Introduction

Enterococci are commensals of the gastrointestinal tract, prevalent in environmental, human and nosocomial settings. {Bello Gonzalez, 2017 #1} Enterococci are equipped with the capabilities to cause infections in the patients within the healthcare facilities; for example the patients with lower immune system and those with serious injuries and trauma, particularly if the patient has previously been treated with antibiotics.^1-3 Enterococcus faecalis and Enterococcus faecium are the two species involved in human nosocomial infections compared to other members of the genus enterococci.⁴ The E. faecium has been proved to cause a number of infections such as, bacteremia, UTI, intra-abdominal infections, skin and soft tissue infections and endocarditis.^5-9 In addition of harboring virulence genes, the development of multi-drug resistance among enterococci further complicates the situation regarding the treatment options and poses a challenge to healthcare professionals. The development of vancomycin-resistance in Enterococcus faecium (VRE) in 1986 followed by the dissemination of this highly resistant and virulent pathogen globally is one of the most challenging aspects of antimicrobial resistance among enterococci.¹⁰ Extensive use of vancomycin within the hospitals worldwide is considered to be the major factor for the spread of nosocomial VRE.^10,11 Vancomycin, resistance (intrinsic and acquired) among enterococci has been studied in detail. Eight types of acquired vancomycin resistance (vanA-vanN) have been reported among enterococci so far, with vanA being the most prevalent genotype worldwide followed by vanB.¹² The difference between expression levels of vancomycin resistance vary and are found to be low in VanB type strains and high in VanA type strains. Because of the low expression vancomycin resistance levels, VanB type strains are characterized by resistance to vancomycin and susceptibility to other glycopeptides like teicoplanin. VanA strains are found to be resistant to all glycopeptides due to the higher expression of vancomycin resistance type. During recent years, there is a significant increase in vanB genotype E. faecium colonization and infections in many European countries.^13,14 Among these outbreaks, a recent outbreak of VanB strains in Sweden reported transferable pRUM-like plasmids harboring vanB2-Tn5382 elements.¹⁵ Furthermore, a mobile genetic element called transposon1546 (Tn1546), which is involved in VanA-type E. faecium vancomycin resistance was reported by French researchers in 1986.¹⁶ Subsequently, this transposon was found to be present in enterococci from humans, animals and the environment.^16-19

The molecular characterizations of vancomycin resistance among E. faecium have been reported in several studies from Saudi Arabia. However, none of the study has been reported from Ha’il region, therefore the current study aimed to characterize vancomycin resistant E. faecium isolated from patients attending Maternity Hospital, Ha’il Saudi Arabia during 2014.

Materials and Methods

Bacterial Isolates

In this study, 26-E. faecium bacterial strains were cultured from clinical specimens collected from patients attending Maternity hospital, Ha’il, Saudi Arabia. The specimens were collected from, axilla (15.4%), blood (11.5%), fistula (23.2%), groin (30.8%), HVS (3.8%), nose (3.8%) and urine (11.5%).

Identification by MALDI-TOF-MS

MALDI-TOF-MS (Bruker Daltonics, Germany), one of the most advanced automated systems was used for identification of bacterial strains.²⁰ In this method, a fresh bacterial colony from overnight culture was smeared on target plate draped with 1 μl of a saturated a-cyano-4-hydroxy-cinnamic acid (HCCA) matrix solution in 50% acetonitrile-2.5% trifluoroacetic acid (Bruker Daltonics) with the help of a sterile toothpick. The target plate was kept at room temperature until dry. The plate was loaded in to the machine and the operation was run. The identification of bacterial strains was performed by using MALDI Biotyper software package (version 3.0).

Identification and Antibiotic Susceptibility by Microscan

Microscan walkaway (Siemens Healthcare Diagnostics, Sacramento, CA, USA); an automated system used for bacterial identification and antibiotic susceptibility test was used for confirmation of identification and antimicrobial susceptibility of the bacterial strains. In this method, a small portion of a well isolated colony was taken and added to a Gram-positive Microscan combo panel. The panel was loaded into the Microscan walkaway machine according to the manufacturer’s protocol. Results were available after 24- 48 hrs.

Whole Genome Sequencing

The sequencing of the bacterial genome for detection of antibiotic resistant genes, virulence factors, plasmids and MLST types was performed by using Illumina methodology using NextEra kit for library preparation.²¹ The fasta files of the sequences were used for the analysis and the ResFinder web server (www.genomicepidemiology.org) and Basespace from Illumina was used to identify acquired antimicrobial resistance genes, MLST types and the presence of different plasmids in the WGS data, using a threshold of 98% identity.

Results

In this study, the characterization of 26-E. faecium isolates collected from the patients attending a hospital in Ha’il region of Saudi Arabia was performed. The antibiotic susceptibility results (Table 1) revealed that 99%, 90%, 83% and 73% of the E. faecium isolates were resistant to Clindamycin, Gentamicin, Oxacillin and Tetracycline respectively. Additionally, 3.8% (1/26) E. faecium were found to be vancomycin resistant. The data revealed that a high percentage of clindamycin, erythromycin, and gentamicin resistance were observed in all E. faecium isolates. Additionally, the resistance of VRE to ciprofloxacin, clindamycin, erythromycin, gentamicin, tetracycline and trimethoprim/sulfamethoxazole was significantly higher in comparison to the vancomycin susceptible E. faecium isolates.

