Dallal, Mohammad Mehdi Soltan PhD*; Saifi, Mahnaz PhD*; Pourshafie, Mohammad Reza PhD†; Eshraghian, Mohammad Reza PhD‡
Urinary tract infection (UTI) is one of the most important causes of morbidity in the general population and is the second most common cause of nosocomial infections.1 Enterococci are second only to Escherichia coli as agent of nosocomial UTIs and third (behind Staphylococcus aureus and coagulase-negative staphylococci) as agents of nosocomial bacteremia.1,2 Enterococci are also frequent inhabitants of the bowel, vagina, and anterior urethra of humans. Several studies have documented that enterococcal infections are most commonly caused by the patient's own commensal flora.3 Among members of the genus enterococci, Enterococcus faecalis and Enterococcus faecium are the most common species isolated from human infections. Enterococcus faecalis is isolated from approximately 80% of human infections and E. faecium from most of the rest.4 Enterococcus faecalis is more common in nosocomial infections than E. faecium, but E. faecium has a great ability to acquire drug resistance.5 Antimicrobial resistance in enterococci is a growing problem at the clinical level. In enterococci; multiresistance is now increasingly common and includes resistance to β-lactams, aminoglycosides (high level), and glycopeptides. Enterococci exhibiting high-level gentamicin resistance (HLGR) have been reported widely as a cause of nosocomial infections in Europe, the United States, and other geographic locations.6 Enterococcus faecium possesses a broad spectrum of natural and acquired antibiotic resistance. This has enabled multiresistant E. faecium to emerge as a severe nosocomial pathogen worldwide.7 Since the E. faecium HLGR appearance in the United States in 1986, there have been a number of reports of the isolation of HLGR enterococci in several other countries. High-level gentamicin resistance in enterococci is mediated mostly by the aac(6′)-Ie-aph(2″)-Ia gene which may be associated with transposable elements located on plasmids or integrated in the chromosome. In the former case, HLGR is often transferable between strains. Enterococci also have the ability to transfer their resistance genes to other bacteria.7,8
Area-specific monitoring studies aimed to gain knowledge about the exact type of pathogens and their resistant profiles may help clinicians to choose the correct empirical treatment. Species identification is a difficult task for enterococci, because of phenotypic and biochemical similarities between many enterococcal species. In such instances, application of other accurate and rapid methods can help researchers to gain the best results. The average turnaround time for polymerase chain reaction (PCR) methods is 48 hours compared with 96 hours for the phenotypic method. Polymerase chain reaction has the potential to reduce both the time and cost of detecting enterococci and can provide valuable information of clinically important enterococci. Accurate identification of enterococci is important for understanding the role of species variation in pathogenicity and resistance pattern of enterococcal infections.
MATERIALS AND METHODS
From November of 2005 to July 2006, 425 urine samples were recovered from inpatient and outpatient cases with UTI attending to 3 hospitals and 4 private clinical laboratories located in Tehran. The urine samples were collected by the midstream "clean catch" method. All of the samples were processed using standard microbiological procedures.
