and high-level aminoglycoside resistance in Enterococcus species

The aim of the study was to investigate vancomycin and high-level aminoglycoside resistance (HLAR) in Enterococcus species by phenotypic and genotypic methods. A hundred Enterococcus strains were included in the study. Antimicrobial susceptibilities of strains were investigated by automated system, betalactamase production was investigated by nitrocefin disks, vancomycin resistance and HLAR were investigated by gradient diffusion method (GDM) and disk diffusion method, respectively. For detection of vancomycin and high-level gentamicin resistance (HLGR) genes, polymerase chain reaction was used. Teicoplanin linezolid, vancomycin, ampicillin, penicillin are the most susceptible antibiotics and strains were detected not to produce beta lactamase. Vancomycin resistance was detected in ten isolates by automated system and in only five isolates by GDM. Five isolates carrying VanA gene were determined. The ratio of HLGR and high-level streptomycin resistance was found 40 and 63% respectively. aac (6’)-1eaph (2’’)-1a gene was detected in 58% of strains. E. faecium strains were found more resistant to the antibiotics than the other species. Beta lactamase was detected in none of strains. The automated system detected vancomycin resistance in more strains than GDM. Therefore it is concluded that strains, which were detected to be resistant to vancomycin, should be confirmed by GDM. The ratio of VanA gene in strains is consistent with other studies. The HLAR ratio was found in about half of strains. The ratio of aac(6’)-1e-aph(2’’)-1a gene, which is the most reported gene in our country and other countries and one of the HLGR genes investigated in our study, was detected 58%.


Introduction
Enterococcus species are detected with increasing frequency in the etiology of nosocomial infections.Enterococcus faecalis and Enterococcus faecium are the most common causes among nosocomial urinary tract infections, surgical site infections and bacteraemia and they lead to serious infections such as endocarditis. 1reatment of enterococcal infections is difficult because they are resistant to many antimicrobial agents by intrinsically and some types of these bacteria show multiple drug resistance.The intrinsic penicillin resistance in enterococci is depended on the presence of penicillin-binding protein 5 (PBP-5) enzyme, which shows low binding affinity to beta-lactam antibiotics.Therefore enterococci are resistant to many beta-lactam antibiotics.The other mechanism of resistance to beta-lactam antibiotics is the production of beta-lactamase.Beta-lactamase-producing enterococci are rarely isolated. 2Beta lactamase-producing E. faecium strain was first identified in 1981 in the United States. 3nterococci develop resistance to aminoglycoside by two different mechanisms.The moderate level of resistance usually develops due to low permeability.This type of resistance can be eliminated by using aminoglycoside with beta lactam group antibiotics, which inhibit cell wall synthesis.High-level resistance (HLR) occurs due to the result of changes in the ribosome binding site of aminoglycosides or the synthesis of enzymes that inactivate aminoglycoside.HLR is often dependent on the production of transferable plasmid-mediated aminoglycoside inactivating enzymes.The most common aminoglycoside-modifying enzyme in enterococci is APH (2'')-AAC (6') and this enzyme is encoded by aac(6')aph(2'') genes and consists of two enzymes fused together.This enzyme is responsible for resistance to all aminoglycosides except streptomycin.Aminoglycoside-modifying enzymes are mainly responsible from high-level streptomycin resistance. 4,5So far detected aminoglycoside resistance genes in enterococci encoding aminoglycoside-modifying enzymes are aac ( 6 As well as, recently major problems have been encountered in the treatment of emerging vancomycin-resistant enterococci (VRE). 4ew pathogens resistant to vancomycin generally lead to difficulty in treatment because they are also resistant to other antibiotics.Resistance to glycopeptide group antibiotics was first reported in 1988 and then high-level vancomycin-and teicoplanin-resistant strains have spread worldwide. 6 studies, glycopeptide resistant enterococci have fairly wide geographical spread and both genotypic and phenotypic heterogenity was determined.So far seven-vancomycin resistance phenotype are defined.These are VanA, VanB, VanC, VanD, VanE, VanG, VanL. 7anA and VanB resistance phenotypes have been described in E. faecalis and E. faecium.VanA resistant strains can be induced and show high-level resistance to vancomycin and teicoplanin.
In this study, the investigation of vancomycin and high-level aminoglycoside resistance (HLAR) in 100 Enterococcus strains isolated from clinical samples (urine, wound, abscess blood) in Microbiology Laboratory of Mustafa Kemal University Hospital between January 2008-August 2011, by phenotypic and genotypic methods was aimed.Identification and antimicrobial susceptibility of the strains were determined by Vitek 2 automated system (bioMerieux, Mercy L'Etoile, France).HLAR was investigated by disk diffusion test using gentamicin (120 µg) and streptomycin (300 µg) disks (Becton Dickinson, Franklin Lakes, NJ, USA).And it was evaluated according to Clinical and Laboratory Standards Institute (CLSI) criteria. 8Vancomycin MIC values of the strains were determined by gradient diffusion method (GDM) using E-test strips (BioMerieux, France) and evaluated according to CLSI criteria. 8Nitrocefin method and the nitrocefin disks (Becton Dickinson, USA) were used to investigate the presence of the beta-lactamase in the strains.

