Determination of vancomycin, linezolid and daptomycin resistance among Eenterococcus isolates from a tertiary care hospital

Introduction

Enterococcus species constitutes normal intestinal microflora in high proportion of healthy adults. Enterococcus faecalis and Enterococcus faecium are the two most common species of enterococci isolated from clinical samples.^[1] E. faecalis are more prevalent than E. faecium, however increase in E. faecium clinical isolation have been reported recently. E. faecium infections are of clinical relevance, due to high percentage of resistant to vancomycin and ampicillin.^[2]

Enterococcus are opportunistic pathogens associated with endocarditis and urinary tract infections, intra-abdominal infections and surgical wound.^[3] These bacteria are intrinsically resistant to low concentrations of β-lactams, trimethoprim–sulfamethoxazole, aminoglycosides, and clindamycin. Furthermore, they readily acquires foreign genetic material with antibiotic resistant or undergo mutation for antibiotic resistance.^[4] Few recent studies have described resistance to higher concentration of penicillins, aminoglycosides, macrolides-lincosamides-streptogramins, fluoroquinolones, rifampin and glycopeptides.^[5], ^[6]

In year 1988, Enterococcus resistance to high levels of vancomycin and teicoplanin was reported for the first time. Since then, number of infections with vancomycin-resistant enterococci has increased in hospitals all over the world. Infectious Diseases Society of America (IDSA) recommends using linezolid or daptomycin for Enterococcus strains resistant to ampicillin and vancomycin.^[7] However, very limited studies are available on antibiotic susceptibility pattern of Enterococcus species to vancomycin, linezolid and daptomycin and these studies results has inter regional and intraregional variations.^[8]

This study was undertaken to determine the antibiotic susceptibility pattern of Enterococcus species to vancomycin, linezolid and daptomycin, in a tertiary care hospital of western Maharashtra.

Materials and Methods

This study was a retrospective study conducted by Department of Microbiology, of a tertiary care hospital. Total of 363 consecutive, non-repetitive clinical isolates of Enterococcus species from different clinical samples were included in the study. The study period was from 01 Jan 2016 to 31 Mar 2019.

Enterococcus species were isolated and identified in accordance with standard procedures.^[9] Antibiotic susceptibility testing (ABST) of all isolate was conducted by both disc diffusion method and Minimum inhibitory concentration (MIC) on automated Vitek (BioMérieux) system.^[10] Enterococcus ATCC strains E. faecalis ATCC 29212 were used as standards for antibiotic susceptibility testing. Strains resistant to vancomycin, linezolid and daptomycin were confirmed further tested by E test strip (Himedia, Mumbai). For automated ABST and E test manufacturer instructions were followed. Minimum inhibitory concentration (MIC) interpreted was as per criteria laid by Clinical Laboratory Standards Institute (CLSI) guidelines.^[11]

Result

A total of 363 Enterococcus were isolated during the study period. Of these, there were 250 (68.87%) isolates were of Enterococcus faecalis and 113 (31.12%) of Enterococcus faecium. No other Enterococcus species were isolated. Clinical samples included urine, blood, pus, sputum and other samples like body fluids etc. Majority of isolates were from urine sample followed by blood and pus ([Table 1]). Among isolates of Enterococcus- 11(3.03%), 18(4.95%) and 334(92.01%) were intermediate, resistant and sensitive to vancomycin respectively. Linezolid intermediate and resistant was identified in 3(0.82%) and 4 (1.10%) isolates of Enterococcus faecium only. Among 2 (50%) linezolid resistant isolates were also resistant to vancomycin, however they were all sensitive to daptomycin ([Table 1]). MIC to vancomycin ranged between 0.25-256 µg/ml, MIC of linezolid ranged between 0.25- 16µg/ml and MIC for daptomycin was less than 1 µg/ml for all the isolates ([Figure 1]) [[Table 2]].

Discussion

In the hospitals, the real challenge in management of enterococcal infection lies in its intrinsic and acquired resistance to numerous antimicrobial agents. Researchers recommend combination therapy for high load enterococcal infection, like endocarditis.^[12] Combination therapy includes ampicillin for susceptible Enterococcus isolates and vancomycin for penicillin resistance isolates. However, many researcher have reported emergence of vancomycin resistance in Enterococcus species, which pose an immense challenge to the clinicians.^[13] For vancomycin resistant clinical isolates, Infectious Diseases Society of America (IDSA) recommends linezolid or daptomycin antibiotics especially in bacteremia.^[14] In views of these recommendation, in vitro susceptibility testing for newer antimicrobials, such as daptomycin and linezolid, is essential for the management of VRE infections.

