Molecular analysis of Enterococcus faecalis isolates collected during a 4-year period

1. Introduction

Enterococcus faecalis is considered an important-acquired pathogen, which has frequently been isolated from all common infections, including urinary tract infection (UTI), bacteremia, neonatal sepsis, endocarditis, and abdominal and pelvic infection ( 1 ). In addition to the resistance to the antibiotics of their choice, especially vancomycin and gentamicin (GM), several virulence factors have contributed to the persistence of enterococci in nosocomial infections, including collagen-binding adhesin of Enterococcus faecalis (Ace), aggregation substance (Asa1), cytolysin (CylA), enterococcal surface protein (Esp), and gelatinase (gelE) ( 2 , 3 ).

Because of the steadily increasing antibiotic resistance of E. faecalis strains within healthcare facilities, it is necessary to perform epidemiological investigations and identify the possible sources of contamination. Recently, multiple-locus variable-number tandem repeat analysis (MLVA) based on variations in the number of repeats at certain variable number tandem repeat (VNTR) loci has been known as a useful method for genotyping purposes of several bacterial pathogens, including E. faecalis ( 4 ). However, in our country, E. faecalis displayed a high frequency, and there are insufficient studies on molecular characteristics of enterococci obtained from pediatric infections ( 5 , 6 ). In the current study, we aimed to determine the virulence genes and MLVA types (MTs) among E. faecalis strains isolated from an Iranian Children's Hospital over four years.

2. Material and Methods

2.1. Bacterial isolates

Sixty-seven pre-identified E. faecalis strains were selected from our previous study ( 7 ). These isolates were collected from clinical samples (urine (n=58), blood (n=4), cerebrospinal fluid (n=2), a wound lesion (n=1), a tracheal secretion (n=1), and a peritoneal fluid (n=1)) of children during December 2011 to July 2014. Most E. faecalis strains originated from outpatients (n=22), emergency (n=9), and urology hospitalized patients (n=8). Antimicrobial susceptibility testing, bacterial genomic DNA extraction, and detection of vancomycin (vanA) and an aminoglycoside (aac(6′)-Ie-aph(2″)-Ia) resistance genes had been performed in our previous study ( 7 ).

2.2. Virulence genes detection

The genes encoding virulence factors (cylA, gelE, esp, ace, asa1) were targeted by means of PCR using pre-extracted DNA by the boiling method ( 7 , 8 ).

2.3. Molecular analysis

On the basis of Titze-de-Almeida study, seven repeat loci (aceB, espA, espC, efa2, efa3, efa5, efa6) were selected for molecular analysis of isolates ( 9 ). Briefly, the PCR protocol consisted of a pre-denaturation step at 95°C for 5 min and a final extension at 72°C for 5 min. Thirty cycles of 95°C for 45 s, 50 s at 66.3°C (for aceB), 56°C (for espA and efa6), 59°C (for efa2), 55.2°C (for efa3), and 49 °C (for efa5) were performed. Amplified amplicons were analyzed on 1% agarose gels and product bands were reflected with KBC power loading dye (GelRed Nucleic Acid Gel Stain, 10,000× in water, Kawsar Biotech Co., Tehran, Iran) under UV illumination. The number of copies in each locus was estimated based on the size of the repeats and the PCR bands. The MLVA type (MT) was given based on one or more band differences; thus, MTs were described as isolates sharing ≥ 85.7% similarity. All results were rounded down and up if they were < 0.5 and > 0.5, respectively, and were considered 0.5 itself if they were = 0.5.

3. Results

The pattern of antibiotic resistance of each strain and the genes involved in vancomycin and GM resistance are shown in Table 1. The prevalence of gelE and aceB virulence genes was high (73% and 62%), followed by asa1, esp, and cylA found in 58%, 31%, and 7% of isolates, respectively.

The results of MLVA typing are shown in Table 1. The MLVA typing revealed 52 variable number tandem repeat (VNTR) patterns belonging to 38 MTs (Table 1). Eighteen isolates were assigned into two common types (13 as MT1 and 5 as MT2). The MT1 isolates were recovered from different parts of the hospital from 2011-2012. It is worth mentioning that, the most common pattern of antibiotic resistance in MT1 isolates was associated with pattern gentamicin, ciprofloxacin, erythromycin, and clindamycin (AP, GM, CIP, E, and CD). Moreover, aac (6')-Ie-aph (2")-Ia resistance gene and gelE+asa1 virulence pattern in MT1 strains were frequent.

4. Discussion

Similar to previous reports, our findings showed a relatively high prevalence of asa1, ace, and gelE among E. faecalis strains ( 1 , 10 ). Studies revealed that antibiotic resistance genes and asa1 are located on a plasmid that can be transferred simultaneously. In addition, more than half of the E. faecalis strains isolated from nosocomial infections harbored genes encoding gelatinase and aggregation substance ( 11 ). The MLVA typing of 67 E. faecalis isolates revealed 52 VNTR patterns and 38 MTs. In a study conducted by Walecka et al., MLVA of 56 E. faecalis isolates revealed 40 VNTR patterns and MTs ( 4 ). In another study in Poland, 111 VNTR patterns and MTs were determined on 153 E. faecalis strains ( 12 ).

This high degree of heterogenicity among isolates may indicate the persistence of enterococci strains. The MT1 strains were isolated from different parts of the Hospital during the first two years of the study. Most of these isolates have GM, CIP, E, CD, and gelE+asa1 pattern, and an aac(6')-Ie-aph(2")-Ia resistance gene, which probably indicates a dominant clone compatible with the Hospital setting in our study Center and was transferred to outpatients referred to the Medical Center. Despite observing these isolates in two years of our study, more samplings from different Wards are required to identify and prevent their spread because, if the MT1 strains are not controlled, they will spread resistance and virulence factors to sensitive isolates. In conclusion, MT1, a common MT in E. faecalis isolates, circulated in different Wards of the Hospital in 2011-2012. Moreover, E. faecalis isolates with multiple resistance were common in our study Hospital.

Acknowledgment

Not applicable.

Authors' Contribution

RB and ME designed the study. FJ and ASM drafted the manuscript. ASM performed data analysis. All authors provided intellectual input to the study and read and approved the final manuscript.

Ethics

The study was approved by the Ethics Committee of Tehran University of Medical Sciences and all methods were performed in accordance with the relevant guidelines and regulations.  Consent to participate is not applicable for this study because the isolates included in the study were obtained from existing clinical collections routinely assembled as part of laboratory practices of university hospitals.

Conflict of Interest

The authors declare that they have no conflict of interest. The authors alone are responsible for the content and writing of the paper.

Funding

This research has been supported by Tehran University of Medical Sciences & health Services grant 97-02-30/38781.

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