Reena Rajan¹  and D Karthikeyan ²

¹Department of  Microbiology ,Vinayaka Mission’s Kirupananda Variyar Medical College Salem,Tamil Nadu-636308.

²Department of Microbiology,Vinayaka Mission’s Medical College Karaikal,Tamil Nadu-609609.

Corresponding Author E-mail: reenarajan83@gmailcom

DOI : https://dx.doi.org/10.13005/bpj/2221

Abstract

Introduction: The combined abilities of colonisation and both inherent and acquired resistance have made Enterococci a significant human pathogen. Aims and Objectives: This study was done to determine the Minimum Inhibitory Concentration of various antibiotics against Enterococci and to correlate the phenotypic and genotypic characteristics of Enterococci with low level and high level drug resistance. Materials and Methods: A total of 774 isolates of Enterococci obtained from various clinical samples were subjected to antimicrobial susceptibility testing by Kirby Bauer Disk Diffusion method. The Minimum Inhibitory Concentration of various antibiotics were determined by Vitek 2 automated system, agar dilution and E test. Results: 15 out of 774 isolates showed the presence of vancomycin resistant genes by Multiplex PCR. 10 (90.91 %) isolates  out of 11  E. faecalis with van A gene showed high level resistance to Penicillin (16-64 µg/ml). 8 (72.73 %) out of 11 isolates showed high level resistance to Gentamicin (512-1024 µg/ml). 6 (54.55 %) , out of 11 isolates were resistant to β lactams. One isolate of E. faecalis from urine with van B gene showed showed high level resistance to Penicillin (32 µg/ml), Linezolid (≥ 8µg/ml), high level resistance to Gentamicin (1024 µg/ml), Fluoroquinolones (≥ 8µg/ml) and Macrolides (≥ 8µg/ml). Conclusion: Isolates of Enterococci resistant to glycopeptides, penicillin, Betalactams and aminoglycosides have important clinical implications in the treatment for infection.

Keywords

High Level  Drug Resistance; Low Level Drug Resistance; Minimum Inhibitory Concentration, Phenotype, Genotype

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Copy the following to cite this article: Rajan R, Karthikeyan D. A Study on High and Low level Drug Resistance Pattern Among Clinical Isolates of Enterococci. Biomed Pharmacol J 2021;14(3) .

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 Introduction

The genus Enterococci  have exhibited the potential to harbour and transfer drug resistant genes and have become an important pathogen in clinical settings. In  Enterococci, lower affinity of  Penicillin Binding Proteins (PBP’s) are responsible for decreased susceptibility to Penicillin’s. The Minimum Inhibitory concentration (MIC) of penicillin  for Enterococci  are higher than  that  for Streptococci and  inhibition of PBP’s does not result in bactericidal activity in Enterococci ¹.

Enterococci display low to moderate  level resistance to aminoglycosides due to slow uptake or permeability of these agents. All Enterococci have innate low level resistance to aminoglycosides with minimal inhibitory concentration ranging from 4 µg/ml to as high as 256 µg/ml. High level resistance to Gentamicin is associated with bifunctional enzyme possessing acetylase (6^’) and phosphotransferase (2^’) activities conferring resistance to all aminoglycosides except Streptomycin ². Enterococci also exhibits acquired resistance via mutations in existing DNA or through acquisition of new DNA and high level resistance are usually due to the transferable plasmid mediated production of aminoglycoside inactivating enzymes ³.

Beta lactamase  producing Enterococci exhibits inducible and constitutive low level resistance ⁴.  In clinical isolates of E. faecium, β lactamase resistance is associated with mutations or overproduction of PBP5 with Ampicillin MIC of >256 mg/L in some strains. Isolates of E. faecium with MIC of Ampicillin ≤64 mg/L may respond to high dose Ampicillin therapy ⁵.

