Introduction

Enterococci are part of the normal faecal flora in humans, but they also colonize the oral cavity, the genitourinary tract and the skin, particularly in the perianal area⁽¹⁾. The main sites of colonization in the hospitalized patients are soft tissue wounds and ulcers and the gastrointestinal tract(2). They were traditionally regarded as low grade pathogens, but have emerged as second leading cause of nosocomial infections and third most common cause of bacteraemia⁽³⁾.
Urinary tract infection (UTI) is the most common cause of nosocomial infection among hospitalised patients⁽⁴⁾. In spite of the fact that enterococci are one of the most frequent aetiology identified, there are still a lot of characteristics that need to be established(5). Intra-abdominal and intra-pelvic abscesses or post surgery wound infections⁽⁵⁾ and blood stream infections⁽³⁾ are also commonly caused by Enterococcus next to UTIs.
Enterococci are classified in five goups, of which only a small part are responsible for producing different pathologies⁽⁶⁾, the most frequent identified species being Enterococcus faecalis and Enterococcus faecium⁽⁷⁾.
Enterococci are considered important especially for their ability of acquiring antibiotic resistance⁽⁷⁾. High Level Aminoglycoside Resistant Enterococci (HLAR) were first reported in France in 1979 and since then they have been isolated from all the continents(8). Enterococcal infections are frequently identified in patients with bacteremia, surgical site infections or others, requiring combinations of antibiotics such as aminoglycosides with beta lactam⁽⁹⁾. Since enterococci that show a HLAR phenotype are not susceptible to aminoglycosides, this antibiotic could not be used for the combination therapy⁽¹⁰⁾.
Resistance to β lactam was first reported in early 1980s. Therefore it necessitates the higher dosage of β lactam and related drugs⁽¹⁾. So, detection of β lactam resistant enterococci should be reported. Resistance to tetracycline, erythromycin and chloramphenicol is well known nowadays. This study was aimed to determine the prevalence of multidrug-resistant uropathogenic Enterococcus.

Materials and method

The study was conducted in “Cantacuzino” National Research Institute, from May 2015 to September 2017. A total of 102 enterococcal isolates were included in the study.

Methodology

UTI was defined as the presence of >105 colony forming units/ml in the culture of an appropriate collected urine specimen. Standard microbiological techniques were used.
The identification of Enterococcus spp. was done using the following parameters: (i) colony morphology on blood agar⁽¹¹⁾, (ii) Gram staining, (iii) catalase, (iv) bile esculin hydrolysis, (v) salt tolerance (growth in 6.5 % NaCl brain heart infusion). Then speciation was performed by sugar fermentation, reduction of tellurite in tellurite blood agar plate and analysis on API Strep kit. All tests were carried out and interpreted as described by Facklam and Collins(12). 

Resistance profile

Antibiotic susceptibility pattern: antimicrobial susceptibility test was performed by Kirby-Bauer disc diffusion method. The peptone water culture standardized to 0.5 McFarland opacity was used for surface seeding on Mueller Hinton agar. After plates were dried, antibiotic discs were placed over the medium and incubated at 37°C for 24 hours. Then the results were recorded according to National Committee for Clinical Laboratory Standards⁽¹³⁾ for the following antibiotic discs: vancomycin (30 μg), ampicillin (10 μg), penicillin (10 units), high level gentamycin (120 μg), ciprofloxacin (5 μg), levofloxacin (5 μg ), chloramphenicol (30 μg), tetracycline (30 μg), nitrofurantoin (300 μg), linezolid (30 μg). E. faecalis ATCC 29212, Staphylococcus aureus ATCC 25923 and E. faecalis ATCC 51299 were used as control strains(13).
In order to detect High Level Gentamicin resistance (HLGR), minimum inhibitory concentration (MIC) of gentamicin was detected for the 27 isolates which showed resistance to high level gentamycin discs (120 μg). The bacterial inoculums of 1.5 x 108 CFU/ml was prepared with brain heart infusion broth, stock solution of gentamycin 5120 μg/ml was prepared with distilled water, to which 9 ml of overnight culture of brain heart infusion broth was added. The method of two fold dilutions up to 1 μg/ml was used. One control culture broth without antibiotic was also kept. After overnight incubation at 37°C with ambient air, the tubes were examined. The point at which there was no visible growth was taken as the minimum inhibitory concentration of gentamycin⁽¹⁴⁾.

Results and discussions

It is important to know the changing trends of the Enterococcus infections and their antimicrobial susceptibility pattern. In our study, 102 enterococcal strains were recovered from urine specimen.
Out of 102 isolates, 44 were from male and 58 were from female patients. About 48.03% of isolates were recovered from patients below 50 years of age. In a study from 2011, Kafayat et al. found that enterococcal infections in the age group of 20-29 years old represented the largest proportion (42.4%), followed by age group of 30-39 years old (23.7%)⁽¹⁵⁾. This was supported by a survey done by Centers for Disease Control and Prevention (CDC) on nosocomial infections, in which Enterococcus accounted for 13.9% infections, being next to Escherichia coli as a causative agent of hospital acquired urinary tract infections⁽¹⁶⁾.
Although the recent studies stated that there is an increase in the isolation of E. faecium and other enterococcal species⁽¹⁷⁾, in our study E. faecalis (98.03%) constituted the major isolate, followed by E. faecium (1.97%). Similar findings were revealed by Facklam et al⁽¹⁸⁾. Enterococcus spp. were isolated in pure culture (90.19%), recovered with other organisms as mixture of two organisms (7.84%) or three organisms (1.96%). Enterococcus faecalis was associated with other enterobacterial species (Table 1).  
E. faecalis isolates were susceptible to vancomycin (94.10%), ciprofloxacin (62.74%), levofloxacin (62.74%), ampicillin (50.98%), penicillin (50.98%), tetracycline (17.65%), chloramphenicol (89.22%), linezolid (97.06%), nitrofurantoin (96.07%) and revealed HLR to gentamycin (25%) (Table 2).
Two strains (E. faecium) showed resistance to ciprofloxacin, one of the most common antibiotic used to treat urinary tract infection, as well as to gentamycin, and were susceptible to vancomycin and linezolid. On the other hand, 49.02% of the E. faecalis isolates showed resistance to ampicillin. HLGR (120 μg/ml) by disk diffusion method was observed in 25% isolates. HLGR was referred as MIC>500 μg/l⁽¹⁹⁾, and was seen in 26.47% of isolates. Sreeja S et al. (2012) found 45% resistance to ampicillin, 50% to ciprofloxacin and 47% to high level gentamicin among Enterococcus isolates⁽²⁰⁾. A study by Kapoor et al. (2005) found 66% HLAR(21), and Vittal P. Prakash et al. (2005) found 43.5% HLGR⁽²²⁾. 
Although E. faecalis (98.03%) was the most common species identified in clinical specimens, there was a deviation towards E. faecium isolation (1.97%). Further, there is an increased isolation of multidrug-resistant Enterococcus with HLGR (26.47%). Thus, definite identification of enterococci at species level is mandatory to assess their variable sensitivity pattern and to treat accordingly.

Conclusions

In our study, the most frequent species of Enterococcus was E. faecalis, of which a large proportion revealed high level resistance to gentamycin. So, infections with E. faecalis can be especially troublesome to treat because of their frequent resistance to multiple antibiotics, including vancomycin, a drug of last resort for many Gram-positive infections⁽²⁹⁾. 
Taking into account their fast capacity of acquiring antibiotic resistance, enterococci may become the most frequent isolated bacteria in hospitalized patients. infecţia tractului urinarEnterococcus faecalisEnterococcus faecium