vanA in vancomycin-resistant Enterococcus faecalis isolated in Baghdad

Enterococcus faecalis has emerged as an important nosocomial pathogen worldwide, infections and outbreaks of Vancomycin-resistant enterococci (VRE) still appear to be rare in Baghdad. In the present study, 20 isolates of E. faecalis were collected from 252 clinical samples (151 urine and 101 blood) from different hospitals in Baghdad and 3 isolates from 50 stool samples from healthy people during the period 1/10/2010 to 1/12/2010. All isolates were identified through morphological, cultural and biochemical tests using Rapid ID-32 strep. Vancomycin-resistance phenotype was determined by the agar diffusion method; results showed that five clinical isolates (25%) and two faecal isolates (66.7%) were resistant to vancomycin. Minimum inhibitory concentrations (MIC) of clinical VRE isolates ranged between 4 to 512 μg/ml and the MICs for isolates of faecal origin were 64 and 128 μg/ml. Seven vancomycin-resistant E. faecalis isolates were examined for their drug resistance and plasmid DNAs. Of the 5 clinical isolates, 2 isolates exhibited resistance to azlocillin (Azl) and ofloxacillin (Ofx). All clinical and stool isolates were resistant to oxacillin (Ox), cefepime (Fep), rifampicin (Ra) and tetracycline (Te). Imipenem and ampicillin were found to be the most effective agents against the isolates. The detection of plasmid DNA by gel electrophoresis showed that some E. faecalis isolates carried a high molecular weight plasmid that was transferred to the recipient by filter mating. The putative presence of vanA gene was examined by PCR, using specific primers. Positive PCR amplifications were obtained in all 7 isolates for vanA gene. Vancomycin resistant E. faecalis increased gradually in Baghdad hospitals and high dissemination of vanA gene, which encoded high resistance level to vancomycin. Continued surveillance is required to prevent further spread of these serious resistances.


INTRODUCTION
Enterococci are known to be opportunistic nosocomial pathogens capable of causing life-threatening infections, such as endocarditis and bacteremia, mostly in immunocompromised patients (Levison and Mallela, 2000) occasionally reported to cause urinary tract infections, septicemia, and endocarditis (Sun et al., 2009).Since Enterococcus spp.are resistant to multiple antibacterial drugs, there are only limited options for effective therapy and prophylaxis of serious infections (Kawalec et al., 2004).The emergence of vancomycin-resistant enterococci (VRE) followed a worst-case scenario for nosocomial pathogens.The isolation of VRE (VanA type) was first reported in 1988 in the United Kingdom (Uttley et al., 1988) and France (Leclercq et al., 1988), and shortly thereafter, it was reported in the United States (Sahm et al., 1989).Since then, VRE have been identified in many countries.
Vancomycin resistance is conferred by one of two functionally similar operons, vanA or vanB.The vanA and vanB operons are highly sophisticated resistance determinant, probably evolved in other species and acquired by enterococci (Rice, 2001).In general, enterococcal isolates with lowered susceptibility to vancomycin can be categorized as vanA, vanB, and vanC.vanA and vanB pose the greatest threat because they are the most resistant and the resistance genes are carried on a plasmid.Since the resistance genes are carried on a plasmid they are readily transferable through conjugation (Paulsen et al., 2003).In addition to the increase in VRE, the potential spread of vancomycin resistance to Staphylococcus aureus or Staphylococcus epidermidis is a serious public health concern.The genes for vancomycin resistance are frequently plasmid-borne and have been transferred in vitro from enterococci to staphylococci (Palazzo et al., 2005).Clinical strains of vancomycin-resistant Staphylococcus haemolyticus and S. epidermidis have already been reported.Spread of vancomycin resistance must be controlled to prevent the development of untreatable staphylococcal infections (Srinivasan et al., 2002).
In Asia, VRE have been isolated from hospitalized patients or food animals in China, Japan, South Korea, Taiwan, and Thailand (Zheng et al., 2007).In particular, they have frequently been isolated in South Korea (Yu et al., 2003) and Taiwan (Lauderdale et al., 2002).In Iraqi hospitals, very rare studies have investigated the incidence of vanA gene among Vancomycin resistant Enterococcus faecalis, The aims of this study were to determine the distribution of Vancomycin-resistant E. faecalis isolates, and studying the vanA gene in this bacteria which isolated from hospitals and healthy persons.

