Vancomycin-resistant enterococci in Saudi Arabia: prevalence, antibiotic resistance and susceptibility array

Vancomycin-resistant Enterococci (VRE) are an important cause of nosocomial infection and may result in increased morbidity and mortality in immunocompromised populations particularly in developing contries. The aim of the present study was to reassess the definition of hospitalacquired Enterococci, with particular emphasis on the relevance of recent hospitalization.Total 228 Enterococci clinical samples were collected from different hospitals of Saudi Arabia. Sampling was performed following sub culturing, strains isolation and antimicrobial susceptibility testing. Out of 228 Enterococci clinical isolates isolated in this study, 186 (82%) were susceptible to vancomycin, 17 (7%) were resistant and 25 (11%) were intermediate. Most of VRE isolated in our study were from Jeddah hospitals 11%. The most common infections caused by Enterococci in current study showed urinary tract infection (56.6%) followed by blood (12.3%) and wound infection (11.4%). Porportion of 87% and 13% Enterococci isolated in this study were from Saudi and non-Saudi patients respectively and were calculated statistically nonsignificant (p-value=0.4). This limited study exhibited a high pervasiveness rate of multiple antibiotic resistant Enterococci infections among hospitalized patients in this environment. There is need for precise observations of clinics and hospitals for Enterococci infections; prudent use and rational prescription of antimicrobials and stringent measures to reduce the prevalence rate by health education on infection control measures such as isolation, cleaning, disinfection and sterilization.

Enterococci are among the most frequent causes of nosocomial infection, particularly in intensive care units (ICU) where they are selected by treatment with cephalosporin and other antibiotics to which they are resistant.They are transmitted from individual to individual primarily on the hand of hospital personnel (hospital acquired), some of whom may carry the organism in their gastrointestinal tracts.Less frequently, Enterococci are transmitted on medical devices.Meningitis and bacteraemia while endocarditis may occur in neonates and adults respectively.Enterococci infection is equally distributed between sexes, although urinary tract infections (UTI) are more common in healthy women than men and in elderly patients due to high incidence of urinary instrumentation [4].Hospital

Objective
The aim of the present study was to reassess the definition of hospital-acquired Enterococci, with particular emphasis on the relevance of recent hospitalization.

Samples collection
A total number of 228 Enterococci clinical isolates were collected in this study from different hospitals of Saudi Arabia.Sampling was performed following urine, wound, high vaginal swab (HVS), bone, blood, eyes, abdominal and placental swabs, pleural fluid, cerebrospinal fluid (CSF), sputum, stool and fetal part.

Sub culturing of specimens and isolation of strains
Specimens were sub cultured on blood and bile esculin agar under complete aseptic conditions and incubated for 24 hrs at 37˚C to grow single pure colonies for performing gram stain and catalase test.All isolated bacteria were preserved at -80˚C in an eppendorf tubes containing l ml brain heart infusion broth along 16% glycerol.

Identification of bacteria and antimicrobial susceptibility testing (AST)
Preserved bacteria were sub cultured in a suitable media and incubated accordingly to obtain pure culture.Ampicillin, gentamycin, vancomycin, tetracycline and ciprofloxacin were used for susceptibility testing.Around 3 ml of clean saline (fluid 0.45% NaCl, pH 7) was put into a reasonable plastic test tube.By then adequate number of morphologically comparative states of immaculate microbes was exchanged to a tube containing the saline to make homogenous suspension with an identical thickness of McFarland (No.0.50-0.63)using calibrated VITEK2 DENSICHEK.The tube was then put in the tape with the recognizable proof card and information passage.To another tube containing 3 ml of saline, 280 ml of the suspension prepared for AST-GP was then transferred.The tube was then put in the anti-infection agents weakness tape, the recognizable proof GP depends on 43 biochemical tests measured carbon source usage, enzymatic actions and resistance.For the minimum inhibitory concentration (MIC) technique, AST card contains 64 micro wells were used.While control well was occupant on all card with the remaining wells containing premeasured measures of particular antimicrobials consolidated with culture medium.MIC values were determined for each antimicrobial contained on the card after a defined period of time (about 18 hrs).

