For more than a century Enterococci have been acknowledged as potential human pathogens, but the latest studies and data reiterate the fact that these organisms are up-and-coming as prominent causes of nosocomial infections. The capability to acquire antibiotic resistance markers and dissemination in hospital set-ups makes the management of certain enterococcal infections more complicated in gravely sick patients. They can cause urinary tract infections, genital tract infections, and even endocarditis due to factors like colonization and resistance to multitude of drugs used for their treatment (1). Enterococci are also known to attain and transmit resistant genes easily which is responsible for emergence of high level aminoglycoside resistance (HLAR) strains, vancomycin resistant Enterococci (VRE) strains, and beta lactamase producing strains. In fact, commonly used antibiotics like aminoglycosides, cephalosporins, aztreonam and semisynthetic penicillins have lost their therapeutic capacities for these organisms because of these resistance mechanisms (2).
Aminoglycoside resistance in Enterococci can be attributed to two main mechanisms the first being moderate level resistance in which the permeability to aminoglycosides is low scale and the modality of synergism with cell wall active agents has been applied to counter this. The beta-lactam antibiotics amply disorganize the cell wall after which the aminoglycosides can enter the cell and target the ribosomes. If the organism attains resistance determinants for aminoglycoside or cell wall active agents (lactams, glycopeptides) this synergism operational for the combination therapy of Enterococci is lost (1). The second mechanism is high level resistance or ribosomally mediated resistance wherein inhibitory enzymes like acetyl transferase and adenyl transferase are formed (3). Adenyl transferase mediates resistance to streptomycin whereas gentamicin resistance is encoded by acetyl transferase. Resistance to gentamycin is a potent indicator of resistance to other aminoglycosides except streptomycin while streptomycin resistance is the sole indicator of resistance to streptomycin alone.
Various hospital outbreaks have been caused by VRE worldwide and this has dramatically augmented in past few years, for the reason that widespread injudicious and irrational use of antibiotics is being practiced allowing the dissemination of these strains (4). This research work was intended to determine the antibiotic susceptibility patterns of Enterococci isolated from diverse clinical specimens with particular emphasis on aminoglycoside and vancomycin. The acquaintance of HLAR strains can help the clinicians to make prudent decisions for the use of antibiotics such as cell wall inhibitor plus aminoglycosides during the commencement of treatment evading the needless use of reserve antibiotics.
A six month study from November 2020 to April 2021 was planned and structured in the department of Microbiology & Immunology at a tertiary care institute. Fifty Enterococcus isolates from various clinical specimens including blood, body fluids (pleural, Peritoneal), CSF, pus, urine, semen, vaginal swab, and throat swab that were sent to the laboratory for culture and sensitivity were included in the study. Without delay the samples were processed post their collection and standard methods of identification were incorporated for the identification of Enterococcus isolates i.e. Gram staining, colony morphology, catalase test, bile solubility, growth in sodium chloride, bile esculin test, and sugar fermentation tests. M100 Clinical and Laboratory Standards Institute 2021 document was referred to analyze the antibiotic susceptibility patterns of enterococcal isolates and for the performance of screening and confirmatory tests used to distinguish particular resistance mechanisms like Penicillin resistance, HLAR and vancomycin resistance (5).
Disc diffusion method was used for detecting the high level aminoglycoside resistance, if present, in the enterococcal isolates (5). In disc diffusion method, high level gentamicin (120 𝜇g) (Himedia, India) and streptomycin (300 𝜇g) discs (Himedia, India) were placed on Mueller –Hinton agar (Himedia, India) medium. The incubation temperature and time of plates was 37ºC for 24 hours and after incubation measurement for the zone of inhibition diameter was done. Observation of no zone or a zone ≤ 6mm implied resistance, inconclusive (7–9 mm) and a zone of diameter ≥ 10mm implied susceptible pattern. A Resistant phenotype meant that the aminoglycoside shows non-synergism with cell wall–active agent (e.g., ampicillin, penicillin, and vancomycin) whereas susceptible indicates synergism with the cell wall inhibitors. Quality control of the test was performed by incorporating Enterococcus faecalis ATCC 29212 (susceptible) and Enterococcus faecalis ATCC 51299 (resistant). If disk diffusion result was inconclusive, verification of the result was done by agar dilution method. An inhibition zone of 7-9 mm was corroborated by the agar dilution method using brain heart infusion agar (BHI) containing gentamicin 500µg /ml as recommended by CLSI guidelines (5). Spot inoculation of 10µl of the 0.5 McFarland suspension bacterial suspension was done on the agar and subject to aerobic incubation at 35±2ºC for a period of 24 hours. Streptomycin 1000 µg/ml was also tested likewise and the incubation was carried out for 24-48 hours. Re-incubation was done if susceptibility was obseved at 24 hours. The interpretation was taken as resistant with > 1 colony seen on screening agar (5).
Vancomycin susceptibility/resistance patterns were reported using vancomycin disc (30µg) (Himedia, India); ≥17millimeter (mm) as sensitive, 15-16 mm intermediate and ≤ 14 mm as resistant. However agar dilution method using BHI agar was also done as per Clinical Laboratory standard institute (CLSI) guidelines. Enterococcus faecalis ATCC 29212 served as the negative control (susceptible) and Enterococcus faecalis ATCC 51299 as the positive control (resistant). Presence of >1 colony indicated presumptive vancomycin resistance. Interpretive criteria were defined as per CLSI: ≤ 4 sensitive, 8-16 intermediate and ≥ 32 as resistant (5).
Microsoft excel was used for data study and analysis. The data extracted was subject to descriptive statistical analyses and descriptive values, which were then recruited as numbers along with percentage frequencies. Independent variables were compared by means of t-test and chi-square test and a value of P <0.05 was considered as statistically significant.