Identification of Isolates
All of the enterococcal isolates were tested for phenotypic characteristics by conventional methods, on the basis of the following criteria: growth on bile esculin agar and in 6.5% NaCl broth, absence of catalase, and presence of pyrolidonyl arylamidase. Species-level identification was performed by biochemical tests including acid fermentation of mannitol, sorbitol, sucrose, arabinose, and raffinose; motility; and arginine hydrolysis.9
After overnight culturing of the strains in 10 mL of brain-heart infusion broth medium, 3 mL of the cultures was centrifuged at 4000 revolutions per minute for 15 minutes, and the pellet was resuspended in TES (Tris HCl 10 mmol/L, EDTA 1 mmol/L, sucrose 50%; pH = 7.5) containing lysozyme (20 mg/mL) and incubated in 37°C for 20 minutes. Twelve microliters of 10% sodium dodecyl sulfate was added to the mixture and mixed vigorously and placed in ice for 10 minutes. After centrifugation at 13,000 revolutions per minute for 15 minutes, the DNA was extracted from the supernatant, with adding 3-times equal volume of phenol-chloroform.10
Detection of Genus and Species by PCR
All strains were screened for genus and species by the PCR method with 3 different primer sets (Table 1). Amplification of both species-specific and genus (rrs) targets produced bands corresponding to their respective molecular size. Polymerase chain reactions were performed in a total volume of 25 μL containing 0.8 mmol/L deoxyribonucleotide triphosphate, 0.5 U of Taq DNA polymerase (Roche, Germany), 2.5 pmol/L of each primer, 2.5 μL of 10× PCR buffer, 1.5 mmol/L MgCl2, and 5 μL of the DNA template. DNA amplification was carried out with the initial denaturation at 94°C for 5 minutes and 30 cycles of amplification (denaturation at 94°C for 1 min, annealing at 54°C for 1 minute, and extension at 72°C for 1 minute), followed by a final extension at 72°C for 7 minutes.11
Detection of aac(6′)-Ie-aph(2″)-Ia Gene by PCR
Total HLGR isolates also were tested for the aac(6′)-Ie-aph(2″)-Ia gene (Table 1). Polymerase chain reaction experiments were performed in a volume of 25 μL with the following content: 2 μL of DNA template, 1.5 mmol/L MgCl2, 0.2 mmol/L of each deoxynucleotide triphosphate, 2.5 μL of 10× PCR buffer, 2.5 U Taq DNA polymerase (Roche), and primers with a 2-pmol/L final concentration. Polymerase chain reaction was performed with an initial denaturation step of 3 minutes at 94°C, 35 cycles of 40 seconds at 94°C, 40 seconds at 55°C, and 40 seconds at 72°C, and a final extension step of 2 minutes at 72°C.10 Polymerase chain reaction products were analyzed by electrophoresis against molecular size markers at 100 V in 1.5% agarose gels stained with ethidium bromide.
Antibiotic Susceptibility Testing
The resistance patterns of isolates were tested by using the disk diffusion method according to the CLSI guidelines.12 The following antimicrobial disks were used: vancomycin (30 μg), ciprofloxacin (5 μg), ampicillin (10 μg), high-content gentamicin (120 μg), trimethoprim/sulfamethoxazole (1.25:22.75), tetracycline (30 μg), erythromycin (30 μg), and nitrofurantoin (300 μg) purchased from BBL (BD BBL, Sparks, Md); quinupristin-dalfopristin (15 μg) and linezolid (30 μg) from Mast Diagnostics Ltd (Bootle, Mersey Side, UK); and teicoplanin (30 μg) from BR (BioRad, Hercules, Calif). Isolates with intermediate levels of susceptibility were classified as resistant.
Enterococcus faecalis ATCC 29212 and E. faecium IP 4107 (Institute Pasteur of Paris microbial collection) were used as quality-control reference strains.
Of 500 urine samples tested, a total of 147 enterococcal isolates were obtained. Distribution of species according to biochemical tests was 104 (70%) E. faecalis and 43 (30%) E. faecium.
Amplification of genus, E. faecalis-specific and E. faecium-specific targets produced 320-, 941-, and 658-base pair bands, respectively (Fig. 1). The species distribution by the PCR method was the same as detected by biochemical tests.
The frequency of the HLGR phenotype was 61.5% in E. faecalis and 79% in E. faecium strains. Polymerase chain reaction results showed that all of the HLGR strains contained the aac(6′)-Ie-aph(2″)-Ia gene (Fig. 2).
The distribution of antimicrobial resistance according to species in HLGR isolates is summarized in Table 2. In the isolates identified as HLGR E. faecium, the prevalence of resistance observed to ampicillin, vancomycin, teicoplanin, nitrofurantoin, ciprofloxacin, cotrimoxazole, and erythromycin was higher than E. faecalis. Vancomycin resistance was detected in 7.9% of E. faecalis and 70% of E. faecium HLGR isolates; 3.8% of E. faecalis strains were resistant to linezolid, as opposed to none of the E. faecium strains.
Among HLGR E. faecium and E. faecalis isolates, 19 (44%) and 9 (8%) multidrug-resistant isolates were recognized, respectively.
There was no significant difference among resistance frequencies between hospitalized and outpatient individuals except for ciprofloxacin, which was higher in E. faecalis HLGR isolates in hospitalized patients (73% vs 45%) (data not shown).
This study investigated the species occurrence and antibacterial resistance pattern of enterococci isolated from UTI in hospitalized and outpatient individuals.