Data analysis
Data was statistically analyzed in Microsoft Excel (ver 2010) by p values.Data was tabulated using a Fisher test (Graph Pad Instat program statistical software).P-values computed < 0.05 were considered as significant.

Results
A total of 228 Enterococci clinical isolates were isolated in this study and tested for vancomycin susceptibility.In this study 186 (82%), 17 (7%) and 25 (11%) Enterococci isolates were found to be susceptible, resistant and intermediate respectively.Clearly, Enterococci were isolated mostly from urine (129 isolates), blood (28 isolates) and wound samples (26 isolates).This indicates that the common infections caused by Enterococci in our study were mainly UTI followed by blood stream and wound infection (Figure 1).Most VRE isolated in our study were from Jeddah (11%) followed by Makkah (9%) and Taif hospitals (3%) (Figure 2).

Figure 1. Distribution of Vancomycin resistant enterococci (VRE) isolates from different samples Figure 2. Distribution of VRE isolates from different cities
Data regarding the VRE isolates from different wards of hospital displayed the higher number of Enterococci (41 isolates) from intensive care unit (ICU), 40 from female medical ward (FMW), 32 from male medical ward (MMW), 18 from surgery, 16 from pediatric, 8 from orthopedic, 5 from nephrology, 4 from hemodialysis, 3 from OBS/Gyn and 1 from antenatal wards.100% Enterococci isolates were found to be sensitive collected from Nephrology, OBS/Gyn, antenatal wards followed by FMW, surgery, ICU, MMW, hemodialysis, pediatric and orthopedic ward, while both the ICU and pediatric wards exhibited 12% Enterococci isolates followed by 9.4% in MMW and 5% in FMW (Figure 3).Current data deduced that out of the 228 Enterococci isolates in this study, 95 were from male patients and 11 (12%) of them were VRE, while 133 were from female patients and 6 (4.5%) of them were VRE, however the mean difference in VRE between genders was quiet statistically nonsignificant (p-value = 0.07).Out of the 228 Enterococci isolated in this study, 204 were from Saudi patients and 14 (7%) of them were VRE, while 24 were from non-Saudi patients and 3 (13%) of them were VRE.
Regarding nationalities statistically non-significant difference was reported (p-value=0.4).About 58.8% and 41.2% of Enteroccoccal infections in our study accounted for age above 50 and less than 50 years respectively.Difference in VRE between age groups was found statistically non-significant (p-value = 1) (Figure 4).Regarding AST it was concluded that 69.75%, 48.2%, 42.6%, 66.6% and 7% of the Enterococci isolates were found resistant to ampicillin, gentamycin, ciprofloxacin, tetracycline and vancomycin respectively (Figure 5).Current study concludes that antimicrobials based drugs like tetracycline articulated higher resistance against Enetrococcci followed by tetracycline, ciprofloxacin, gentamycin and vancomycin while increased activity of sensitivity was analyzed in antimicrobials viz; vancomycin followed by ciprofloxacin, gentamycin, tetracycline and ampicillin against Enterococci.Based upon the inference it is concluded that antimicrobial vancomycin has the higher sensitivity with least resistance against Enterococci.The role of these drugs according to their resistance and sensitivity levels can be manipulated and wrought by clinicians against Enterococci based infections in future.

Conclusion
Introduction Enterococci are hardy, facultative anaerobic Gram positive cocci in pairs or short chains that are capable to grow and survive in many environments [1].They are part of normal intestinal flora of humans and animals but may be responsible for serious infections.Of the 20 Enterococcus species [2], 2 species are particularly pathogenic to man; Enterococcus faecalis causes 85-90% of Enterococci infections while Enterococcus faecium causes 5-10% [3].

Figure 3 .
Figure 3. Distribution of VRE isolates among different hospitals wards