Molecular epidemiology of vancomycin-resistant Enterococcus faecium clinical isolates in a tertiary care hospital in southern Thailand: a retrospective study

Introduction

Vancomycin-resistant enterococci (VRE) are nosocomial pathogens that are responsible for causing a large number of hospital-acquired infections worldwide. These organisms affect the bloodstream, urinary tract, and surgical wounds and cause intra-abdominal and intra-pelvic abscesses (Reyes, Bardossy & Zervos, 2016). Hospitalized patients who have been using certain antibiotics or are on dialysis for a long time are at an increased risk for VRE colonization and infections (Arias, Contreras & Murray, 2010; Howden et al., 2013). Enterococci VRE are resistant to commonly used antibiotics, particularly aminoglycoside, ampicillin, vancomycin, and others that affect cell walls (Billington et al., 2014; Puchter et al., 2018).

With the increase in VRE infections among hospitalized patients, the control of VRE prevalence in healthcare units has become a significant concern for public health experts and for our hospital.

Enterococcus faecium and E. faecalis are the most common enterococcal species causing nosocomial infections (Reyes, Bardossy & Zervos, 2016). According to Thailand’s National Antimicrobial Resistance Surveillance Center, the prevalence of E. faecium increased from 0.8% to 9.9% between 2012 and 2019, but the occurrence of E. faecalis has been stable for 10 years (0.4% in 2018) (NARST, 2020).

Using molecular techniques, genes associated with enterococcal virulence can be used for identifying, controlling, and preventing E. faecium and E. faecalis transmission, as opposed to using biochemical tests that are expensive and time-consuming (Bhatt et al., 2015; Fang et al., 2012; Ulu-Kilic et al., 2016). These virulence factors include asa (aggregation substance), gel (gelatinase), cyt (cytolysin), esp (enterococcal surface protein), hyl (hyaluronidase), cpd (sex pheromone determinant), and ebp (endocarditis and biofilm-associated pilus subunit A).

Esp from E. faecalis contributes to biofilm formation and colonization (Pillay, Zishiri & Adeleke, 2018), and hyl influences nasopharynx and lung inflammation (Jackson, Fedorka-Cray & Barrett, 2004). The molecular techniques used for the identification of Enterococcus species are multilocus sequence typing, pulsed-field gel electrophoresis (PFGE) (Nasaj et al., 2016), and enterobacterial repetitive intergenic consensus-polymerase chain reaction (ERIC-PCR), which is a simplified typing method for hospital-based epidemiology (Shahi et al., 2020).

No reports are available on the molecular analysis or epidemiology of E. faecium and E. faecalis, and there are limited data on the possible relationship between the prevalence of virulence. This study investigates the incidence rate and genetic relationship of E. faecium and E. faecalis. We analyze the prevalence of resistance genes using clinical samples collected from a tertiary care hospital in southern Thailand during an 8-year period.

Materials & Methods

Results

Detection of van determinants and virulence factor genes in VRE

Ninety VRE strains (100%) harbored the vanA gene, whereas the vanB, vanC, vanD, vane, and vanG genes were not found in any of these isolates. The esp and hyl genes were found in 82 (91.1%) and 5 (5.6%) isolates, respectively. However, gel, cyt, cpd, and ebp genes were absent from all isolates. The amplicons of the VRE isolates and resistance-gene distribution are shown in Fig. 1.

ERIC-PCR analysis

The ERIC-PCR patterns of the 90 VRE isolates are shown in Fig. 2. The number of bands in each varied from one to nine, and the ERIC fragment sizes ranged from 100 bp to 1.5 kb. Genomic typification by ERIC-PCR revealed that 51 VRE isolates (56.7%) were grouped into 14 clusters (A–N) at a similarity level of 95%. The predominant A genotype contained 12 isolates; B genotype, six isolates; C–E genotypes, four isolates; F–H genotypes, three isolates; and I–N genotypes, two isolates. Thirty-nine isolates were uniquely genotyped. MDR and XDR isolates were distributed among all scattered patterns, except for the three genotypes of the XDR pattern (J, L, and N) (Table 6). The heterogeneity among the isolates obtained from urinary tract infections was stronger than those from other infection sites.

Table 6:

ERIC-PCR pattern, source, hospital collection location, antibiogram profile, and isolation date of multidrug-resistant (MDR) and extensively drug-resistant (XDR)-VRE strains from hospitalized patients at Songklanagarind Hospital, southern Thailand.

