Babak Asghari1, Hamid Reza Sadeghi2* and Mohammad Jalilian3

1Department of Microbiology, Faculty of Medicine, Hamadan University of Medical Sciences, Hamadan,Iran.
2Department of Medical Physics, Faculty of Medicine, Hamadan University of Medical Sciences, Hamadan, Iran.
3Imam Khomeini Hospital, Tehran University of Medical Sciences, Iran.

ABSTRACT

Vancomycin-resistant enterococci (VRE) remain as one of the important cause of nosocomial infections. Reports of the VRE carriers are increasing worldwide. In this study, a total of 220 patients was screened for vancomycin-resistant enterococci colonization, of which 36 (16.3%) were VRE carriers. Among the VRE isolates, 17 were vanA positive. The distribution of VRE carriers in different wards that pose high risks for healthcare infection to hospitalized patients emphasizes applying suitable infection control strategies to prevent the dissemination of the organism. This is the first report from Iran in which a vanA-containing enterococci were isolated from intestinal colonization of patients. Strict measures are required to control the further spread of VRE strains in the Iranian patients.

Keywords: Vancomycin-resistant enterococci, Antibiotic resistance, Colonization surveillance, Hospital infection control, Patients.

Introduction

Vancomycin-resistant enterococci (VRE) have been known as one of the most important nosocomial pathogens worldwide, and their colonization has occurred in the hospital setting.  The screening of patients at high risk of VRE colonization is recommended to prevent transmission of VRE 1. Carriage of VRE by patients is important from two aspects: (1) the source of Enterococci infections could be endogenous, and carriage of VRE may predispose the carrier to Enterococcal infections. (2) Carriers can serve as the reservoir for vancomycin-resistant enterococci and spread these to other hospitalized patients 1,2, 3. In Iran, no study has been performed to evaluate the prevalence of VRE intestinal tract colonization in the hospitalized patients. We have tried to fill this knowledge gap and have undertaken this study to determine the prevalence of intestinal colonization and antibiotic resistance profile of VRE strains in patients, in a hospital affiliated to Tehran University of Medical Sciences, in Iran.

METHODS

This cross-sectional study was performed among 220 admitted patients from January to December 2015. These patients admitted to hospital were screened for gastrointestinal carriage of VRE. A fecal sample was taken during the hospitalization. Stool specimens were inoculated onto the bile-esculin agar plates and into the bile-esculin broth. Colonies growing on bile-esculin agar with the dark brown halo were identified by Gram staining; absence of catalase; and growth on 40% bile, in 6.5% sodium chloride, and for further identification was based on the conventional biochemical tests. Antibiotic sensitivity of the enterococci isolates was investigated by disk diffusion and agar dilution methods. All tests were performed and interpreted according to Clinical and Laboratory Standards Institute guidelines. Vancomycin resistance was determined  by using two methods, including vancomycin disk diffusion test (≤14 mm indicated VRE) and vancomycin MICs (≥32 mg/ mL indicated VRE) 4. Additionally, vanA, vanB and vanC genes were determined by polymerase chain reaction 5. The protocol was approved by the local Ethical Committee of Tehran University of Medical Sciences, and informed consent was taken from all subjects. Data were analyzed using descriptive statistics.

RESULTS

A total of 220 patients (hospitalized more than ten days) out of 492 (the total number of hospitalized patients) participated in the study. The prevalence of intestinal carriage of enterococci was 25.4% (56), of which 9.1% (20) and 16.3% (36) were vancomycin-sensitive enterococci and VRE, respectively (Table 1). Polymerase chain reaction testing of 36 VRE isolates identified 17 (47.2%) positive for the vanA gene. The MICs for vancomycin was between 32 to 256 mg/mL and 0.25 to 1 mg/mL for VRE and VSE strains, respectively. Table 2 shows the distribution of VSE and VRE carriage in different wards. VRE carriers were seen in hematology, dialysis unit, internal medicine ward, infectious diseases ward, surgical ward and intensive care unit. Dialysis unit, intensive care unit and surgical ward showed the highest rate of VRE carriage. The HICPAC Guidelines recommended for the management of the infection control measures to reduce cross transmission among hospitalized patients: these included restriction of vancomycin use; education of hospital staff (including hand washing with an antiseptic soap or a waterless antiseptic agent); routine screening for vancomycin resistance among clinical isolates; contact isolation for patients with VRE 20. Table 3 represents the antibiotic resistance pattern of Enterococci isolates (VRE and VSE). All VRE isolates were sensitive to linezolid and tigecycline. 7 (35%) of 20 VSE and 29 (80.5%) of 36 VRE isolates were resistant to tetracycline. The MICs for teicoplanin against resistant VRE strains were >16 mg/mL and for the resistant VSE strains were >4 mg/mL.