Investigation of vancomycin and high-level aminoglycoside genes by polymerase chain reaction DNA isolation
Bacterial DNA isolation was performed with commercial DNA extraction kit [High Pure polymerase chain reaction (PCR) Template Preparation Kit (Roche, Basel, Switzerland)] according to manufacturer's directions.

Demonstration of PCR products
The PCR products were analyzed in a 2% (w/v) agarose gel in 1X Tris Borate EDTA (TBE) (Wisent, Canada).Ethidium bromide stained DNA amplicons were visualized using a gel imaging system (Wealtec, Dolphin-View, NV, USA).To determine the expected bp lengths, DNA marker with defined molecular weights in the range 100-3000 bp were used.

Statistical analysis
Data were analyzed using Statistical Package for Social Sciences.Comparison for categorical variables was calculated using chisquare test.A P-value <0.05 was considered statistically significant.

Article
There was no difference between HLSR and HLGR in different species (P>0.05).On the other hand E faecalis strains were found more susceptible to erytromycin (0.031), nitrofurantoin (P<0.001),levofloxacin (P=0.035),linezolid (P=0.028),teicoplanin (P=0.003),ampicillin (P<0.01),penicillin (P<0.01),HLS (P=0.02) but more resistant to tetracycline (P<0.001)than the other strains.MIC range of vancomycin was found from 0,5 to 256 µg/mL by GDM.And MIC 50 and MIC 90 value of vancomycin were found 1.5 and 3 µg/mL respectively.MIC 50 and MIC 90 values were in the range of sensivity limits.Ten strains were found resistant to vancomycin by automated system.Eight of these strains were E. faecalis and two of them were E. faecium.The rate of vancomycin susceptibilities of Enterococcus spp.determined by automated system in this study is shown in Table 3.
The five strains were determined resistant to vancomycin by GDM.All of these strains were E. faecium.The rate of vancomycin susceptibilities of Enterococcus spp.determined    by GDM in this study is shown in Table 4.
VanA gene was detected in five strains.Gel image of the five strains containing VanA gene is shown in Figure 1A.VanB and Van C genes were detected in none of the strains (Table 5).
All of the strains carrying VanA gene were E. faecium.All of the strains carrying VanA gene were found to be resistant to vancomycin, nitrofurantoin, teicoplanin, ampicillin and penicillin (P<0.001).MIC values of vancomycin and MIC values of teicoplanin in five VanA positive strains were found higher than MIC values in 95 VanA negative strains (P<0.001).