Code of isolates	ID	source	ward	Male (M) Female (F)	age	Collection date (day/month/ year)	Resistance genes	Resistance patterns	ERIC types
5	874-3-PSU	rectal	surgical ward	M	70	05032014	vanA, esp	MDR	A
20	1949-PSU	rectal	medical ward	M	60	28072011	vanA, esp	MDR	A
22	1114-PSU	blood	surgical ward	F	64	07082014	vanA, esp	MDR	A
24	198-PSU	body fluid	surgical ward	M	67	02082014	vanA, esp	MDR	A
26	1949-PSU	rectal	medical ward	M	60	13082014	vanA, esp	MDR	A
29	1007-PSU	rectal	medical ward	M	87	06082014	vanA, esp	MDR	A
34	4129-PSU	tissue	intensive care units	F	53	28022012	vanA, esp	XDR	A
40	5056-PSU	urine	medical ward	F	88	29032012	vanA, esp	XDR	A
79	5296-12br-PSU	body fluid	medical ward	M	73	24122018	vanA, esp	MDR	A
82	2484-1u-PSU	urine	medical ward	F	74	11012019	vanA, esp	MDR	A
83	7028-10u-PSU	urine	surgical ward	M	70	30102018	vanA, esp	MDR	A
84	2079-4u-PSU	urine	medical ward	F	44	11042018	vanA, esp	MDR	A
1	3044-PSU	rectal	medical ward	M	73	17032014	vanA, esp	MDR	B
2	3576-PSU	rectal	surgical ward	M	90	20032014	vanA, esp	MDR	B
11	3180-8-PSU	urine	medical ward	F	38	19082015	vanA, esp	MDR	B
42	696ii-PSU	blood	medical ward	F	63	04122013	vanA, esp	MDR	B
49	580-PSU	body fluid	intensive care units	F	21	04022013	vanA, hyl	XDR	B
77	5296-PSU	body fluid	medical ward	M	73	24122018	vanA, esp	MDR	B
3	3374II-PSU	blood	medical ward	F	23	20022014	vanA, esp	MDR	C
8	3384-PSU	rectal	surgical ward	F	71	19032014	vanA, esp	MDR	C
9	3576-PSU	rectal	surgical ward	F	90	20032014	vanA, esp	MDR	C
16	2725-PSU	tissue	medical ward	M	67	15032016	vanA, esp	XDR	C
37	1879-PSU	blood	medical ward	M	86	10032011	vanA, esp	XDR	D
69	874-PSU	rectal	surgical ward	M	70	05032014	vanA, esp	MDR	D
80	940-1u-PSU	urine	medical ward	F	85	04012019	vanA, esp	MDR	D
85	5005-12u	urine	medical ward	F	83	23122018	vanA, esp	MDR	D
53	762-PSU	body fluid	medical ward	M	37	05022014	vanA, esp	XDR	E
54	176-PSU	blood	gynecology ward	F	25	01112013	vanA	MDR	E
58	1243-PSU	MSU	gynecology ward	F	70	08052013	vanA, esp	XDR	E
63	135-PSU	body fluid	operating room	F	64	01052012	vanA	XDR	E
74	3784-PSU	rectal	medical ward	M	46	21032014	vanA, esp	MDR	F
75	32-PSU	urine	medical ward	F	79	01062012	vanA, esp	XDR	F
76	5552-1 I-PSU	blood	intensive care units	F	0.25	24012019	vanA, esp	MDR	F
52	1134-PSU	rectal	gynecology ward	F	52	07112013	vanA, esp	MDR	G
59	3236-PSU	MSU	medical ward	F	50	18102016	vanA, esp	XDR	G
60	977-PSU	MSU	medical ward	F	90	6102016	vanA, hyl	XDR	G
61	1630-PSU	body fluid	medical ward	F	74	10092013	vanA, esp	MDR	H
62	4399-PSU	urine	surgical ward	F	28	29042012	vanA, esp	XDR	H
65	3093-PSU	rectal	gynecology ward	F	52	18112013	vanA, esp	MDR	H
50	4215-PSU	urine	medical ward	F	68	22042012	vanA, esp	MDR	I
51	5324-PSU	body fluid	surgical ward	M	66	30082012	vanA, esp	XDR	I
12	3672-PSU	urine	surgical ward	F	79	20022015	vanA, esp	MDR	J
33	1329-PSU	rectal	medical ward	M	74	08082014	vanA, esp	MDR	J
46	4006-PSU	urine	medical ward	F	73	24122013	vanA	MDR	K
48	3209-PSU	body fluid	medical ward	M	66	19082012	vanA, esp	XDR	K
41	910-PSU	MSU	medical ward	F	85	06082012	vanA, esp	MDR	L
43	2000-PSU	MSU	gynecology ward	F	42	11112013	vanA	MDR	L
32	198-PSU	body fluid	surgical ward	M	63	02082014	vanA, esp	MDR	M
57	5245-PSU	pelvic	operating room	F	19	28092016	vanA, esp	XDR	M
13	3398-PSU	urine	surgical ward	M	78	18092015	vanA, esp	MDR	N
15	4190-PSU	tissue	medical ward	F	38	25042017	vanA, esp	MDR	N

