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Khan M. A, Al-Rashid F, Meqdam M, Al-Ajlan H, Al-Mogbel M. Whole Genome Sequencing of Vancomycin Resistant Enterococcus Faecium Isolated from Saudi Arabia. Biosci Biotech Res Asia 2018;15(4).
Manuscript received on : 16-Dec-2018
Manuscript accepted on : 28-Dec-18
Published online on:  28-12-2018

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Whole Genome Sequencing of Vancomycin Resistant Enterococcus Faecium Isolated from Saudi Arabia

Mushtaq Ahmad Khan1, Fauwaz Al-Rashid2, Mamdoh Meqdam1, Hisham Al-Ajlan3 and Mohammed Al-Mogbel1

1Molecular Diagnostic and Personalized Therapeutic Unit, College of Applied Medical Sciences, University of Ha’il, Kingdom of Saudi Arabia.

2King Khalid Hospital, Ha’il, Kingdom of Saudi Arabia.

3Prince Sultan Military Medical City, Riyadh, Kingdom of Saudi Arabia.

Corresponding Author E-mail: drmushtaqkhan9@gmail.com

DOI : http://dx.doi.org/10.13005/bbra/2687

ABSTRACT: Enterococcus faecium are one of the most prevalent species cultured from humans and they have become increasingly common cause of infections in the hospital settings globally. The objective of current study was to characterize 26 E. faecium isolates collected from different patients attending Maternity Hospital in Ha’il, Saudi Arabia. The bacterial isolates were identified by MALDI-TOF-MS and the antibiotic susceptibility was performed by Microscan. Whole genome sequencing of a single vancomycin resistant E. faecium (VRE) was performed using MiSeq. The results of antimicrobial susceptibility revealed that, 99%, 90%, 83% and 73% of isolates were resistant to Clindamycin, Gentamicin, Oxacillin and Tetracycline respectively. One (3%) among 26 E. faecium isolates was found to produce resistance to vancomycin. The WGS analysis of VRE showed that it belonged to ST280 and was found to harbor vanB gene cassette. 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 dissemination of VRE shall be implemented in the healthcare facilities all across Saudi Arabia.

KEYWORDS: Multi-Drug Resistant; VanB; Virulence; VRE; Whole Genome Sequencing

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Khan M. A, Al-Rashid F, Meqdam M, Al-Ajlan H, Al-Mogbel M. Whole Genome Sequencing of Vancomycin Resistant Enterococcus Faecium Isolated from Saudi Arabia. Biosci Biotech Res Asia 2018;15(4).

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Khan M. A, Al-Rashid F, Meqdam M, Al-Ajlan H, Al-Mogbel M. Whole Genome Sequencing of Vancomycin Resistant Enterococcus Faecium Isolated from Saudi Arabia. Biosci Biotech Res Asia 2018;15(4). Available from: http://www.biotech-asia.org/?p=32483

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.

Antibiotic Resistance %
Augmentin 3.08
Ampicillin 4.55
Ciprofloxacin 28.79
Clindamycin 98.44
Daptomycin 0
Erythromycin 62.5
Fosfomycin 0
Fucidin 14.06
Gentamicin 89.23
Levofloxacin 25.76
Moxifloxacin 28.79
Nitrofurantoin 3.03
Oxacilin 82.81
Penicillin 7.94
Rifampicin 26.56
Teichoplanin 6.25
Tetracyclinee 72.73
Trimethoprim/Sulfamethoxazole 36.36
Vancomycin 4.69

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

Gene %Identity Alignment Length DB Allele Length Gaps Best Match
adk 100 437 437 0 adk_1
atpa 100 556 556 0 atpa_1
ddl 100 465 465 0 ddl_3
gdh 100 530 530 0 gdh_1
gyd 100 395 395 0 gyd_1
psts 100 583 583 0 psts_1
purk 100 492 492 0 purk_1

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

Resistance gene % Identity Allele/ Alignment length Contig ID Position in Contig Phenotype Accession number
Aph(3’)-III 100 795/795 NODE_339_length_2181_COV_140.196243 356..1150 Aminoglycoside resistance M26832
aadE 100 864/509 NODE_331_length_1301_COV_195.631058 823..1331 Aminoglycoside resistance KF421157
lnu(B) 99.88 804/804 NODE_171_length_6626_COV_45.661785 1348..2151 Lincosamide resistance AJ238249
msr(C) 98.99 1479/1479 NODE_363_length_5831_COV_19.333733 1825..3303 Macrolide,Lincosamide and Streptogramin B resistance AY004350
Erm(B) 100 738/738 NODE_37_length_1642_COV_111.984169 272..1009 Macrolide resistance AF299292
Tet(M) 96.46 1920/1920 NODE_127_length_20046_COV_45.256859 11316..13235 Tetracycline resistance EU182585
Tet(L) 100 1377/1377 NODE_127_length_20046_COV_45.256859 13429..14805 Tetracycline resistance M29725
vanH-B 99.49 972/972 NODE_41_length_30848_COV_24.928553 2001..2972 Vancomycin resistance AF192329
vanW-B 97.58 828/828 NODE_41_length_30848_COV_24.928553 2969..3796 Vancomycin resistance AF192329
vanX-B 96.72 609/609 NODE_41_length_30848_COV_24.928553 366..974 Vancomycin resistance AF192329
vanY-B 100 807/807 NODE_41_length_30848_COV_24.928553 3814..4620 Vancomycin resistance AF192329
vanS-B 99.85 1344/1344 NODE_41_length_30848_COV_24.928553 4791..6134 Vancomycin resistance AF192329
vanR-B 99.25 663/663 NODE_41_length_30848_COV_24.928553 6134..6796 Vancomycin resistance AF192329
vanB 99.13 1029/1029 NODE_41_length_30848_COV_24.928553 980..2008 Vancomycin resistance AF192329

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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