DISCUSSION

Hospitalized patients are at higher risk of the acquisition of VRE. Risk factors for VRE colonization included vancomycin use, hospitalization, ICU stays, receipt of antibiotic, anemia, leukocytosis, diabetes mellitus, gastrointestinal procedures and acute renal failure 16.  In this study, out of 220, 56 (25.4%) of subjects were enterococci carriers, of which 36 (16.3%) were VRE carriers. Askarian et al., in Shiraz Namazi Hospital reported that 99 out of 700 patients (14%) were colonized with VRE 6. The estimated prevalence in this study correlates with reported ranges. The rate of VRE colonization varies widely in different studies. In the study of Wisplinghoff et al. was shown that the prevalence of vancomycin resistant enterococci in hospitalized patients was 2 % and 60 % for E. fecalis and E. faecium, respectively 12. The prevalence of intestinal colonization of VRE in patients at Rawson Hospital (12.20%) was similar to that reported by Coque et al. 13 in hospitals in the USA, and was higher than that reported by Endz et al. 14 in Europe (4.9%). Zanella et al. 15 reported vanA Enterococcus clinical isolates from colonized patients obtained during a nosocomial outbreak in a hospital in São Paulo, Brazil.

The important finding of this study is that the VRE carriers were mainly those who hospitalized prolonged in the wards in which the risk of nosocomial infection is relatively high (Table-1). Similar findings have been reported in other studies 7.

VRE strains isolated in the present study shown three patterns of MIC values for vancomycin. Five VRE isolates were vanA-gene positive and had MIC values of 256 mg/mL. One VRE isolate was vanA-gene positive and had the MIC value of 32 mg/mL for vancomycin. Two isolates were vanA-gene-positive (MIC: 16 mg/mL) enterococci strains.

Morris et al., 17 found, that the restriction of vancomycin use, cannot lead to reducing the rate of colonization with VRE in a hospital where VRE were endemic. Nonetheless, use of vancomycin was probably the crucial factor in the initial emergence of VRE, and use of vancomycin, cephalosporins, and other antibiotics probably maintains the selective pressure for VRE.

Transmission of VRE is a major concern because the pathogen may develop phenotypic resistance during a course of antibiotic therapy 8,9. VRE can colonize the gastrointestinal tract and the skin, thus producing an epidemiological risk similar to that of nosocomial gut flora (eg, antibiotic-resistant gram-negative bacilli) and nosocomial colonizers (eg, methicillin-resistant Staphylococcus aureus). Moreover, because environmental contamination occurs frequently, it is associated with an epidemiological risk similar to that of Clostridium difficile.

Furthermore, because colonization seems to be persistent in the gastrointestinal tract, persistently colonized patients can be a reservoir from which VRE can be continually spread 19.

The high resistance rate for most used antibiotics (eg, Ciprofloxacin) was observed among VRE isolates. None of the isolates tested in our study were resistant to tigecycline  and linezolid. However, other studies have reported the colonization of linezolid-resistant enterococci strains among patients 10.

Unexpectedly, in our study, resistance to quinupristin/dalfopristin was observed in both VSE and VRE isolates. Werner et al., 18 suggested an association of quinupristin/dalfopristin resistance with the use of virginiamycin as a feed additive and indicate the possibility of transfer to humans via the food chain.

Our study shows that VRE colonization among patients in a University hospital in Iran is around 16.3%. Secondly, this unrecognized silent carriage of VRE could be one of the most important factors leading to spread of VRE to others in the hospital. Therefore, patients who were colonized with the VRE strain in our hospital were subjected to isolation precautions, even in the case of repetitive negative follow-up cultures.

In conclusion, the distribution of VRE carriers is more likely in certain high risk wards like ICU hence suitable application infection control strategies is very important to prevent the spread of the organism. The occurrence of the VRE strains demonstrates the need for using suitable approaches for treatment and diagnosis of VRE infections. More studies are necessary to reveal the relative contributions of patient-related factors, antibiotic treatment, and characteristics of VRE colonization 11. The routine surveillance cultures are one way to identify asymptomatic VRE-colonized patients. Infection prevention and control strategies include improving compliance with hand hygiene, enhancing environmental cleaning, ensuring antimicrobial stewardship, and identifying and isolating VRE carriers to interrupt transmission and reduce VRE infections. Strict measures will be required to control the further spread of VRE pathogens in hospital settings. More attention should be paid to the efficacy of prevention of VRE colonization in patients.

 

 References

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 Table 1
Type of antibiotic(s) received and days of hospitalization among Vancomycin-resistant enterococci strains isolated from intestinal tract of patients

Patient no.
Clinical data
Enterococcus spp
Resistance

Phenotype

Days of hospitalization
Type of antibiotic(s) received
1
Septic arthritis
E. faecium
VanA
23
None
2
Diabetes mellitus
E. faecium
VanA
24
Vancomycin, Cephalexin
3
Community acquired pneumonia
E. faecalis
VanA
21
Vancomycin, imipenem, Ciprofloxacin
4
Postsurgery

 

