|Year : 2015 | Volume
| Issue : 5 | Page : 80-86
Molecular characterization of Enterococcus spp. clinical isolates from Cairo, Egypt
YA Hashem1, AS Yassin2, MA Amin2
1 Department of Microbiology and Immunology, Faculty of Pharmacy, Modern Sciences and Arts University, Giza, Egypt
2 Department of Microbiology and Immunology, Cairo University, Cairo, Egypt
|Date of Submission||27-Oct-2013|
|Date of Acceptance||14-Apr-2014|
|Date of Web Publication||6-Feb-2015|
A S Yassin
Department of Microbiology and Immunology, Cairo University, Cairo
Source of Support: None, Conflict of Interest: None
Purpose: Enterococci are responsible for serious diseases such as bacteraemia, endocarditis and urinary tract infections. The ability of enterococci to cause such diseases is due to acquisition of certain virulence factors such as haemolysin, gelatinase and enterococcus surface protein. This study has been conducted to investigate the occurrence of virulence factors and resistance to various antibiotics with emphasis on vancomycin in the Enterococcus spp. Materials and Methods: Clinical specimens were collected and isolates were identified by proper microscopic, culture and biochemical tests. Susceptibility and degree of resistance of the isolates to various antibiotics were determined. Virulence factors were examined by phenotypic tests followed by molecular methods. Bioinformatics analysis was used to detect regions in the genomes that might have originated from horizontal gene transfer. Result: The presence or absence of virulence genes did not affect the pattern of antimicrobial resistance in Enterococcus isolates; consequently, no relationship was found between virulence factors and resistance to different antibiotics used. Bioinformatics analysis showed that the virulence genes were mainly transferred by transposons. Conclusion: Among the enterococci, environmental factors may interfere in the expression of virulence factors. Horizontal gene transfer plays an important role in the spread of resistance and virulence genes.
Keywords: Enterococcus, Egypt, resistance, urinary tract infections, virulence
|How to cite this article:|
Hashem Y A, Yassin A S, Amin M A. Molecular characterization of Enterococcus spp. clinical isolates from Cairo, Egypt. Indian J Med Microbiol 2015;33, Suppl S1:80-6
|How to cite this URL:|
Hashem Y A, Yassin A S, Amin M A. Molecular characterization of Enterococcus spp. clinical isolates from Cairo, Egypt. Indian J Med Microbiol [serial online] 2015 [cited 2020 Apr 1];33, Suppl S1:80-6. Available from: http://www.ijmm.org/text.asp?2015/33/5/80/148836
| ~ Introduction|| |
Enterococci are Gram-positive commensal bacteria that make up an important part of the intestinal flora of man and animals.  For many years, enterococci were believed to be harmless to humans. Moreover, Enterococcus species have been used widely over the last decade as probiotics and in the food industry as starter cultures. 
Recently, enterococci have become one of the most common nosocomial pathogens, with patients having a high mortality rate.  Enterococci are capable of causing various serious diseases such as endocarditis, bacteraemia, urinary tract infections (UTIs), and central nervous system infections. Most of these clinical infections are attributed to either Enterococcus faecalis (E. faecalis) or Enterococcus faecium (E. faecium).
Several virulence factors may contribute to the ability of enterococci to cause such diseases. Adherence of enterococci to host cell is the first step in the process of infection, one of the adhesion factors is enterococcal surface protein (Esp).  Enterococci also secrete cytolysin (Cyl), a bacterial toxin that shows haemolytic activity against erythrocytes of human, rabbit and horses.  Another secreted molecule is gelatinase (GelE) which has the ability to hydrolyze gelatin, collagen, casein, haemoglobin and other small biologically active peptides.  Aggregation substance (Agg) is a virulence factor that causes the bacterial cells to aggregate or clump and, hence, facilitate plasmid transfer; it also plays a role in enterococcal endocarditis and UTI by supporting the adherence of bacteria to cardiac vegetations and renal epithelial cells. 