Discussion and Conclusions
Enterococci are situated between troubled bacteria because of observed in the increasing rate in nosocomial infections and carrying clindamycin, fluoroquinolones, trimethoprimsulfamethoxazole, low level penicillin, lowlevel aminoglycoside and transferring of genetic material or acquired tetracycline, erythromycin, rifampin, chloramphenicol, nitrofurantoin, fusidic acid, and beta-lactam HLAR, fluoroquinolones and vancomycin resistance by mutation. 4itially, the beta-lactamase-producing strains of enterococci were reported as rare in the United States but nowadays isolates all over the world. 4In our study, no beta lactamase production was determined.Many studies conducted in our country, beta-lactamase-producing strain did not appear as similar to our study. 11,12Beta-lactamase production in enterococci has not been reported in many studies conducted in foreign countries as similar to studies in our country. 13,14artial or complete beta-lactam resistance is characteristic in Enterococcus species.E. faecalis is 10-100 times less susceptible to penicillin than other streptococcal species.E. faecium is 4-16 times less susceptible to penicillin than E. faecalis. 4In our study, it was determined 30 (30%) of the strains are resistant to ampicillin and penicillin.The strains of E. faecalis were more susceptible to ampicillin and penicillin.In our country, Kacmaz and colleagues found that penicillin and ampicillin resistance rates of all strains were respectively 27 and 26%. 11In other countries, D'azevedo and colleagues determined that only 14 strains were high-level ampicillin resistant in their study with 455 enterococci. 15In our study, nitofurantoin resistance rate was 35%.In other countries, Moaddab and colleagues found that nitrofurantoin resistance was 1,5%, Akhtar and colleagues found 5%. 13,16n our study, tetracycline resistance has been identified in 64% of strains.][19] In our study, erythromycin resistance was found to be 69%.Sirin and Adiloglu detected that erythromycin resistance rate was 38%. 18omert and colleagues have found that all of six VRE strains were resistant to erythromycin in their study. 20Kirdar and colleagues found that all of 12 VRE strains were resistant to erythromycin in their study. 19xazolidinone's are new member of the synthetic antibiotics group with activity against Gram-positive.In our study, linezolid resistance was found to be 7%.Ak and colleagues found that resistance rate for E. faecalis was 10.2%, for E. faecium was 9.1% in their study. 21hese rates were lower in other countries.Akhter and colleagues detected that linezolid resistance rate was 4% in their study. 16rotonotario and colleagues detected that linezolid resistance rate for E. faecalis and E. faecium were respectively 0.3%, 1.6% in their study. 22nterococcus infections carrying resistance genes against glycopeptides, penicillin and aminoglycoside group of antibiotics lead to serious problems.The number of enterococci strains which are resistant glycopeptide antibiotics such as vancomycin and teicoplanin gradually increase. 2Glycopeptide antibiotic resistance in enterococci group was first reported in 1988. 6Then high-level of vancomycin-and teicoplanin-resistant strains has spread all over the world.Efe and colleagues have isolated 21 (18.8%)VRE strains in 112 patients. 23Sirin and Adiloglu determined that all strains were susceptible to vancomycin and teicoplanin except one strain moderately susceptible to vancomycin in 100 enterococci strains in their study. 18Ak and colleagues detected five vancomycin-resistant, one moderately vancomycin resistant strains with automated systems in their study. 21n our study, ten vancomycin resistant strains were detected by automated systems, but just five vancomycin resistant strains were identified by GDM.In our country, Karaca and colleagues did not find vancomycin resistance in enterococci by GDM. 24Protonotario and colleagues found vancomycin resistance rate of 0.5% for E. faecalis, 9.6 % for E. faecium by automated systems in their studies containing 1498 E. faecalis and 625 E. faecium strains. 22ouain and colleagues examined vancomycin and teicoplanin susceptibility of 153 enterococci strains by the disk diffusion and GDM method.The vancomycin or teicoplanin resistance was determined in only one E. gallinarum strain, which confers moderate resistance against vancomycin. 