E. gallinarum
VanA
23
Vancomycin, imipenem
5
Polymyositis
E. faecium
VanA
57
Vancomycin, imipenem, piperacillin-tazobactam, amoxicillin, ciprofloxacin, gentamicin
6
Septic arthritis
E. faecalis
VanA
47
Vancomycin, imipenem, Ciprofloxacin
7
Chronic myeloid leukemia
E. faecium
VanA
27
Cefotaxime
8
Postcardiac surgery
E. faecium
VanA
37
Vancomycin, Cefotaxime, Ciprofloxacin
9
Ischemic heart disease
E. faecium
VanA
54
Vancomycin, piperacillin-tazobactam, amoxicillin-clavulanate
10
Septic arthritis
E. faecium
VanA
12
Vancomycin, piperacillin-tazobactam, ciprofloxacin
11
Ischemic heart diseas
E. faecium
VanA
22
Azithromycin, amoxicillin-clavulanate,
12
Postcardiac surgery
E. gallinarum
VanA
27
Vancomycin, gentamicin,  azithromycin, trimethoprim
13
Chronic myeloid leukemia
E. gallinarum
VanA
31
Amoxicillin, Ciprofloxacin
14
Ischemic heart diseas
E. faecium
VanA
35
Vancomycin, Azithromycin, amoxicillin-clavulanate
15
Diabetes mellitus
Enterococcus Spp
VanA
33
Vancomycin, Azithromycin, amoxicillin-clavulanate
16
Ischemic heart disease
Enterococcus Spp
VanA
34
Vancomycin, imipenem
17
Ischemic heart disease
E. faecalis
VanA
21
Gentamicin, Ciprofloxacin
18
Polymyositis
E. faecium
VanB
12
None
19
Septic arthritis
E. faecium
VanB
11
Ciprofloxacin
20
Community acquired pneumonia
E. gallinarum
VanC
22
Gentamicin, azithromycin, trimethoprim
21
Community acquired pneumonia
E. gallinarum
VanC
20
None
22
Septic arthritis
Enterococcus Spp
VanC
23
Piperacillin-tazobactam, ciprofloxacin
23
Septic arthritis
Enterococcus Spp
VanC
22
None
24
Ischemic heart disease
E. faecium
34
None
25
Ischemic heart disease
E. faecium
22
Ciprofloxacin
26
Diabetes mellitus
E. faecium
12
None
27
Chronic myeloid leukemia
E. faecium
10
Vancomycin, azithromycin, amoxicillin-clavulanate
28
Septic arthritis
E. gallinarum
14
None
29
Chronic myeloid leukemia
E. faecium
16
Gentamicin, azithromycin, trimethoprim
30
Septic arthritis
E. gallinarum
15
Ciprofloxacin, gentamicin
31
Septic arthritis
E. gallinarum
20
None
32
Diabetes mellitus
E. faecalis
19
None
33
Chronic myeloid leukemia
E. faecium
11
Vancomycin
34
Septic arthritis
E. faecalis
12
Piperacillin-tazobactam, ciprofloxacin
35
Septic arthritis
Enterococcus Spp
24
Vancomycin, imipenem
36
Ischemic heart disease
E. hirae
10
Ciprofloxacin, gentamicin

 

 Table 2
Prevalence of intestinal carriage of VRE and VSE strains among patients in relationship with hospital wards

Ward
Carriage, n (%)
With VRE, n (%)
With VSE, n (%)
Hematology
7 (12.5)
2 (28.6)
5(71.4)
Dialysis
13 (23.2)
9 (69.2)
4(30.8)
Internal
4 (7.1)
4 (100)
Infectious
4 (7.1)
2 (50)
2(50)
Operating room
Clinic
Surgery
10 (17)
8(80)
2(20)
Angiography
1 (1.7)
1(100)
Cardiology
Cardiac care unit
Emergency
Intensive care unit
17(30.3)
11 (64.7)
6(35.3)
Total
56 (25.4)
36 (16.3)
20 (9.1)

 

Table 3
Antibiotic susceptibility profiles of
enterococci strains isolated from the intestinal tract of patients by disk diffusion method

Antibiotic VRE (N = 36), n (%) VSE (N = 20), n (%)
Susceptible Intermediate Resistant Susceptible Intermediate Resistant
Erythromycin 6 (16.6) 4 (11.2) 26  (72.2) 12 (60) 6 (30) 2 (10)
Ampicillin 23 (63.8) 4 (11.2) 9 (25) 9 (45) 6  (30) 5 (25)
Vancomycin 36 (100) 20 (100)
Teicoplanin 1 (2.8) 35 (97.2) 20 (100)
Tetracycline 6 (16.6) 2 (5.5) 28 (77.7) 10 (50) 2 (10) 8 (40)
Doxycycline 13 1 22 16 (80) 1 (5) 3 (15)
Ciprofloxacin 6 (16.6) 9 (25) 21 (58.4) 7 (35) 4 (20) 9 (45)
Levofloxacin 14 (39) 6 (16.6) 16 (44.4) 11 (55) 2 (10 ) 7 (35)
Nitrofurantoin 34 (94.4) 2 (5.6) 20 (100)
Rifampicin 3 (8.3) 2 (5.5) 31 (86.1) 2 (10) 9 (45) 9 (45)
Linezolid 36 (100) 20 (100)
Tigecyclin 36 (100) 20 (100)
Quinupristin/dalfopristin 12 ( 33.3) 1 (2.7) 23 (63.8) 8 (40) 5 (25) 7 (35)