The severity of enterococcal infections has increased due to emergence of strains with multiple antimicrobial resistances. Resistance can be either intrinsic, such as resistance to low level of aminoglycosides, cephalosporins and penicillin, or acquired, such as resistance to glycopeptides, e.g., vancomycin and teicoplanin.  Vancomycin-resistant Enterococcus (VRE) infections are serious because glycopeptides are considered the last treatment available for life-threatening infections; therefore, it may lead to an increase in mortality rate.  The resistance of Enterococcus to vancomycin is mediated via group of genes (vanA, vanB, vanC, vanD and vanE). In the presence of vancomycin, these genes are transcribed and cell wall precursors with low affinity to vancomycin are synthesized. 
UTIs encompass infections of the kidney, ureters, bladder or urethra and are amongst the most common bacterial infections worldwide.  Enterococci are considered to be among the most common pathogens associated with uncomplicated UTIs. 
The aim of the present study is to investigate the patterns of UTIs caused by enterococci among Egyptian patients and to analyze their antibiotic resistance profiles as well as their virulence factors, both phenotypically and molecularly. In addition, we attempt to investigate the possibility of spread of virulence and resistance genes by horizontal gene transfer by applying bioinformatics analysis of the available enterococci genomes. Studying resistance and virulence patterns of pathogens among patients admitted to local hospitals in Egypt can be used as a measure for understanding mechanisms leading to the spread of resistance, as a large number of the population rely on these hospitals due to socioeconomic factors.
| ~ Materials and Methods|| |
Statement of ethical approval
All experiments were performed in accordance and approval of the ethical committee at Cairo University, Cairo, Egypt. In addition, all personnel who contributed any samples did this according to their written informed consent.
Clinical urine samples were collected from 100 patients admitted to the outpatient clinics of 'Abou-Elreesh' and 'El-Demerdash Hospitals' (University hospitals, belonging to Cairo University and Ain Shames University, respectively, in Cairo, Egypt). Clean-catch midstream urine was collected in sterile tubes and streaked on cysteine lactose electrolyte deficient agar or (CLED agar) and Enterococcsel agar. Isolates belonging to Enterococcus spp. were identified and stored using agar slants at 4°C; glycerol stocks were made and stored at −70°C. Reference standard of E. faecalis ATCC 29212 was obtained from (Naval Medical Research Unit-3, Cairo, Egypt).
Differentiation of isolates was carried out via Gram stains and cell morphology. Isolates which appeared Gram-positive cocci or coccobacilli cells in pairs or short chains when viewed through a microscope were suspected to be Enterococcus.
Biochemical and cultural identification
Catalase-positive reaction was indicated by a continuous bubble formation when hydrogen peroxide was introduced in bacterial colonies. Identification of Enterococcus isolates was confirmed by growth on 6.5% NaCl broth salt. Culture media used were Enterococcosel Agar HiCrome, E. faecium Agar, MacConkey's no. 2 agar and CLED agar. All media were from (Oxoid, UK) and were prepared by following the instruction of the manufacturers.
Detection of cytolysin production and gelatinase activity
Brain-heart infusion agar (Oxoid) supplemented with 5% sheep blood was used for the detection of cytolysin activity. Pure isolates were cultivated on blood agar plates, and the plates were incubated at 37°C for 24 h. Cytolytic activity was observed as (β) haemolysis surrounding bacterial colonies (complete haemolysis appeared as clear zones). 
Gelatinase assay was carried out by adding an inoculum from a pure culture into tubes containing 12% gelatin in 0.8% Nutrient Broth. Tubes were incubated for 24-72 h at 37°C and then placed in the refrigerator for approximately 30 min. The liquefaction of gelatin was considered as a positive result. 