The whole genome sequencing results of the sole VRE are presented in Table 2 and Table 3. The data showed that VRE from current study exhibited VanB type of resistance with MLST type 280. Additionally, this VRE revealed the aminoglycosides, macrolides, tetracycline and vancomycin resistant genes. It also exhibited acm a virulence factor (collagen binding protein) similar to adhesion-associated protein EfaA (E. faecalis endocarditis antigen A).

Table 1: Antibiotic susceptibility patterns of 26-E. faecium isolates.

Table 2: Sequence Type: ST280 (MLST Scheme: E. faecium) of 1- vancomycin resistant E. faecium.

Table 3: Different antibiotic resistance genes of vancomycin resistant E. faecium using NGS.

Discussion

E. faecium is a member of normal flora of the gastrointestinal tract of humans and animals. The E. faecium are prevalent in human, environmental and healthcare settings, {Bello Gonzalez, 2017 #1}and generally are not virulent.^2,3 Multi-drug resistant E. faecium have become increasingly common in the hospital settings and it poses a challenge to clinicians. MDR- E. faecium including VRE have been reported worldwide including Saudi Arabia.^1,22-24 The aim of current research was, molecular characterization including whole genome sequencing of E. faecium isolates collected from patients attending a hospital in Ha’il, Saudi Arabia. This study is important because of capability of E. faecium specifically VRE to produce outbreaks and severe infections in hospital settings, which put the patients and the staff at a high healthcare risk. The E. faecium has been proved to cause a number of infections such as, bacteremia, UTI, intra-abdominal infections, skin and soft tissue infections and endocarditis.^5-9 The development of multidrug resistance among these bacterial isolates further complicate the treatment options and one of the most important clinically significant antimicrobial resistance is development of vancomycin-resistant E. faecium (VRE). VRE is highly resistant to large number of available antibiotics and this potentially difficult-to-treat pathogen is now being reported all across the world.¹⁰ In our study, molecular characterization of 26-E. faecium isolates (including a single VRE) was performed. The antibiotic susceptibility revealed that 99%, 90%, 83% and 73% of the E. faecium isolates were resistant to Clindamycin, Gentamicin, and Tetracycline respectively. Additionally, 3.8% (1/26) E. faecium were found to be vancomycin resistant. The data revealed that a high percentage of clindamycin, erythromycin, and gentamicin resistance were observed in all E. faecium isolates. Additionally, the resistance of VRE to ciprofloxacin, clindamycin, erythromycin, gentamicin, tetracycline and trimethoprim/sulfamethoxazole was significantly higher in comparison to the vancomycin susceptible E. faecium isolates. Although, there are several studies about the molecular characterization and prevalence of VRE conducted in Saudi Arabia, there is no report of prevalence of VRE from Ha’il region. The studies conducted in this aspect used PCR, MLST and PFGE approaches,^22-24 with a recent paper from Makkah region using the whole genome sequencing.¹ As per our knowledge we report the first VRE from Ha’il region. We performed the characterization of the only VRE in our study by whole genome sequencing using MiSeq platform. The whole genome sequencing result of E. faecium revealed that a genome size of 2861776 bp with 874 contigs was successfully sequenced. The MLST data revealed that the VRE from our study belong to ST type 280. MLST 280 is part of cluster of E. faecium MLST genotype called clonal complex CC17, which has been found to be associated with global nosocomial infections.^22-25 Additionally, the res finder showed that our VRE contained the genes which exhibited the resistance to aminoglycosides, macrolides, tetracycline and vancomycin. Furthermore, the result of current study showed that the VRE harbored vanH-B, vanW-B, vanX-B, vanY-B, vanS-B vanR-B and van-B genes, which are known to confer vancomycin resistance. VanB type of VRE has been previously reported from Saudi Arabia.24 The vanB-type genes have been found to confer resistance to vancomycin and not to teicoplanin in comparison to vanA-type genes which confer resistance to both teicoplanin and vancomycin. Thus, the prevalence and dissemination of resistant enterococci, specifically VRE is a challenging health care problem faced by health care professionals in all geographical areas.

Conclusion

This is the first report of VRE from the Ha’il region of Saudi Arabia. VRE may act as a reservoir for multidrug resistant genes and other important virulence factors that favor the dissemination of antimicrobial resistance. Therefore, the surveillance studies to prevent the dissemination of VRE shall be implemented in the healthcare facilities all across Saudi Arabia.

Acknowledgements

The authors would like to thank King Abdulaziz City for Science and Technology (KACST), General Directorate of Research Grants for funding this study.

Conflict of Interest

There is no Conflict of Interest

Funding

This study was funded by King Abdulaziz City for Science and Technology (KACST), General Directorate of Research Grant number 143-34.

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