Quinolone resistance occurs by mutation in the quinolone resistance determining regions of the genes that encodes gyrase and topoisomerase IV and have been observed in clinical  isolates of Enterococci. These mutations prevent efficient binding of the antibiotics to the enzymes which enable DNA replication to continue despite the presence of antibiotics. The second mechanism contributes to quinolone resistance are Multidrug resistance efflux pumps (MDRs) on bacterial chromosomes. Inactivation of homolog of quinolone resistance (Qnr) identified in E. faecalis resulted in modest decrease in resistance to quinolones and over expression of gene results in increased resistance ⁶.

The most common form of acquired resistance to macrolides is production of an enzyme (erm B gene) that methylates a specific adenine in the 23 S rRNA of the 50S ribosomal subunit there by reducing the binding affinity of this drug for the ribosome, which also reduces the binding of Lincosamide and Streptogramin to the ribosome. An efflux pump encoded by transferrable mefA gene is known to pump macrolides out of the cell and confers low level resistance in Enterococci. ⁷

Linezolid has been reported to cure VRE endocarditis and other serious intravascular infections, bacteremia, UTI and skin and soft tissue infections ⁸. Tigecycline displays broad spectrum of activity and potency against Staphylococcus, Streptococci and Enterococci with MIC ₉₀ value of ≤ 12µg/ml.Tigecycline may play a role in combination therapy with bactericidal agents such as Vancomycin, Gentamicin, Rifampicin or Daptomycin. Combinations of Daptomycin (8 mg/kg) plus Ampicillin plus Gentamicin and Daptomycin plus Gentamicin plus Rifampicin have been reported to be successful in cases of Vancomycin resistant endocarditis ⁹.

One of the major concerns that physicians face during treatment of Enterococcal infection is the probability of developing resistance during therapy, which may lead to therapeutic failures and contributes to patient mortality. Monitoring the resistance pattern of clinical isolates of Enterococci is a useful tool to obtain information on the prevalence of multidrug resistant isolates and thereby limiting the spread of bacterial resistance.

Aim and Objectives

To determine the Minimum Inhibitory Concentration of various antibiotics  against Enterococci  by Vitek 2 automated system.

To detect High Level Gentamicin Resistance among Enterococci by various phenotypic methods.

To correlate phenotypic and genotypic characteristics of Enterococci with low level and high level drug resistance.

Materials and Methods

A total of  774  isolates of  Enterococci obtained from various clinical samples  were subjected to antimicrobial susceptibility testing on Muller Hinton Agar  by Kirby-Bauer disk diffusion method and the plates were incubated for 16-18 hrs at 37⁰C.The diameter of the zone of inhibition was measured and zone size interpreted according to CLSI standards ¹⁰

The antibiotic disks used were Penicillin (10 U), Amoxicillin Clavulanic acid (20/10μg),

Erythromycin (15μg), High Level Gentamicin (120 μg), Linezolid (30μg), Teicoplanin (30 μg), Ciprofloxacin (5μg), Levofloxacin (5 μg) and Nitrofurantoin (300μg) .Zones of inhibition were measured and recorded and the organism was interpreted as sensitive or resistant as per the recommendations from CLSI guidelines using Enterococcus faecalis ATCC 29212 as control. Screening for   isolates resistant to  Vancomycin and High Level Gentamicin was done on Brain Heart Infusion agar containing 6µg/ml of vancomycin and 500 µg/ml of Gentamicin respectively. Inoculum of 10 μL of 0.5 McFarland’s was spot inoculated. Presence of more than 1 colony was interpreted as a resistant strain. ¹¹ Multiplex PCR was performed to detect the  presence of vancomycin resistance genes., vanA and van B using the following primers (Sigma Aldrich, USA).¹²

Gene: VanA (732 bp)

Primer: F (+) – GGGAAAACGACAATTGC (position: 176–192)

R (-) – GTACAATGCGGCCGTTA (position : 907–891)

Gene: VanB (647 bp)