Determination of antibiotics susceptibility (by Disc diffusion method)
The susceptibility of Enterococcus isolates to different anti-microbial agents was measured in vitro by disc diffusion method. From a pure culture, 5-6 colonies of the organism were transferred to a test tube containing 0.9% saline, and the suspension was standardized by 0.5 McFarland Standard. The entire surfaces of Mueller-Hinton agar (MHA) plates were inoculated with the cultures evenly; antibiotic discs were applied to their surfaces and the plates were incubated at 37°C for 24 h. After incubation, the diameters of the zone of complete inhibition were measured in mm. Isolates were classified as susceptible, intermediate or resistant in accordance with the Clinical and Laboratory Standard Institute (CLSI, 2011).
Determination of minimum inhibitory concentration of Vancomycin
MIC was determined by micro-broth dilution test using sterile 96-well microtitre plates. Antibiotic stock solution was prepared by dissolving vancomycin powder in sterile distilled water, and the concentration was adjusted to 512 μg/ml. A 1:10 dilution of 0.5 McFarland Standard was used; 50 μl each of antibiotic dilutions and organism suspension were mixed and incubated at 37°C for 24 hrs. The highest dilution which inhibited growth was considered MIC. MIC ≥32 μg/ml was considered to be indicative of resistant isolates.
Polymerase chain reaction amplification of virulent and resistance genes
PCR was carried out in a final reaction volume of 25 μl. A master mix containing green buffer, 1.5 mM MgCL 2 , 200 μM of each deoxyribonucleotide, 10 pmol of each primer and 0.5 U Taq polymerase for a minimum of 10 samples was prepared and aliquoted in 22.5 μl quantities in individual PCR tubes. An amount of 1μg sample of DNA was added in each tube, and the final volume was adjusted to 25 μl. A list of primers used is in [Table 1]. ,,
The thermocycling conditions were initial denaturation at 94°C for 2 min, then 30 cycles of denaturation at 94°C for 30 s, annealing (48°C-54°C), depending on the primer pairs, for 30 s and extension at 72°C for 30 s with final extension cycle for 2 min (Techne Gradient, UK). Products were detected by agarose gel electrophoresis using agarose 1.5% W/V gel in 1X TAE buffer. Products were purified by the aid of AxyPrep PCR Clean-up Kit (Axygen, USA) according to manufacturer's instructions in order to be sequenced. DNA sequencing was carried out on five E. faecium and five E. faecalis PCR-positive samples using the Big Dye Terminator v3.1 cycle sequencing kit and Centri-Sep™ spin columns (Applied Biosystems, CA, USA) for cycle sequencing and products purification according to the manufacturer's protocols. Sample electrophoresis was then performed using automated sequencer ABI PRISM 310 Genetic Analyzer, (Applied Biosystems, CA, USA), followed by sequencing analysis using Sequencher software (Genes Codes Inc., MI, USA).
The DNA sequences for gelE, agg, yl, esp and vanA genes were retrieved from the NCBI database ( http://www.ncbi.nlm.nih.gov/ ). The sequenced PCR products were saved in FASTA format for various applications. Multiple sequence alignments were done by the aid of NCBI Blast ( http://blast.ncbi.nlm.nih.gov/Blast.cgi ). Genome wide analysis for pathogenic islands in enterococci was done using IslandViewer  ( http://www.pathogenomics.sfu.ca ), detection of prophages in sequenced bacterial genomes was done by ACLAME prophinder tool  ( http://aclame.ulb.ac.be/Tools/Prophinder ) and cumulative GC skew analysis was done by Genometrics analysis  ( http://www2.unil.ch/comparativegenometrics/ ).
| ~ Results|| |
Screening and isolation of enterococcus isolates
Urine samples were collected from 100 patients admitted to Abu-El Reesh and El-Demerdash hospitals. Out of 100 isolates collected, 73 isolates were identified as Enterococcus sp. Preliminary identification of the suspected isolates was conducted by Gram staining; the isolates showed a Gram-positive cocci or coccobacilli arranged in pairs and short chains. The identification of positive Enterococcus isolates was confirmed by other biochemical tests including catalase test; all the isolates were catalase negative. The ability of the isolates to grow in nutrient broth containing 6.5% NaCl was also confirmed.