Bacterial isolates
A total of twenty (20) E. faecalis isolates were collected from 252 clinical samples (151 urine and 101 blood) from different hospital in Baghdad and 3 Isolates from 50 stool samples from healthy people during the period 1st October to 1st December, 2010.For each isolate, the clinical source and limited demographic data were documented

Bacterial identification
All isolates have been identified by morphological, cultural and biochemical tests such as API 20 STREP test (bioMérieux, France) and Rapid ID-32STREP system (bioMérieux, France).All isolates were identified using the agar supplemented by potassium tellurite reduction, motility, and pigment production tests (Macfaden, 2000;Forbes et al., 2002).

Plasmid DNA extraction
The plasmid DNA of the clinical and stool isolates was isolated by using alkaline method according to Klaenhammer (1984) and analysed on 0.8% agarose gel.

Conjugation experiments
Filter mating was performed as described previously (Ike et al., 1983;Dahl et al., 2000).A 100 μL sample of mating mixture containing donor strains and the recipient strain was spread on a 45 μM nitrocellulose membrane filter (Millipore) placed on top of brain heart infusion (BHI) agar.After 18 h incubation at 37°C, cells were resuspended in 1 ml BHI broth and spread on BHI agar containing antibiotic.

PCR-based detection of vancomycin resistance genes
A PCR assay was used to detect genes coding for vancomycin resistance in enterococci (van A and van B).Primer pairs used to amplify fragments of the van A (F = GGGAAAACGACAATTGC and R=GTACAATGCGGCCGTTA). Primer pairs used to amplify fragments of the vanB (F=AAGCTATGCAAGAAGCCATG and R= CCGACAATCAAATCATCCTC).Four to five well-isolated colonies obtained after overnight growth on trypticase soy agar were resuspended in lysis buffer (50 mM Tris-HCl [pH 8.0], 50 mM NaCl, 5 mM EDTA [pH 8.0]).Proteinase K was then added to a final concentration of 1 mg/ml, and the suspension was incubated at 50°C for 60 min, followed by incubation at 100°C for 10 min.Processed samples were used immediately for PCR or stored at 4°C until they were used.
The PCR amplification mixture consisted of reaction buffer (final concentration, 2 mM KCl, 3 mM MgCl 2 , 10 mM Tris-HCl [pH 8.3]), 400 mM (each) dATP, dCTP, dTTP, and dGTP, 0.1 mM vanA primer pair, 0.5 mM vanB , 50 U/ml of Taq polymerase (Promega/USA), and 1.0 ml of enterococcal DNA in a total volume of 10.0 ml.DNA amplification was carried with a Gradient PCR System (TechNet-500 /USA).The cycling conditions consisted of an initial denaturation step at 95°C for 4 min and then 30 cycles of 95°C for 45 s, 54°C for 1 min, and 72°C for 1 min.In the final cycle, the extension step was carried out at 72°C for 7 min.A reagent blank (containing all the components of the reaction mixture with water instead of target DNA), were run in every PCR procedure.Amplified PCR products were detected by agarose gel electrophoresis with 1.5%.A DNA marker (Promega/USA) was run with each gel, and the VRE genotype was determined by the size of the amplified product.

RESULTS
Clinical isolates were obtained mostly from urine 17 isolates (11.25 %), followed by blood 3 isolates (2.97 %).Five clinical isolates (25%) and two stool isolate (66.7%) were resistant to vancomycin.Minimum inhibitory concentrations for Vancomycin were determined, VR E.faecalis clinical isolates that had concentrations between 4-512 µg/ml and the MICs for stool isolates (2 isolates) were 64 and 128 µg/ml.The resistance patterns for the VRE.faecalis isolates are shown in Table 1.All VRE.faecalis isolates were resistant to Oxacillin ,Cefepime ,Rifampicin and Tetracycline.In addition, four clinical isolates were resistant to streptomycin, two isolates were resistant to ofloxacin, and one stool isolate was resistant to Ampicillin.All VRE.faecalis isolates were susceptible to Imipenem.The detection of plasmid DNA by gel AL-Marjani 117 electrophoresis showed that some E. faecalis isolates carried a high molecular weight plasmid that was transferred to the recipient by filter mating.The detection of vanA gene by PCR (Figure 1) showed the high dissemination of vanA gene which encoded to high resistsnce level to vancomycin between the local VRE.faecalis isolates in comparison to vanB gene .