Primer: F (+) – ACGGAATGGGAAGCCGA (position: 169–185)

R (-) – TGCACCCGATTTCGTTC (position: 815–799)

Minimum Inhibitory Concentration for Gentamicin  was determined by agar dilution method  and Epsilometer test. Agar dilution was performed by on Brain heart infusion agar by using  500 μg , 1000 μg  and 2000 μg of Gentamicin. To detect Gentamicin resistance by E test,MIC strips coated with Gentamicin in a concentration gradient of 0.064-1024 μg/ml was used  . MIC was read when the ellipse intersects MIC scale on the strip.¹¹

Turbidometrically controlled bacterial pure growths were suspended into sterile physiological saline and this suspension was used to fill Vitek 2 Compact system and antimicrobial susceptibility testing cards.¹³ For biochemical identification  of isolates in Vitek system, the following parameters were used: Growth in 6.5 % NaCl, β- glucuronidase, Trehalose, Arginine dihydrolase, D-sorbitol, Urease, Raffinose, D-galactose, D-mannitol, Sucrose, β-galactosidase, Salicin, L-pyrrolidonyl arylamidase, D-xylose, D-maltose, Methyl- β-D-glycopyranoside, D-ribose, α-glucosidase, α- mannosidase, Phosphatase etc. ¹⁴

Minimum inhibitory concentrations for the following antibiotics were tested: Penicillin, Erythromycin, Vancomycin, Teicoplanin, Ciprofloxacin, Levofloxacin, Tigecycline, Nitrofurantoin, Linezolid and Daptomycin.

Results

Out of 774 samples studied ,  726 (93.80 %)  isolates were Enterococcus faecalis, followed by Enterococcus faecium, 33 (4.26 %), Enterococcus avium, 12 (1.55 %) and Enterococcus durans, 3 (0.39 %).

Out of 30 Vancomycin resistant isolates obtained by agar screen method from 774 samples, resistant phenotype was detected in 18 isolates by Vitek 2 automated system.15 out of  18  isolates showed the presence of  vancomycin resistant genes by multiplex PCR.

10 (90.91 %) isolates out of 11 E. faecalis with van A gene showed high level resistance to Penicillin (16-64 µg/ml), 8 (72.73 %) out of 11 isolates showed high level resistance to Gentamicin (512-1024 µg/ml), 6 (54.55 %) , out of 11 isolates were resistant to β lactams due

to the modification of Penicillin binding proteins [Table 1]. One (9.09 %) out of 11 isolates were resistant to Linezolid, 10 (90.91 %) isolates showed resistance (≥ 8µg/ml) and one isolate showed intermediate resistance (2 µg/ml) to Ciprofloxacin.

Nine (81.82 %) isolates showed resistance (≥ 8µg/ml) and 2 (18.18 %) showed intermediate resistance (4 µg/ml)  to Levofloxacin, 9 (81.82 %) isolates showed resistance (≥8µg/ml) and 2 (18.18 %) showed intermediate resistance (1-4 µg/ml)  to Erythromycin. 6/11 (54.55 %) showed resistance and  2 (18.18 %) were with intermediate resistance to Nitrofurantoin.

One isolate of E. faecalis from urine with van B gene showed high level resistance to Penicillin (32 µg/ml), Linezolid (≥ 8µg/ml), high level resistance to Gentamicin (1024 µg/ml), Fluoroquinolones (≥ 8µg/ml) and Macrolides (≥ 8µg/ml). One E.faecium isolate with van A gene showed susceptibility to Penicillin with high level resistance to Gentamicin (1024 µg/ml). One E. faecalis isolate from pus  with both van A and van B gene showed high level resistance to Penicillin (32 µg/ml), Linezolid (≥ 8 µg/ml), high level resistance to Gentamicin (1024 µg/ml),Fluoroquinolone(≥ 8µg/ml) and  intermediate  resistance to macrolides(4 µg/ml).