DOI: 10.7717/peerj.11478/table-6

Notes:

MSU

mid-stream urine

BioSample accessions: SAMN18201951, SAMN18201952, SAMN18201953, SAMN18201954, SAMN18201955, SAMN18201956, SAMN18201957, SAMN18201958, SAMN18201959, SAMN18201960.

Discussion

Enterococci significantly contribute to nosocomial infections. These infections most often occur in health care settings, particularly in intensive care units (ICU). They can spread by person-to-person contact or from contaminated medical devices (Olawale, Fadiora & Taiwo, 2011). Control measures to prevent the spread of enterococci infections are essential. To monitor the spread of enterococci infections at our hospital, samples collected from patients are routinely screened by conventional biochemical tests for nosocomial enterococci (resistant or sensitive to vancomycin). The prevalence of VRE among the nosocomial enterococci isolates in our study was 0.2%–4.3%, resulting in an average prevalence of 1.2%.

The prevalence of VRE isolates in our study was relatively low compared with that found in India (7.0%) (Bhatt et al., 2015) and in Egypt (6.3%) (Said & Abdelmegeed, 2019), indicating that risks of VRE infections may be associated with geographic variability. Blanco et al. (2017) reported in 2017 that relative humidity and geographical location were factors affecting VRE colonization rates in adult patients admitted to ICUs in the United States. Increasing humidity resulted in a greater VRE colonization rate, and VRE colonization was lower in southern states. In developing countries, hospitals could inadvertently be increasing the risks of VRE colonization and infections because of the lack of oversight procedures for transferring patients from hospitals with VRE outbreaks (Kang et al., 2014; Resende et al., 2014).

Risk factors for severity of the patient’s condition, hemodialysis, and central venous catheter usage and in univariate analysis include male gender, alcohol use, and antibiotics. Hospitalization in certain units of the hospital and lack of isolation measures are primary factors that affect VRE acquisition (Fossi Djembi et al., 2017). Our study demonstrated similar results after our 90 patients, whose test samples were positive for VRE, were paired with demographic and clinical data from the patient database. Nearly 50% of VRE carrier patients were ≥65 years old, with a median age of 64 years, and were admitted to the medical ward. In a 2019 study by Mathis et al., older patients had the highest infection during VRE outbreak in 2013–2014 at a teaching hospital in Lyon, France (Mathis et al., 2019).

In addition, positive VRE samples were detected in urine specimens (28.9%), followed by those obtained from body fluid (20.0%) and the rectal area (20.0%). These results were similar to a Brazilian study of VRE infection, where the highest VRE concentrations were isolated from patients with urinary tract infections (Resende et al., 2014). Vancomycin-resistant E. faecalis is a significant cause of nosocomial infections stemming from the urinary tract, surgical wounds, and catheter use and affecting the bladder, prostate, and kidneys (Zalipour, Esfahani & Havaei, 2019).

Because VRE are multidrug-resistant organisms with limited therapeutic alternatives that result in increased morbidity and mortality in patients (Buetti et al., 2019; Zalipour, Esfahani & Havaei, 2019), we examined the 90 VRE isolates in our study for antimicrobial susceptibility. The findings showed resistance to β-lactam, glycopeptide, and fluoroquinolone antibiotics in most of the tested isolates, with the highest resistance rate to ampicillin (74.4%) and imipenem (62.2%).