Isolates appeared as yellow colonies on CLED agar. Cultivation of the isolates on Enterococcosel agar showed brownish-black colonies surrounded by a black zone, while cultivation on MacConkey no. 2 showed small, intensely red colonies. Identification to species level was done by cultivation on HiCrome E. faecium agar. Twenty-six isolates were identified as E. faecium, producing green colonies along with yellow colouration to the medium and 47 isolates were identified as E. faecalis, producing blue colonies on the media.
Phenotypic identification of virulence factors
Cytolysin production was screened by cultivation on blood agar, nine isolates produced complete (β) haemolysis, eight isolates produced partial (α) haemolysis and 56 isolates did not produce haemolysis on blood agar (g haemolysis). Gelatinase assay by gelatin hydrolysis test revealed that none of the isolates phenotypically expressed gelatinase gene as they could not liquefy gelatin media.
Antimicrobial susceptibility pattern and MIC of enterococcus isolates to various antibiotics
The Enterococcus isolates were tested for resistance to different classes of antibiotics by using disc diffusion method. The antibiotic resistance profiles of the entire isolates collection against 11 antimicrobial agents are summarized in [Figure 1]. MIC values were determined for sensitive and resistant Enterococcus isolates to vancomycin. MIC ranged from 0.25 μg/ml to 256 μg/ml. However, most of the isolates had MIC from 1 μg/ml to 2 μg/ml. MIC results revealed that that two isolates were highly resistant to vancomycin.
|Figure 1: The antimicrobial resistance pattern of Enterococcus isolates expressed as percentage of the total number of collected isolates|
Click here to view
Amplification of virulence and resistance genes of enterococcus isolates by PCR
Genes of virulence and resistance including gelatinase (gelE), aggregation substance (agg), cytolysin (cyl), Enterococcal surface protein (esp) and vancomycin resistance (vanA) of 50 Enterococcus isolates were amplified by PCR. The presence of the virulence and resistance genes detected in 50 Enterococcus isolates is revealed in [Figure 2]. Two isolates carried the four virulence genes. Five isolates carried three virulence genes, 24 isolates carried two of the four virulence genes, 13 isolates carried one of the four virulence genes, two isolates carried the VanA gene and six isolates did not carry any of the virulence or resistance genes. From the 18 isolates that carried the Cyl gene, only nine isolates produced complete (β) haemolysis on blood agar. The gelatinase activity could not be monitored phenotypically in all isolates carrying the gelE gene. The occurrence of the gelE, cyl and agg genes was higher in E. faecalis isolates, while the esp gene was more prevalent in E. faecium isolates. The VanA gene was present only in E. faecium isolates.
|Figure 2: Presence of the virulence and resistance genes detected in E. faecalis and E. faecium separately as percentage of occurrence|
Click here to view
Genometric characterization of horizontally transferred genes
Based on the concept that bacterial genomes tend to naturally have differences in sequence composition such as GC% and codon bias, regions were observed within a genome that has abnormal sequence composition as they could indicate that it had originated from another genome and was horizontally transferred. ,, The genomes of Enterococcus spp. can virtually carry virulence genes that were transferred by either transposon or phage through horizontal gene transfer. The genomes of the standard strains, E. faecalis V583 and E. faecalis 62, were used as model Enterococcus genomes for this purpose (as it was economically prohibitive to sequence the entire genomes of all our clinical isolates). The IslandViewer program was used to identify segments in the genomes that might have originated from horizontal gene transfer and revealed possible candidates as shown in [Table 2] and [Table 3].