Table 1: Resistance profile of Vancomycin resistant Enterococci

Sample	Isolates (n=15)	Genotypes	Resistance profile (MIC)
			Penicillin	HLG	β lactams
Urine	E. faecalis (11 isolates )	vanA	8-64 (R) 11 (100 %)	512-1024 8 (72.73 %)	Positive 6 (54.55 %)
Pus	E. faecalis	vanA & vanB	32 (R)	1024 (R)	–
Urine	E. faecalis (1 isolate )	vanB	32 (R)	1024 (R)	–
Urine	E. faecium	vanA	2 (S)	1024 (R)	–
Urine	E. durans	vanA	16 (R)	1024 (R)	–

No significant association was found between Vancomycin resistant  genotypes of Enterococci  and  their high and low  levels of resistance to Penicillin, Gentamicin and β lactams (p value >0.05) [Table:2, 3 and 4].

Table 2: Relation between van genotypes and high and low level Penicillin resistance.

Isolate	Genotype	Low Level Resistance ≤8µg	High Level Resistance  (16-64 µg)
E. faecalis (11)	vanA	1(9.09)	10 (90.9)
E. faecium (1)	vanA	0 (0.00)	0 (0.00)

Table 3: Relation between van genotypes and high and low level Gentamicin resistance.

Isolate	Genotype	Low Level Resistance  (64-512 µg)	High Level Resistance  (512-1024µg)
E. faecalis (11)	vanA	0 (0.00)	8 (72.73)
E. faecium (1)	vanA	0 (0.00)	1 (100)

Table 4: Relation between van genotypes and high and low level resistance to β lactams

Isolate	Genotype	Low Level Resistance  (Penicillin Binding Protein modification)	High Level Resistance  (β lactamase)
E. faecalis (11)	vanA	6 (54.55)	0 (0.00)
E. faecium (1)	vanA	0 (0.00)	0 (0.00)

Discussion

Enterococci possess remarkable ability to acquire antibiotic resistance determinants. The ability of these organisms to colonise the gastrointestinal tract  of  hospitalised  patients for  prolonged  period  is a crucial factor  that influences  the development of drug  resistance. With the increased use of Vancomycin and wide spectrum antibiotics in the health care setting, these organism have been identified as common etiology of nosocomial infection .

Enterococci exhibit decreased susceptibility to Penicillins and Ampicillins as a result of expression of low level Penicillin Binding Proteins (PBPs). Isolates of Enterococci susceptible to Penicillin are predictably susceptible to Ampicillin, Amoxicillin, Ampicillin- Sulbactam, Amoxycillin-Clavulanate, Piperacillin and Piperacillin-Tazobactam for non β

lactamase producing Enterococci. Isolates of Enterococci susceptible to Ampicillin cannot be assumed to be susceptible to Penicillin ¹⁰.In the present  study, 82.17 % isolates were resistant to Penicillin by disk diffusion method. About.19.90 % (154/774) isolates were with  MIC range of 16-64 µg. In a similar study from Delhi, S Jain et al., 2011 have reported 100 % isolates resistant to Penicillin by disk diffusion method ¹⁵. In the present study, out of E. faecium isolates, 33.33 % showed MIC of ≥ 16 µg and 19.42 % of E. faecalis were resistant to Penicillin with MIC ≥ 16 µg. In a study from Chandigarh by V Gupta et al., 2007, 14 % isolates were with MIC of > 16 µg/ml for Penicillin ¹⁶. In a study reported by Triveda , 2016 from South India, Penicillin resistance was found to be higher among E. faecium (64.51 %) isolates compared to E. faecalis (48.45 %) ¹⁷. In the present study no significant difference in resistance to Penicillin was found between E. faecalis and E. faecium isolates (p value >0.05).