DISCUSSION
Infection or colonization with VRE results in a wide spectrum of possible deleterious effects for patients in health care settings.Studies have demonstrated that VRE can cause serious infections, and the attributable mortality of bacteremia caused by VRE has been estimated to be nearly 40% (Werner et al., 1999).The percentage of the clinical isolates in the present study was slightly in a good agreement with percentage results in other study in Baghdad (Alsaadi, 2007;Al Kafaji, 2005).Enterococci account for approximately 110 000 urinary tract infections, 25 000 cases of bacteremia, 40 000 wound infections, and 1 100 cases of endocarditis annually in the United States (Emori and Gaynes, 1993).
In our study, 25% of clinical isolate and 66.7% of stool isolate were resistant to vancomycin, these results agreed with those of other results (Al Kafaji, 2005), but in disagreement with that of (Alsaadi, 2007) which showed that all isolates of E. faecalis were sensitive to vancomycin but in the study of (Kandela, 2006) the resistance was higher the current study.Kühn et al., (2005) studied the occurrence of VRE in 2 580 samples collected in different geographical regions in Europe, high proportions of VRE-positive urban sewage samples were found in Sweden, Spain, and the United Kingdom (52 to 90%), apparently reflecting a human source rather than an animal source; despite the fact that the consumption of antibiotics in hospitals in Sweden (population, 9 million) is among the lowest in Europe.Thus, it is possible that antibiotic-resistant enterococci present in the human population are enriched in hospitals, released from there into municipal sewers to become established, and then further released via sewage treatment plants into recipient water and transferred back into humans.However, 7% of isolates from Germany and 16.7% of isolates from Switzerland and Greece were vancomycinresistant enterococci (Bouchillon et al., 2004).
Vancomycin-resistant E. faecalis tend to be multidrug resistant against a large number of currently available antimicrobial agents, compromising treatment options and increasing the likelihood of inadequate antimicrobial therapy and increase in morbidity and mortality (Kawalec et al., 2004).In the present study, VRE.faecalis showed resistance to a wide range of antimicrobial agents.Our results illustrated that, Imipenem and Ampicillin has a good activity against both clinical and stool isolates.
Close to this result was reported in a study carried out by (Alsaadi, 2007).Our results detected that all vancomycin resistant E. faecalis isolates were resistant to Oxacillin, Cefepime, Rifampicin and Tetracycline.Multiple-drug-resistant enterococci and vancomycinresistant enterococci (VRE), in particular, are opportunistic pathogens and major causes of nosocomial infections in immunocompromised patients (Zheng et al., 2009).
Since the first report of vancomycin-resistant enterococci (VRE) in 1988, VRE have emerged as an important cause of hospital-acquired infections, particularly in the United States.According to the National Nosocomial Infection Surveillance System of the Centers for Disease Control and Prevention, over a period of 10year the percentage of VRE has increased from 0.4 to 25% among enterococcal isolates from patients in intensive care units (ICUs) (Patel, 2003).Regarding the origin of VRE, European studies demonstrated that the carriage of VRE in the community was probably caused by the use of antibiotics as growth promoters in animals (Bates, 1997).VRE are usually transmitted by contact.No skin found that VRE could survive for at least 1 h on gloved and ungloved fingertips and for 5 to 7 days on environmental surfaces (Noskin et al., 1995).Other investigators have shown that VRE can exist in stool specimens of a carrier for up to 2 years, providing a source for environmental spread (Roghmann et al., 1997).Results of PCR detection showed that vanA gene was present in all the tested isolates regardless of their origin, if they are clinically or stool.Elsewhere in South Africa, four clinical isolates of E. faecalis vanB and one strain of Enterococci gallinarum were isolated in a hospital in Bloemfontein, Free State, in 1995 (Derby et al., 1998).In a hospital in Cape Town, an E. faecium vanA strain was isolated in 1993 from a clinical specimen (Derby et al., 1998).
The vanA gene results in high levels of vancomycin resistance, as well as, resistance to teicoplanin.The MICs of vancomycin for enterococci with the vanB gene may be from low to very high, but these enterococci remain susceptible to teicoplanin.Both these classes of vancomycin resistance (VANA and VANB VRE) have been detected mainly in E. faecium and E. faecalis.These types of resistance are acquired and encoded by genetic elements, which are transferable.To date, all published outbreaks of VRE have been restricted to these two classes of vancomycin resistance (Toye et al., 1997).In 1992, Handwerger et al. reported that the occurrence of glycopeptide-resistant enterococci with a VanA phenotype was rare in the United States.The VanB phenotype shows variable levels of vancomycin resistance but remains susceptible to teicoplanin and is inducible by vancomycin alone.The genes encoding the VanB resistance phenotype are more commonly chromosomal but can also be transferred by conjugation.
In conclusion, Vancomycin Resistant E. faecalis increased gradually in Baghdad Hospitals and high dissemination of vanA gene, which encoded high resistance level to vancomycin.Continued surveillance is required to prevent further spread of these serious resistances.

Table 1 .
Susceptibility of the 20 isolates of vancomycinresistant E. faecalis to 10 antibiotics.
Figure 1.Presence of vanA gene in VRE.faecalis isolates.