Enterococci have intrinsic resistance to several classes of antibiotics that inhibit cell wall synthesis, such as β-lactams and glycopeptides, and they exhibit the ability to acquire new mechanisms of resistance, particularly to penicillin/ampicillin, aminoglycosides (high-level resistance), and glycopeptides (Cetinkaya, Falk & Mayhall, 2000; Gagetti et al., 2019; Werner et al., 2008). Results also showed susceptibility to meropenem in 10 of 15 tested isolates and 7 of the 21 tested isolates were susceptible to gentamicin. Meropenem and gentamicin are β-lactam and aminoglycoside antibiotics, respectively, and combinations of meropenem and aminoglycosides could function as alternative therapies for multi- and VRE infections. Similarly, there is evidence that β-lactams in combination with daptomycin reduced bacterial activity (Smith et al., 2015). Moreover, this combination of antibiotic therapy was successful in the treatment of endocarditis/severe MDR enterococcal infection (Antony et al., 1997; El Rafei et al., 2018).

E. faecium and E. faecalis are most frequently detected, and E. durans, E. avium, E. casseliflavus, E. hirae, E. gallinarum, E. raffinosus, and E. muntdii are less commonly detected (Agudelo Higuita & Huycke, 2014; Zaheer et al., 2020). In this study, all the detected VRE isolates were identified as E. faecium, a species rarely found in Songklanagarind Hospital. Other studies have found that vancomycin-resistant genetic elements can transfer between enterococcal species (Murray, 1998). Most VRE isolates were MDR, and only six VRE isolates were extensively drug-resistant (XDR). All VRE isolates in this study were vanA E. faecium strains, whereas vanB, C, D, and E were not detected, similar to the findings from a 2014 report from (Resende et al., 2014).

Enterococcal virulence that results in pathogenicity is considered a multifactorial process (Comerlato et al., 2013). Virulence factors promoting the emergence of hospital-acquired enterococcal infections have been reported in a number of studies. In our study, VRE strains originally isolated from urine specimen expressed a high frequency of the esp gene (91.1%), confirming the important role of esp in enterococcal colonization and biofilm formation in urinary tract infections (Comerlato et al., 2013). This finding was comparable to that of other investigators who reported that the incidence levels of esp in E. faecium isolates were 71.5% in northwest Iran (Sharifi et al., 2012) and 90% in China (Yang et al., 2015) but rather low in southwest Iran (57.5%) (Arshadi et al., 2018).

Other virulence factors, hyl, asa, gel, and cyt genes, were also analyzed. The hyl gene was detected in only five (5.6%) VRE isolates in our study, similar to the findings from Haghi et al. who found the hyl gene in 3 of 79 (3.8%) VRE isolates in northwest Iran (Haghi, Lohrasbi & Zeighami, 2019). We did not detect the asa, gel, and cyt genes, confirming that esp and hyl are primary virulence factors among clinical strains of vanA E. faecium and play an important role in pathogenesis.

Molecular techniques for typing bacteria can vary when comparing standardization, cost, reproducibility, discriminatory power, and interpretation. We found that ERIC-PCR is a straightforward procedure for distinguishing organisms that are closely related. This method is less expensive and faster than many other methods, such as PFGE and amplified fragment length polymorphism typing (Borgmann et al., 2007).

The ERIC-PCR results identified 14 clusters among the 51 VRE isolates. The majority of the isolates were clustered in the A genotype, mostly from urine samples, and collected from the medical ward, and these were resistant to ampicillin, gentamicin, and carbapenem, similar to a report from Columbia (Corredor et al., 2019). We identified 39 unique traits of VRE isolates in our study, implying a circulation of these strains in the Songklanagarind Hospital for a period of time prior to the present study. These findings may suggest a hospital epidemiology stemming from the spread of MDR and vancomycin-resistant E. faecium with endemic vanA VRE. These bacteria may contribute to antibiotic resistance and virulence through horizontal gene transfer.

Conclusions

This was the first study conducted in a tertiary care hospital in southern Thailand to show a high prevalence of MDR and XDR-VRE in patients, which could lead to pathogenic effects on public health. The results of this study suggest that overused antibiotics appear to be mediated mainly by the vanA gene, which carries one or two virulence genes that create a high resistance level to vancomycin. However, molecular techniques are highly effective in detecting antibiotic-resistant genes, which would allow monitoring to control VRE infection in hospitals.

Supplemental Information