High level resistance to β lactam antibiotics are seen in E. faecium due to the over production of low affinity Penicillin Binding Proteins (PBP’s). A variety of point mutation in PBP has been described in E. faecalis and E. faecium ¹⁸. In our study, by vitek 2 automated system, resistance to β lactams due to modification of Penicillin Binding Proteins was detected in 40.70 % isolates. A similar study by S Sivasankari et al., 2013 have reported 72.70 % E. faecium isolates resistant to Ampicillin compared to E. faecalis (24.30 %) ¹⁹ .In a North Indian study, C S Aher et al., 2014 have reported 80.70 % Enterococci isolates resistant to Ampicillin ²⁰

The prevalence of Enterococci with high level Gentamicin resistance are increasing and varies by geographical region and are generally resistant to all aminoglycosides with the occasional exception of Streptomycin. Aminoglycoside Modifying Enzymes (AME’s) are the most important mechanism mediating high level resistance. These enzymes confer high level resistance to more than one aminoglycoside. A single strain of Enterococci may acquire several AME’s. Both E. faecalis and E. faecium may acquire 6’- adenyl transferase which confer HLR to Streptomycin ³.  By disk diffusion method, 53.49 % showed high level Gentamicin resistance. In a  study from North India, D K Mendritta et al., 2008 have reported, 46 % isolates resistant to both high Level Gentamicin(HLG) and High Level Streptomycin(HLS) by both disk diffusion and agar dilution method and combined resistance to both HLG and HLS was higher in E. faecium compared to E. faecalis ²¹.In the present study no significant difference in combined resistance to HLG and HLS was observed between E. faecalis and E. faecium (p value >0.05) where as HLGR was higher among E. faecium isolates. In our study 15.89 % isolates showed high level resistance to Gentamicin (2000 µg/ml) by agar dilution method and 33.66 % isolates showed resistance to both Penicillin and High Level Gentamicin.

By E test 18.22 % isolates showed MIC of 512 µg/ml and 16.67 % were with MIC ≥ 1024 µg/ml for Gentamicin. In the present study the results of Minimum Inhibitory Concentration for HLG were in concordance with disk diffusion method. A similar finding was reported in a study by V P   Prakash, 2005 ²².In a South Indian study P Jyothi et al., 2014 have reported 40 % isolates from urine showed high level resistance to Gentamicin and Streptomycin by both disk diffusion and Enz MIC strip method ²³ .In a South Indian study by E Padmasini et al., 2014 among clinical isolates of Enterococci  ,42.7 % were HLGR (MIC ≥ 512 µg/ml) by E test ²⁴.In a study report by S Mittal et al., 2016, high level gentamicin resistance was more common among Enterococci isolated from urine sample (41.50 %) compared to other clinical samples and HLGR was found to be more common in VRE isolates compared to VSE ²⁵where as our study showed no significant association between VRE and HLGR (p value >0.05).

Quinolone resistance in Enterococci is due to mutation in the quinolone resistance determining regions of the genes that encodes gyrase and topoisomerase IV enzymes. In the present study, 417 (57.44 %)  E. faecalis isolates were resistant to Ciprofloxacin (MIC ≥ 8 µg/ml) and 414 (57.02 %) isolates to Levofloxacin (MIC ≥ 8 µg/ml). About 57.23 % isolates of E. faecalis were resistant to both Ciprofloxacin and Levofloxacin. 72.73 % E. faecium isolates were resistant to the fluoroquinolones tested. Among the fluoroquinolone resistant isolates, no significant difference was observed between E. faecalis and E. faecium isolates (p value >0.05). In a study from Tamilnadu S Sivasankari et al., 2013, 73.10 % E. faecalis and 81.80 % E. faecium isolates were resistant to Ciprofloxacin by disk diffusion method ¹⁹. In a North Indian study, N Gangurde et al., 2014 have reported 80.5 % E. faecalis and 86.2 % E. faecium resistant to Ciprofloxacin ²⁶.