Other confirmatory tools used for detection of horizontally transferred genes
The ACLAME prophinder tool was able to identify seven phage or phage-like regions in E. faecalis V583 genome [Figure 3] and [Table 4] which suggests that they originated from horizontal gene transfer. In silico analysis measuring GC skewness indicated four regions (grooves) in the genome (data not shown). These regions indicate different in GC content, suggesting that they originated from a horizontal gene transfer process.
|Figure 3: Prophages predicted in bacterial genome of E. faecalis V583. The boxes represent the location of this phage and phage ID is given to each identified phage|
Click here to view
| ~ Discussion|| |
The increasing incidence of enterococcal infections in recent years suggests that the acquisition of certain virulence factors might play a role in increasing the pathogenesis of these organisms. The present study aimed to investigate the relation between the presence of virulence and resistance factors and the susceptibility of isolates to different antimicrobial agents as well as to explore the possibility of horizontal gene transfer for these genetic elements.
Genotypic detection of virulence genes was done by PCR. A total 36% of isolates were positive for the cyl gene giving a band appeared at 186 bp, although (β) haemolysis was observed in only 18% of the isolates. These findings compare favourably with previous studies where the presence of phenotypic characteristics, such as cytolysin, was lower than that expressed genotypically.  The occurrence of the cyl gene was higher in E. faecalis (39%) isolates than in E. faecium isolates (29%).
PCR detection of the gelE gene revealed that 84% of the isolates (highest percentage) were gelatinase-positive, yielding a band that appeared at 419 bp; however, none of the isolates expressed gelatinase phenotypically. Similar results were previously reported in which none of the gelE gene positive Enterococcus isolates were found to produce gelatinase phenotypically.  The occurrence of this gene was higher in E. faecalis isolates (93%) compared to E. faecium isolates (64%), as shown previously when E. faecalis isolates predominantly harboured the gelE gene (80%) while the gene was less predominant in E. faecium isolates (31.9%). 
The lack of phenotypic activity of the cyl and gelE genes may be explained by low levels or down regulation of gene expression or an inactive gene product. Environmental factors also are known to influence gene expression.  It was suggested by Creti et al., (2004)  , that the presence of genes that are expressed only under in vivo conditions, to the presence of undetected gene mutations or to the fact that detection by PCR of a single gene inside an operon, as is the case of the cylA gene for cytolysin production, may overlook the absence of other genes that are necessary for phenotypic expression.
Genotypic detection of Agg and Esp revealed that the agg gene was present in 3% of the isolates yielding a band that appeared at 1553 bp while the esp gene was present in 42% of the isolates with band appeared at 933 bp. Although the role of the esp gene as virulence factor has been demonstrated,  all the isolates did not express the esp gene, in accordance with previous studies. 
The presence of the gelE, cyl and agg genes was higher in E. faecalis as previously reported, , where E. faecalis isolates were shown to harbour a broader spectrum of virulence determinants compared to E. faecium isolates. The esp gene was more prevalent in E. faecium than in E. faecalis isolates, as observed in previous studies. 
Antibiotic susceptibility tests revealed that 82% of the isolates had multiple antibiotics resistance with resistance to more than three of the antibiotics tested. Although Enterococcus strains harboured multiple antibiotic resistance, there was an elevated sensitivity rates to β-lactams and glycopeptides which deserves particular attention.  Two isolates were highly resistant to vancomycin with MIC equal to 256 μg/ml, while the rest of the isolates had MIC ranged from 1 to 2 μg/ml.
