Iranian Journal of Medical Microbiology

Isolation of E. faecium EntfacYE
Twenty-five clinical enterococci were isolated from patients' blood from 2019 to 2020. Sampling was carried out in a university teaching hospital, Tehran, Iran (ethics approval no. IR.TUMS.SPH.REC.1397.139). Of all the enterococcal isolates, one E. faecium strain isolated from a clinical blood sample that three various bacteriophages were isolated on it, was selected in this study (E. faecium EntfacYE) (12). The bacterial strain was cultured on trypticase soy agar (TSA) media for purification. Furthermore, the isolate was verified using morphological, biochemical, and molecular techniques. Assays such as Gram staining, catalase, growth in 6.5% NaCl, and glucose utilization were used for the isolate as well.
Furthermore, the isolates were assessed for resistance to common antibiotics using the disk diff-usion method. The isolate was cultured on Mueller-Hinton agar media and exposed to the selected antibiotics at 37°C for 24 h. The assessed antibiotics included cefoxitin, ceftriaxone, clindamycin, erythr-omycin, linezolid, and vancomycin. After incubation, inhibition zone diameters were measured, and a bacterial resistance pattern was reported.

Sanger Partial Sequencing

Enterococcus faecium EntfacYE Genome Analysis

The E. faecium EntfacYE genome was extracted using ethanol and propanol (precipitation method). Then, the extracted genome was wholely sequenced using Illumina Hiseq platform (Novogene, China) and de novo genome assembly as well as SPAdes algorithm. The bacterial genome was compre-hensively analyzed using RAST (rapid annotation using subsystem technology) online sequence analysis service (https://rast.nmpdr.org).

In recent decades, the resistance of most pathogens such as enterococci to available antibiotics has broadly increased. Enterococci cause a variety of infections, including opportunistic infections in hospitalized patients. In fact, E. faecium is one of the most important causes of nosocomial infections. Based on the reports, the mortality rate due to enterococcal bacteremia is estimated as 15–35% (14). In the present study, 23% of the bacterial genome was covered by subsystems, including various categories (Figure 1). The highest proportion was linked to the carbohydrates and protein metabolism groups, similar to a recent study on E. faecium R.A73 genome by Jeni et al. (2020) (15). Results from the present study demonstrated that the E. faecium EntfacYE strain isolated from a clinical blood sample was resistant to vancomycin, erythromycin, clindamycin, cefoxitin, and ceftriaxone and included various antibiotic-resistance genes. In recent decades, enterococcal resistance to vancomycin has increased, causing serious problems in treating infections (16, 17). In 2015, O’Toole et al. reported significant increases in vancomycin-resistant E. faecium isolates from Royal Hobart Hospital of Australia. Their study presented a shifting epidemic-ology pattern of VREfm from sporadic to endemic. They also showed the utility of isolates from asymptomatic patients to link epidemiological gaps between the patients (18). Lytsy et al. compared whole-genome sequences of vancomycin-resistant enterococci in three suspected outbreaks in Sweden during 2013–2015 using PFGE and MLST. They concluded that WGS-ANI was an easy method for this purpose. It was a user-friendly method, compared to MLST and PFGE. They recommended using WGS-ANI instead of PFGE for epidemiological outbreak investigations (19).

 
 
Figure 1. EntfacYE genome subsystem analysis using RAST

 
Based on the results from molecular studies, E. faecium is more resistant to routine antibiotics compared to E. faecalis, in which more than half of the enterococcal pathogens are resistant to common antibiotics such as vancomycin, ampicillin, and aminoglycosides (20). In this study, vancomycin resistance in EntfacYE isolate was due to the presence of van genes in the bacterial genome. A study in Iran showed that 12 out of 22 E. faecium isolates were resistant to vancomycin (21). In another study by Arbabi et al. (2016), more than half of E. faecium isolates were resistant to vancomycin (22). Active efflux pump is a common mechanism responsible for erythromycin resistance (23); thus, the presence of genes encoding efflux proteins in EntfacYE might lead to erythromycin resistance. In a study in Iran, 64% of enterococcal isolates, including E. faecalis and E. faecium, demonstrated resistance to erythromycin (24). Enterococci are intrinsically resistant to clinda-mycin; however, no relevant mechanisms have already been described (25). In this study, clindamycin resistance was associated with the ABC protein family. In a study in 2020, two genes involved in clindamycin resistance were identified in Bacillus licheniformis and Bacillus paralicheniformis (26). In the current study, resistance to cefoxitin and ceftriaxone was due to the presence of beta-lactamase genes. In a study by Rengaraj et al. (2016), 49% of S. aureus isolates were resistant to cefoxitin (27). In the present study, fosfomycin resistance genes were detected. This antibiotic is an alternative antibiotic against multi-drug-resistant bacteria (28). In a study on Pseudo-monas stutzeri, Soltani et al. (2015) reported that soils contaminated with heavy metals were potential sources for isolating metal-resistant strains (29). As heavy metals’ level increases in the environment due to modern industrial and agricultural activities, bacteria develop strategies to decrease intracellular concentrations of these toxic contaminants. There-fore, environments contaminated with heavy metals naturally contain microorganisms capable of resisting these metals (30). Plasmids mostly carry resistance-related genes and could spread resistance genes between bacteria through MGEs (e.g., the spread of plasmid genes from cadmium-resistant bacteria to other bacteria). In fact, heavy metal resistance is often associated with plasmids. However, this resistance is sometimes chromosomally originated (31). Copper homeostasis subsystem in E. faecium EntfacYE was reported in Salmonella enterica and Mycobacterium tuberculosis in 2019 (32).
Furthermore, bile salt hydrolysis determinants in E. faecium EntfacYE were investigated in Enterococcus strains isolated from artisanal dairy products in a study by Nami et al. (2019) (33). The presence of heavy metal resistance genes such as copper-zinc, cadmium, and mercury in the bacterial genome could be due to the gene transfer through plasmids or other MGEs. Accurate identification of Enterococcus species and their antibiotic resistance patterns is critical in providing effective treatments and appropriate medications (28, 34).

In this study, E. faecium EntfacYE genome analysis was carried out, focusing on antimicrobial resistance encoding genes. Further analysis of bacterial genomes, especially antibiotic resistance genes, can open new horizons for discovering novel medications other than common antibiotics and efficiently prevent the further spread of bacterial resistance.

 

The authors thank all the staff within the Microbiology Laboratory, School of Public Health, Tehran University of Medical Sciences for their help.
 

None.

 

Conflicts of Interest

The authors declared no conflict of interest.