Enterococci exhibiting HLGR have been reported widely as a cause of nosocomial infections in Europe, United States, and other geographic locations.6 Gentamicin is one of the most commonly used aminoglycosides against enterococci.13 Enterococci have intrinsic low-level resistance to aminoglycosides and, in addition, have acquired aminoglycoside resistance genes.14 Along with the rapid rise in incidence of resistance in enterococci to traditional antibiotics, including aminoglycosides, transmission of multiresistant and HLGR strains has been reported.15
In the present study, E. faecalis (70%) and E. faecium (30%) were the most prevalent species colonizing the urinary tract of patients. We had a clear dominance of E.faecalis as expected, but the frequency of E. faecium isolates was more than that in other reports.3,5,16
The difficulty in phenotypic identification of enterococcal strains is not unexpected and points to the need to go beyond the conventional biochemical tests, but also to do the fundamental additional tests necessary for genus and species identification of enterococci. The results obtained from PCR and phenotypic assays showed a high rate of agreement with conventional tests for both species. The procedure for biochemical reactions is time consuming, whereas the PCR method provided rapid and exact identification of enterococcal isolates and can be recommended. Indeed, the correct definition of enterococcal species enabled us to assess species-specific antibiotic susceptibility patterns in our area.
Earlier studies in Iran have reported the prevalence of HLGR strains in clinical samples to be about 52%17 and in other reports range from 1% to 49%.18 In this study, the prevalence of HLGR enterococci was significantly higher (61.5% of E. faecalis and 79% of E. faecium strains).
The aac(6′)-Ie-aph(2″)-Ia gene is the most clinically important gene among HLGR isolates,10 and the high frequency of its presence in the present study indicates widespread dissemination of this resistance determinant.
The high resistance to ciprofloxacin and cotrimoxazole seen in the present study may be due to the widespread usage of these antibiotics for UTI as a first-line treatment in Iran. The higher resistances to ciprofloxacin among isolated HLGR E. faecalis from hospitalized patients also may be due to this point. There are also reports of increasing resistance of enterococci to ciprofloxacin.19 Most ciprofloxacin resistance was noted in the studied enterococci which also showed HLGR.
Vancomycin-resistant enterococci have received increasing attention since the late 1980s. Vancomycin resistance is most commonly found in E. faecium, but it is increasingly seen in the more predominant enterococcal species in humans, E. faecalis.20 The prevalence of resistance to vancomycin in E. faecium isolated in the present study was significantly higher than E. faecalis, and this was in concordance to teicoplanin resistance frequency.
Ampicillin resistance in E. faecium isolates was significantly higher, and this finding is similar to the report of Jureen et al.21 According to their study; this kind of resistance in E. faecium may be due to the production of low-affinity penicillin-binding protein 5.
Iran is a country where antibiotic prescription policies are very relaxed, so here we have multiresistant strains of some pathogenic bacteria. With the raised prevalence of aminoglycoside resistance, in particular, HLGR strain, among clinical enterococcal isolates, the usage of gentamicin for synergistic combination therapy has been limited. This problem will be solved by application of some new-generation aminoglycosides or other new drugs such as quinupristin-dalfopristin or linezolid. Quinupristin-dalfopristin and linezolid are new antibiotics and have a spectrum of in vitro activity against enterococci.20,21 In the present study, both species showed low resistance to linezolid, and most of the E. faecium isolates were sensitive to quinupristin-dalfopristin.
According to our results; it seems that nitrofurantoin can be considered a good alternative therapy in enterococcal UTIs because of the lower resistance especially in E. faecalis strains. Because of the great ability of enterococci to transferring their antibiotic resistance elements to other bacteria, the serious problem was not only for enteromcoccal infection treatment, but also for all important common pathogens which can accept these different resistance genes.
Accurate identification at the species level of enterococci isolated from clinical specimens is considered to be necessary as it provides a quantitative evaluation of their resistance to ampicillin, vancomycin, and teicoplanin and high-level resistance to gentamicin and streptomycin.22 It is also necessary to distinguish the low-virulence enterococcal species with constitutive low-level resistance to vancomycin and gentamicin from the species that are more frequently isolated from clinical specimens such as E. faecalis and E.faecium which in some countries can often show high-level inducible and transmissible resistance to glycopeptides and aminoglycosides.23
Due to the predilection of resistance in enterococci, species identification can provide an important clue for choosing an appropriate antibiotic therapy.
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