Enterococci: Emerging Drug Resistant Bacteria In Hospital Acquired Infections At Hospital Kuala Lumpur, Malaysia
R Ibrahim, M Mohamad, M Rahman
antibiotic susceptibility, enterococci, polymerase chain reaction, resistance, vanocomycin
R Ibrahim, M Mohamad, M Rahman. Enterococci: Emerging Drug Resistant Bacteria In Hospital Acquired Infections At Hospital Kuala Lumpur, Malaysia. The Internet Journal of Microbiology. 2010 Volume 9 Number 2.
Enterococci are the most common pathogens in hospital acquired infections. Some of them are resistant to Vancomycin(VRE) and some are susceptible to Vancomycin (VSE). The present study was carried out to identify enterococci from clinical cases and to illustrate their clinical features and drug resistance characteristics. Antibiotics susceptibility of the identified bacteria was determined by disk diffusion method
Enterococci normally present in the human intestines and in the female genital tract and are often found in the environment. Recent National Nosocomial Infections Surveillance1 reveals that these enterococci remain in the top 3 most common pathogens that cause nosocomial infections. These are: urinary tract infections, bloodstream infections, and wound infections in hospitalized patients. Enterococci infections typically occur in very ill debilitated patients who have been exposed to broad-spectrum antibiotics. According to NNIS2 data from January 2003 through December 2003, more than 28% of enterococci isolates were found to be associated infections in incentive care unit (ICU) of 300 participating hospitals.
The acquisition of vancomycin resistance by enterococci has seriously affects treatment and infection control program which leaves clinicians treating VRE infections with limited therapeutic options (Fraser, 2010)
NNIS2 reported that more than 25% of health care-associated enterococcal infections were associated with organisms resistant to vancomycin. In Malaysia 2006, a confirmed case of vancomycin resistant
Materials and Methods
The study was carried out during May 2007 to April 2008 at Hospital Kuala Lumpur (HKL), Malaysia. The hospital consists of 3000 beds and 90 wards and provides services and acts as a reference centre for the hospitals of other states, in Malaysia.
Study population and sample collection
A total number of
Information on clinical data
Information on patient’s profiles :age, diagnosis, risk factors such as duration of hospitalization (prolonged >2 weeks), catheter use, usage of antibiotics like cephalosporin, carbapenem, vancomycin, recurrent admission and others such as malignancy, use of steroids were obtained from their medical records.
Identification of enterococcus spp
Enterococcal genus identification was based on Gram staining that showed gram positive cocci in pairs or short chains, growth and blackening of bile-esculin agar after overnight incubation , growth in the presence of 6.5% NaCl, absence of catalase (catalase negativity) and presence of pyrrolidonyl arylamidase (PYR). Streptococcal group antigens were also detected using group D antisera (Slidex Strepto-Kit; Bio Merieux, France). API 20Strep (BioMerieux, France) was used for species identification.
Antibiotic Susceptibility test
Antibiogram of the isolates was determined by disk diffusion method, testing for vancomycin (30µg), teicoplanin (30µg), ampicillin (10µg), high concentration of gentamicin (120µg) and linezolid (30µg). The tests were performed using the methodology recommended by the Clinical and Laboratory Standards Institute 4
Disk Diffusion Method
Susceptibility test of enterococci to ampicillin, high level gentamicin, vancomycin, teicoplanin and linezolid was determined by a Kirby-Bauer disk diffusion method as per CLSI criteria 4
E-test of vancomycin
Minimum inhibitory concentration (MIC) for vancomycin was determined by E-test as per the procedure of CLSI4 An isolate is considered susceptible to vancomycin if the MIC is ≤ 4µg/ml and resistant if MIC ≥ 32 µg/ml 4
Two QC strains
Molecular detection of vanocomycin resistance genes
Vanocomycin-resistance genes were detected by PCR using specific primers as per the procedure of Boyd 5et al. Briefly, PCR conditions consisted of a pre-denaturation step at 94°C for 5 min, followed by 30 cycles of 45 sec denaturation at 94°C, 45 sec annealing at 54°C and 45 sec extension at 72°C. A final extension step was performed at 72°C for 5 min. Amplified products were analyzed by electrophoresis on 1.5% agarose gel.
All statistical analyses were carried out using SPSS version 12.0.
Ethics Committee approval
The research work was approved by Ethics committee after finalization of the project proposal.
Enterococci of both vancomycin resistant and susceptible were isolated and identified from different clinical conditions (Table-1). It was observed that 21.5% enterococci were isolated from urinary tract infections, 18% from end stage renal failure, 12.2% from neurological problem, 12.3% from sepsis, 8.2% from malignancy, 7.4% from surgical problem, 4.1% from diabetes mellitus, 2.9% from motor vehicle accident with head injury, 2.5% from respiratory infections, 1.6% from neonatal infections, .8% from skin infections and .4% from nephritic syndrome
Enterococci identified from the patients of different wards and department at hospital KKL have been listed (Table-2). In the study 39% Enterococci were identified from the patients of nephrology ward, 23% from medical ward, 12% from surgical ward,13% from neurology ward,5% from pediatric, 3% from oncology/radiotherapy,2% from maternity, 1% from burn and 2% from out patient department(OPD)
Clinical features that were recorded for the infections of vancomycin resistant and susceptible patients were analyzed.
Out of all enterococci isolated 6 were found to be resistant to vancomycin.