In our study, Vancomycin resistance was noted among 10.61% Enterococcus faecium and 2.40% Enterococcus faecalis isolates. The prevalence of VRE in our study is 4.95% which similar to other published studies.^[15], ^[16] Hospitals laboratory should monitor for any evidence of creeping MIC, which indicates heteroresistance.  These heteroresistant isolates often leads to treatment failure.^[17]

Six different types of vancomycin resistance are shown by Enterococcus: Van-A, Van-B, Van-C, Van-D, Van-E and Van-G. Van A confers high degree of resistance to both vancomycin (MIC≥64μg/ml) and teicoplanin (MIC≥16μg/ml), whereas Van B and Van E confers varying level resistance to vancomycin (MIC 4 -1000 μg/ml), but are susceptible to teicoplanin.^[18] Van A is the most common mechanism for resistance among clinical isolates, so identification of any intermediate susceptibility to vancomycin warrants detailed molecular investigation.^[19], ^[20] In this study, Vancomycin intermediate susceptibility was noted among 11 (3.03%) isolates, which is in concordance with other studies.^[19], ^[20]

Linezolid resistance was detected in 4 (1.10%) Enterococcus isolates, whereas 3(0.82%) isolates demonstrated intermediate susceptibility. Linezolid resistance in Enterococcus has been reported earlier by other researchers also.^[21] In this study, prevalence of linezolid resistance is relatively lower compare to other publisher report, due to robust hospital antibiotic policy. Further, in this study, two Enterococcus isolates demonstrated combined linezolid and vancomycin resistance. Combined resistance to both vancomycin and linezolid is very rare.^[21], ^[22] These results are in concordance with findings reported by other researchers.^[21], ^[22] In- vitro detection of linezolid resistance in  Enterococcus, has poor correlation between various methods. Therefore, in our study, disc diffusion method was initially used, which was followed by MIC based automated antimicrobial susceptibility testing by Vitek 2 system (bioMérieux) and confirmation by the E-test method.^[23]

Daptomycin is lipopeptide antibiotic used in the treatment life-threatening infections caused by gram-positive organisms. Daptomycin activity in media requires presence of divalent cations, especially calcium ions. E- strips of daptomycin with supplemented calcium ions can be applied directly on Mueller Hinton Agar. In our study, calcium supplemented culture media was used for daptomycin activity and all isolates were sensitive to daptomycin. Daptomycin resistance in Enterococcus isolates, is rare and reported in cancer patients.^[22], ^[24] Researchers have reported association of daptomycin resistant in Enterococci species with earlier exposure to daptomycin and vancomycin resistance. Enterococcal strains with vancomycin hetero-resistance, may fail to respond to daptomycin therapy, despite in vitro susceptibility to daptomycin.^[25]

Recommended therapy for serious enterococcal infections consists of synergistic combination of an aminoglycoside and a cell wall–active agent. However, many E. faecium isolates are intrinsically resistant to cell wall active antibiotics like penicillin and some acquires resistance to vancomycin. Further, this resistance is mediated by plasmids, which are easily transferable between bacterial species. Newer antibiotics were developed for treatment Enterococci infections resistant to ampicillin, vancomycin, or the aminoglycosides. These antibiotics include linezolid, daptomycin, and tigecy­cline. However soon after clinical usage of linezolid, resis­tance to linezolid has also emerged, which is steadily rising.^[26]

Present recommendation for Enterococcus susceptible to ampicillin, but resistant to aminoglycosides is a combination of ampicillin plus daptomycin or linezolid. For Enterococcus isolates that are resistant to ampicillin and susceptible to amino­glycosides, aminoglycoside combined with vancomycin should be used. However, if the isolate is resistant to both ampicillin and aminoglycosides, management should include newer antibiotics daptomycin, linezolid, or vanco­mycin combined with another susceptible antimicrobial agent.^[24], ^[27]

Conclusion

Our study demonstrated relatively low prevalence of Vancomycin and Linezolid resistance among clinical isolates of Enterococcus. However, persistence of vancomycin pressure on hospital flora and emergence of isolates Enterococcus species with combined resistance to newer antibiotics, is a cause of concern. Linezolid and daptomycin are effective antibiotics against VRE. Strict implementation of hospital antibiotic policy with judicious use of antibiotics is a key to prevention of emergence of multidrug resistant strains of Enterococcus species.

Source of Funding

None.

Conflict of Interest

None.