Genotypic detection for the vanA gene using PCR revealed that two isolates were positive for the gene, yielding a band at size 732 bp. The isolates that were positive for the vanA gene also showed a high level of resistance to vancomycin by MIC. This result indicates a low presence of vancomycin resistance in Enterococcus isolates. In a previous study conducted in Egypt,  a low presence of vancomycin resistance (4.2%) among enterococci was demonstrated. It was noticed that the isolates carrying the vanA gene were resistant to all antibiotics used; this result compares favourably with a study conducted in Italy,  in which vancomycin-resistant isolates were also multidrug-resistant. This is of concern, as vancomycin resistance may be transferred to more pathogenic microorganisms. In our study, the presence of the vanA gene was accompanied by the presence of the cyl, esp and gelE genes and absence of the agg gene; however, this is not always true as the vanA gene was previously linked with the presence of the agg gene.  The VanA gene was present only in E. faecium isolates but not in E. faecalis isolates, as observed in other studies. ,
The presence or absence of virulence genes did not affect the pattern of anti-microbial resistance in Enterococcus isolates. Hence, no relation was found between the presence of virulence genes and multiple antibiotics resistance used as previously mentioned.  Sequencing of virulence genes, including cyl, agg, gelE and esp, and resistance gene (vanA) of Enterococcus clinical isolates indicated no mutations in the sequenced fragments.
The data retrieved from sequence analysis revealed that the vanA gene that was present in Enterococcus spp. was also present in Staphylococcus aureus (S. aureus), as both possessed the same sequence. This result suggests that vancomycin resistance was transferred from enterococci to staphylococci since it was previously shown that enterococci transferred the vanA gene to vancomycin-resistant S. aureus (VRSA). 
Enterococci are noted for their capacity to exchange genetic information by conjugation,  and these processes are known to take place in the gastrointestinal tract.  Together with transmissible antibiotic resistance plasmids, virulence factors, such as cytolysin production, and the capacity for adhesion is known to be transmissible by highly efficient gene transfer mechanisms. 
We suggest that the shift of enterococci from a normal inhabitant of human gastrointestinal tract, that is considered medically unimportant to a pathogen causing serious diseases such as endocarditis, bacteraemia, UTIs and central nervous system infection, along with the increase in strains resistant to multiple antibiotics, especially glycopeptides, such as vancomycin, is a result of the fact that certain genes of virulence and resistance were transferred to enterococci. Consequently, we used bioinformatics tools and analyzed the genomes of standard model Enterococcus trains to investigate the possibility of gene transfer.
Bacterial genomes contain clusters of genes that are acquired by horizontal transfer called genomic islands (GIs) that are capable of integration into the chromosome of the host, excision and transfer to a new host by transformation, conjugation or transduction. A GI can code for many functions, can be involved in symbiosis or pathogenesis and may help an organism's adaptation. GI associated with pathogenesis is often called a pathogenicity island (PAI). These 'islands' are characterised by their large size (>10 Kb) and a different G + C content compared with the rest of the genome. Some GIs can excise themselves spontaneously from the chromosome and can be transferred to other suitable recipients.  Detection of GIs was aided by the use of Island Viewer software which showed that the virulence genes in our current study were mainly transferred by transposons.
The presence of phage or phage-like regions was aided by the use of ALCAME prophinder tool that detected seven phage or phage-like regions in E. faecalis V583 genome. By comparing the co-ordination ranges of phage or phage-like regions with those of virulence and resistance genes, we found that these genes were not transferred by prophages. The drawback of this tool is its inability to detect other mobile genetic elements.
GC skewness measurement of E. faecalis V583 genome showed four regions (grooves), indicating different GC content in these four regions thus suggesting horizontally transferred genes. Although this method is a fast tool for detection of horizontally transferred genes, the results it presents are not completely reliable since the skew pattern deviation is detected manually through observation of the grooves along the genome curve, and person-to-person variations may arise.
| ~ Conclusion|| |
genes including Cyl, GelE, Agg and Esp were present in the majority of Enterococcus spp. isolates but only two isolates expressed the four virulence genes. Presence of genes was not usually associated with expression, however, other environmental factors may interfere. Antibiotic resistance was relatively high in clinical isolates with elevated sensitivity to vancomycin. No relation was found between the presence of virulence factors and resistance to different antibiotics used. Bioinformatics analysis of possible gene transfer revealed that transposons are the main elements responsible for horizontal gene transfer among enterococci.
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[Figure 1], [Figure 2], [Figure 3]
[Table 1], [Table 2], [Table 3], [Table 4]