PCR results revealed that all the 6 vancomycin resistant enterococci possess Van A gene(In press)
Enterococci have both an intrinsic and acquired resistance to antibiotics,which make them important nosocomial pathogens.
Of all the enterococci infections analyzed (Table-3), out of 244 patients, 50 (21%) presented with urinary tract infections, 41 (17.2%) end stage renal disease, 29 (12.2%) neurological problems, 20 (8.4%) malignancy, 18 (7.4%) had surgical intervention and 10 (4.2%) had diabetes mellitus. Amongst the factors that described in this study to favor the infections, majority of them (29%) had recurrent hospitalization and catheter usage, followed by prolonged hospitalization, 21.4%. Although recurrent hospitalization and catheter usage were more frequent risks described in the enterococci infections, these risk factors were not statistically significant. Other risk factors like carbapenem and cephalosporin usage were also not statistically significant. Enterococci are rated as the second leading cause of urinary tract infections (UTI) and comprised about 10% of nosocomial UTI6 and this study showed that UTI was the most common clinical manifestation of enterococci infections. Instrumentation like urinary catheter and urinary tract abnormality (urethral stricture, hydronephrosis)might have promoted infection with enterococci especially in elderly patients. In neurology units, most of the patients had limited mobility especially patients with cerebrovascular accidents and they usually used urinary and venous catheters that might have promoted enterococcal colonization and infections.
For patients with end stage renal disease, usually hospitalized frequently for dialysis. Some patients had dialysis via venous catheter (internal jugular catheter and/or femoral catheter) and this catheter was probably prone to be colonized with enterococci with the help of biofilm production. This group of patients had catheter related bloodstream infections and usually treated with intravenous antibiotics (cephalosporin, vancomycin, carbapenem) requiring prolonged hospitalization. The presence of an invasive device was identified previously as a strong clinical risk factor for VRE invasive infections7. It is unclear whether catheters served as the actual conduit through which VRE infection acquired, whether they were just markers of debilitation, prolonged hospitalization and severe co-morbidities. Patients with prolonged hospital stay were more susceptible to cross-transmission of pathogens and to a greater use of antimicrobials with selective pressure; therefore they had a greater chance of acquiring resistant pathogens. A previous study found that the duration of hospitalization was considerably longer to pick up infections of the VRE groups (57.7 days) compared to VSE group (29 days)8
Among the 6 VRE cases, 4 (66%) isolates were identified as
The two remaining VRE isolates were obtained from urine specimens. The first urine specimen was taken from a 55 year old man with interstitial lung disease and respiratory failure and complicated with cardiac event. He had been hospitalized more than one month and also had recurrent admission to intensive care unit and prolonged intubation. He was put on piperacillin-tazobactam for more than 3 weeks because the tracheal aspirate and urine grew
It has been shown that vancomycin use was a major risk factor for the VRE infection or colonization10. An increased risk of VRE infection and colonization had been associated with non-ambulatory status11 , receipt of antibiotics and hospitalization12. In this study, the use of vancomycin was the significant risk factor for the development of VRE by statistical analysis. The RR (relative ratio) represents the effect of vancomycin use on promoting resistance to vancomycin in
In previous studies, VRE colonization was associated with prolonged length of hospital stay, previous admission to an intensive care unit (ICU), and severe underlying disease13. In addition, Huh et al.14 showed evidence that longer hospitalization and ICU stay are possible risk factors to get colonized with multiple clones of VRE. Extensive or multiple hospitalizations show a correlation between the subsequent development of VRE infections, prior antibiotic treatment, prolonged stay within intensive care units and, in some instances, even intrahospital transfers10.
Other investigators have shown that ongoing vancomycin use creates and maintains an intraluminal environment favorable to VRE growth, thus increasing the likelihood of VRE entry into bloodstream10. A study conducted from 2003 to 2004 showed that prior use of antimicrobial therapy, including vancomycin and cephalosporin, has been shown to be associated with acquisition of VRE15. Also, the presence of underlying disease was significantly associated with an increased risk for VRE colonization. Of note, impaired renal function and hemodialysis have been previously implicated as risk factors in VRE outbreaks12. The use of vancomycin increased the risk of colonization 2.5-fold and third generation cephalosporins 2-fold10,11. A study showed that vancomycin use is the major risk factor for VRE bacteremia . Since 1990s, vancomycin and ceftazidime have been used as first line empirical therapy for line related sepsis in the renal unit (HKL), however with the emergence of VRE, it has been changed to cloxacillin and ceftazidime
Other risk factors that have described in this study like cephalosporin usage, use of carbapenem, malignancy, instrumentations like catheter and previous hospitalization were not statistically significant. Edmond et al16 studied VRE bacteremia on an oncology ward found that the use of antimicrobial agents with activity against anaerobes (metronidazoles, clindamycin and imipenem) determined a risk factor for the development of vancomycin-resistant bacteremia. In the present study, all the 6 confirmed VRE cases were further analysed by polymerase chain reaction (PCR) and all of them showed
The findings of the study provided first Malaysian data on the prevalence of VRE and VSE in patients reported to HKL, Malaysia. Clinical features of the patients infected with enterococci infections were provided for the identification of the clinicians. The results would serve as an alert to the clinicians of the emergence of infections by VRE and VSE and encourages implementation of appropriate infection control measures in order to curb further rise in prevalence.