25Hallgren and colleagues   [26][27][28] VanA and VanB are the most common resistance genotypes, although there are seven different glycopeptide resistant genotypes found in enterococci.In United States and Europe VanA resistance phenotype is more common than others. 29,30When compared with other types of enterococci, VanA resistance phenotype occurs more frequently.In our study, VanA type resistance have been identified in five of strains by PCR.All of these five strains were E. faecium.First time in Turkey VanA phenotypes of E. faecium was isolated in 2001. 31Then, in 2002, outbreak of VanA phenotype E. faecium was reported. 32Coskun and colleagues identified VanA and VanB genes in thirty and five strains respectively. 33These are the first VanB positive E. faecium strains in our country.And also Kirdar and colleagues also found VanA gene in 12 vancomycin resistant E. faecium isolated from patients with hematologic malignancies. 19In a study in 2000 with the participation of our country, the prevalence of vanA VRE was found highest in the United Kingdom (2.7%), while the prevalence of vanB VRE was highest in Slovenia (2%). 34The prevalence of VanC VRE was highest in Latvia and Turkey, where rates were 14.3 and 11.7%, respectively.The highest prevalence of high-level gentamicin-resistant enterococci was seen in Turkey and Greece. 34ontrast to VanA type, which is common worldwide, VanB type is much less frequently encountered but VanB type have been reported from different countries. 35Nelson and colleagues found that seven E. faecalis strains have VanA phenotype in their study, which includes vancomycin resistant 144 (93.5%)E. faecium, seven (4.5%) E. faecalis and three (2%) E. gallinarum. 36It was determined that remaining six strains have VanA phenotype and 138 of 144 E. faecium have VanB phenotype.Van C-1 gene was amplified in one of three E. gallinarum strains.Descheemaeker and colleagues reported that 46.1% of 601 vancomycin resistant strains was carrying VanA gene while enterococci carrying VanB gene were not detected. 30ow-level aminoglycoside resistance in enterococci, due to a reduction of the permeability of the cell wall.The high-level resistance is mediated by ribosomal or inactivating enzymes.A synergistic beta-lactam-aminoglycoside bactericidal effect is eliminated in the presence of high-level aminoglycoside resistance.High-level aminoglycoside-resistant enterococci are important because they can be resistant to other antibiotics.In our study HLGR rate was 40%, HLSR rate was determined as 63%.No difference was found between Enterococcus species according to HLGR and HLSR rates.High-level aminoglycoside resistance rate was repoted as 16% in the study from our country. 11Sirin and Adiloglu also reported HLGR and HLSR rate as 23 and 16% respectively. 19Another two studies HLAR rates were reported as 39.7 and 54.5% in E. faecalis, 9.1 and 36.3% in E. faecium. 11,18LAR rate was reported 48.1% in Turkey by European vancomycin-resistant enterococci study group. 34][39] The addition of aminoglycoside to an antibiotic, which inhibits cell wall synthesis, plays a significant role in increasing effect of both drugs.However acquired genes through plasmid and transposon lead to release aminoglycosides modifying enzymes and high level aminoglycosides resistance.Thus, the combination treatment loses its synergistic effect. 4,5he most common aminoglycoside modifying enzymes in enterococci is APH (2'')-AAC (6 ') enzyme, which is consisting of two enzymes and responsible for resistance to all aminoglycosides except streptomycin. 4aac(6')-Ieaph(2'')-Ia gene, which encoding bifunctional aac (6')-Ie-aph (2'')-Ia enzyme is the most common clinically.
Treatment options are limited in high-level aminoglycoside-resistant enterococci because of disappeared beta-lactam-aminoglycoside combination for synergistic bactericidal effect.For these reasons, accurate identification of enterococci, determination of antimicrobial resistance state in time, a different resistance pattern and to reveal the resistance mechanisms is important.
Enterococcus strains were included in the study isolated from clinical samples (urine, wounds, abscess, blood) between January 2008 and August 2011 in Microbiology Laboratory of Mustafa Kemal University Hospital.Staphylococcus aureus ATCC 25923 and Enterococcus faecalis ATCC 29212 were

Table 5 . The number of strains containing VanA, VanB, VanC gene.
determined vancomycin resistance in rate of 3.9% by GDM in Sweden, Paberzo and colleagues determined 20% by GDM in Lithuania, Udo and colleagues determined 3% by GDM in Kuwait.