The most common form of acquired resistance to macrolides is production of enzymes that methylase a specific adenine in the 23S rRNA of 50 S ribosomal subunit which reduces binding affinity of macrolides to ribosomes .77.27 % isolates of E. faecalis and 72.73 % E. faecium isolate were resistant to Erythromycin (MIC ≥ 8µg/ml) in this study. In the present study no statistically significant difference in Erythromycin resistance observed between E. faecalis and E. faecium (p value >0.05). Various Indian studies have reported Erythromycin resistance higher among E. faecium compared to E. faecalis ^{19, 26}.

Nitrofurantoin is active against E. faecium and E. faecalis and is effective in the treatment of VRE infection associated with urinary tract. A study conducted by G Zhanel et al., 2001 have  reported that Nitrofurantoin is active against urinary isolates of E. faecalis and E. faecium ²⁷. In the present study from urinary isolates, 14.93 % E. faecalis were resistant to Nitrofurantoin with MIC of 128 µg/ml. The  MIC for 1.81%  isolates  of E.  faecalis  and 20.00 %  E. faecium  were  256 µg/ml and 128 µg/ml respectively. About 2.07 % isolates showed resistance to Nitrofurantoin in a study from Maharastra by S Bose et al., 2012 ²⁸

A similar study by D Atray et al., 2016 have reported 80 % urinary isolates susceptible to Nitrofurantoin ²⁹.

Linezolid resistance may be due to the presence of transferable plasmid borne cfr gene encoding methyl transferase or due to selective pressure imposed by antibiotic treatment. Linezolid is used in the treatment of infections caused by resistant gram positive bacteria particularly Vancomycin – resistant Enterococcus faecalis. Linezolid inhibits protein synthesis but at a different site from other agents that target the ribosome (Chloramphenicol, Macrolides, Lincosamides, Streptogramin, Aminoglycosides, Tetracycline). As a result, existing mechanisms of resistance to these agents do not confer cross-resistance to Linezolid ³⁰.  Our study reports 1.16 % isolates resistant to Linezolid. In a North Indian study by G Reena et al., 2013, 95 % isolates were susceptible to Linezolid ³¹. In  a  South  Indian  study , I Praharaj et al., 2013  have reported isolates with 100 %  susceptibility  to  Linezolid ³².  In  a similar study by  K Archana Rao et al., 2014,  12 %  isolates  showed   resistance  to Linezolid ³³.

Tigecycline shows in vitro bacteriostatic activity against Vancomycin resistant Enterococci. Tigecycline was found to be the most effective drug against Enterococci in our study with 100 % susceptibility. Similar finding were reported from Indian studies ^{31, 32}

As a result of mutation of chromosomal genes, Daptomycin resistance following therapy has been observed in clinical isolates of Enterococci. Daptomycin synergy has been described in vitro with Ampicillin, Cephalosporins, Imipenem, Rifampin and Gentamicin ³⁴. In our study,

96.12 % isolates showed susceptibility to Daptomycin with MIC ≤ 4 µg/ml.

The overall antimicrobial pattern of the isolates showed significantly higher
(p value < 0.05)  percentage resistance to Penicillins, High level aminoglycosides, Macrolides, Fluoroquinolones and Linezolid among the clinical isolates  of  Enterococci.

Conclusion

The present study reports significantly higher percentage resistance to Penicillins, High level aminoglycosides, Macrolides, Fluoroquinolones and Linezolid among the clinical isolates of Enterococci. The most effective drug against Vancomycin Resistant Enterococci was found to be Tigecycline. No significant association was found between Vancomycin resistant genotypes of Enterococci  and high and low  levels of resistance to Penicillin, Gentamicin and β lactams.

Acknowledgement

We thank  Dr. V .Anandi, Former Professor, Department of  Microbiology, Vinayaka Mission’s Medical College  Karaikal,Tamil Nadu, for guiding us in our research work. We are grateful to Biogen Care Research Centre ,Chennai for the technical help and support.

Conflicts of Interest

None

Sources of funding

None

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