Fifty strains of Enterococcus spp. were isolated over a period of six months from different clinical specimens. Pus samples yielded a maximum of 20 (40%) strains, followed by urine 15 (30%) and blood 9 (18%). The rest of the strains were isolated from peritoneal fluid and Bronchoalveolar lavage (BAL); 4(8%) and 2 (4%), respectively. The maximum number of isolates were those having high-level gentamicin (HLGR) (54%), whereas the isolates with high-level streptomycin resistance (HLSR) comprised 32% of the isolates. Of the isolates, 14% were positive for both HLGR and HLSR (Table 1). Of Enterococcus faecium isolates, 61.7% were HLGR, and 43.75% of Enterococcus faecalis isolates were HLSR (Table 2). maximum majority of the pus samples demonstrated HLGR (41.5%) with blood and urine samples showing 12% of this resistance pattern. HLSR was as well more frequent in pus samples (12%) with 10% similar resistance morphology shown by urine samples (Table 3), while 70% of isolates were sensitive (zone diameter >17 mm) to vancomycin and 8% isolates showed Intermediate (zone diameter 15-16 mm) and 22% isolates showed resistance to vancomycin. The agar dilution method revealed that 74% of isolates with a vancomycin MIC < 4 µg/mL and 12% isolates with MIC ≥ 32 µg/mL are therefore resistant. 25% vancomycin resistance (maximum) was detected in isolates from pus samples (Table 4).
Table 1. Distribution of high-level gentamicin and high-level streptomycin resistance among the Enterococcal isolates (n=50)
HLAR | No. of Isolates (%) |
HLGR | 27 (54%) |
HLSR | 16 (32%) |
HLGR+HLSR | 7 (14%) |
HLAR- High level aminoglycoside resistance, HLGR-High level gentamicin resistance, HLSR-High level streptomycin resistance
Table 2. High-level aminoglycoside resistance among E. faecalis and E. faecium (n=50)
Enterococcal species | Total Isolates No (%) |
HLGR No (%) |
HLSR No (%) |
Resistant to both HLGR And HLSR No (%) |
E. faecalis | 16(32%) | 6(37.5%) | 7(43.75%) | 2(12.5) |
E. faecium | 34(68%) | 21(61.7%) | 9(26.4) | 5(14.7) |
HLGR-High level gentamicin resistance, HLSR-High level streptomycin resistance
Table 3. The pattern of High-level Aminoglycoside resistance in Enterococcus spp. from various clinical specimens (n=50)
Clinical Specimen | HLGR | HLSR | ||
No. | % | No. | % | |
Pus | 11 | 22% | 6 | 12% |
Urine | 6 | 12% | 5 | 10% |
Blood | 6 | 12% | 2 | 4% |
Peritoneal fluid | 3 | 6% | 1 | 2% |
BAL | 0 | 0% | 0 | 0% |
Total | 26 | 52% | 16 | 32% |
HLGR-High level gentamicin resistance, HLSR-High level streptomycin resistance
Table 4. Vancomycin Susceptibility among isolates of Enterococcus by using disc diffusion and agar dilution methods.
Disc Diffusion Method (n=50) | Agar dilution method (n=50) |
|||||||||
Disc Content (30 µg) | MIC (µg/mL) | |||||||||
>17(S)* mm |
15-16(I)# mm | <14(R)** mm | 0.5 | 1 | 2 | 4 | 8 | 16 | 32 | |
35 (70%) | 4 (8%) | 11 (22%) | 3 (6%) | 12 (24%) | 20 (40%) | 2 (4%) | 2 (4%) | 5 (10%) | 6 (12%) |
*S- Sensitive, **-Resistant, #-Intermediate
When VRE was compared to VSE, the rate of HLSR was detected to be 4.64% in VRE, while it was 32.55% in VSE; the rate of HLGR was noted to be 11.62% in VRE, and it was 41.87% in VSE. The association of HLGR with HLSR, namely high-level aminoglycoside resistance (HLAR), was 2.32% in VRE, and 13.95% in VSE strains, respectively (Table 5).
Table 5. High-level aminoglycoside resistance rate between VRE and VSE (n=43).
Variable | VRE(6) | VSE(37) | P value |
GEN R-STREP R | 1(2.32%) | 6(13.95%) | 0.977 |
GEN S- STREP S | 0(0%) | 11(25.58%) | - |
GEN R-STREP S | 4(9.30%) | 12(27.90%) | 0.10 |
GEN S- STREP R | 1(2.32%) | 8(18.6%) | 0.78 |
*7 Enterococcal Isolates with Intermediate Vancomycin Resistance ( 8-16 µg/mL)
VRE-vancomycin resistant Enterococci, VSE- Vancomycin sensitive Enterococci, GEN- gentamicin, STREP- Streptomycin, R- Resistant, S - Sensitive
Multiple drug-resistant Enterococci pose a grave concern in hospital settings, leading to therapeutic failure in patients. As a significant number of clinical Enterococci were positive for both HLGR and HLSR, there is a need to treat infections with high-level aminoglycoside resistance in optimal dosage and duration. It is, therefore, imperative for laboratories to endow with precise antimicrobial resistance phenotypes for Enterococci so that efficient remedies and measures for infection control can be initiated. Also, drugs like vancomycin and daptomycin should be judicious, and linezolid-like antibiotics should be administered when enterococcal infections are proved by culture.
All the facilities for this study were kindly provided by the Department of Medical Microbiology, Shri Guru Ram Rai Institute of Medical and Health Sciences.
Nil.
The study protocol was approved by the Ethical Committee Shri Guru Ram Rai Institute of Medical and Health Sciences.
Conflicts of Interest
The authors declare no conflict of interest.
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