Introduction

Enterococci are one of the important cause of infections, especially in the hospitalised patients. They are normal commensals found in the intestines of the humans. They are intrinsically resistant to majority of the commonly used antibiotics in the hospital like cephalosporins, aminoglycosides, clindamycin etc. Therefore, these bacteria can successfully cause nosocomial infections. (1)

There are various known species of enterococci which can cause infections in humans. E. faecium infections are generally found to be more difficult to treat compared to other species of enterococci. In this study, we determined the proportion of the enterococcal infections due to the E. faecium species and the differences in the epidemiology and susceptibility profiles of the E. faecium isolates compared to E. faecalis isolates in a tertiary care hospital in Mangalore, in southern India.

Methods

This prospective cohort study was conducted at Father Muller Medical College Hospital, Mangalore for a period of six months from June to November 2023, after ethical clearance from the Father Muller Institute Ethics Committee (FMIEC/CCM /297 /2023).

Inclusion criteria: Non repetitive clinical isolates of Enterococcus species from various clinical specimens like urine, exudates and blood received in the microbiology laboratory.

Exclusion criteria: The isolates of enterococci from the stool and respiratory sample were excluded from the study.

Procedure:

The samples were cultured using 5% sheep blood agar (HiMedia Laboratories Pvt. Ltd., Mumbai, India) and MacConkey agar (HiMedia Laboratories Pvt. Ltd., Mumbai, India Bac T/Alert aerobic culture bottle (bio Mérieux, France) was used for blood culture. All the suspected enterococcal isolates were identified and speciated by standard biochemical tests. The small translucent colonies on the 5% sheep blood agar and corresponding pinpoint magenta pink colonies were selected for colony smear and Gram staining. The isolates were tested for production of catalase enzyme. The Gram-positive cocci in pairs, which did not produce catalase enzyme, were further tested for hydrolysis of bile esculin, fermentation of mannitol, arabinose and motility. The isolates which hydrolysed bile esculin were identified as Enterococcus species. The non motile isolates were speciated as E. faecalis or E. faecium based on fermentation of arabinose. The confirmation of identification of enterococci was done by Bruker Daltonics Microflex LT/SH MALDI-MS System (Bruker Daltonics, Germany).

Antibiotic susceptibility testing was done by modified Kirby Bauer disc diffusion method according to Clinical Laboratory Standards Institute (CLSI) guidelines (3). Antibiotic discs from HiMedia Laboratories Pvt. Ltd., Mumbai, India were used. Enterococcus faecalis ATCC strain 29212, Staphylococcus aureus ATCC 25923 and Escherichia coli ATCC 25922 were used as quality controls for antibiotics. Ampicillin 10µg, high level gentamicin 120 µg, nitrofurantoin 300 µg, ciprofloxacin 5 µg, levofloxacin 5 µg, vancomycin 30 µg, teicoplanin 30 µg and linezolid 30 µg discs were used for antibiotic susceptibility testing.

Statistical Analysis:

The statistical analysis was done using SPSS version 23, IBM, USA. The categorical variables have been expressed in terms of percentages and frequencies. The continuous variables are expressed as mean or median based on the type of distribution. The chi square test or Fishers exact test has been used as test of significance between the categorical variables. Independent t test or Man Whitney U test were used for the analysis of continuous variables.

Results

In the present study, 132 isolates of the enterococci from the urine, exudates and blood were included. The median age of the patients with the enterococci in clinical samples was 59 years.

Among the isolates included in the study, sixty-six (50%) each were from males and females. On speciation, 68/132 (51.51%) isolates were identified as E. faecalis, 61/132 (46.21%) as E. faecium and2/132 (1.53%) isolates as E. gallinarum and 1/132 (0.75%) as E. raffinosus.

Fig 1: Species distribution of the enterococcal isolates

Fig 2: Sample wise distribution of the various enterococcal species.

Ninety-one (68.9%) of the enterococcal isolates were from urine samples, 34 (25.8%) of the isolates were from exudates and 7 (5.3%) of the isolates were from blood culture samples as shown in Fig 2.

Ninety-six (72.72%) of the enterococcal isolates were from patients admitted to the hospital, whereas 36 (27.27%) of isolates were from the outpatient department. The overall susceptibility testing of all the enterococcal isolates is as in the Table 1. The highest rate of resistance was against fluoroquinolones, high level gentamicin, ampicillin, nitrofurantoin, vancomycin and teicoplanin. No resistance was detected in our isolates against linezolid.

The characteristics of E. faecium compared with E. faecalis are shown in Table 2. On statistical analysis, it was found that E. faecium was more significantly isolated from the urine samples, whereas E. faecalis was significantly more in the exudates. Also, E. faecium was isolated significantly more in the samples from the inpatient department. E. faecium was found to be significantly more resistant to ampicillin, high level gentamicin, nitrofurantoin and vancomycin.

Discussion

Enterococci are important cause of infections in a wide range of patients. They cause urinary tract infections, blood stream infections and skin and soft tissue infections. These infections tend to be resistant to some of the common antibiotics used in the hospitals. E. faecalis and E. faecium are the 2 common species isolated from the clinical samples.

The enterococcal infections occurred in all the age groups of patients in the hospital. The median age of the patients with enterococcal isolates was 59 years. This is in contrast to studies by Yadav and Agarwal, and Mussadiq et al, where the maximum number of isolates from age group 20-29 years and 21-40 years respectively. (4,5) The isolates were equally distributed among males and females. In contrast, Yadav and Agarwal observed that the enterococci were isolated more among the female patients. (4)

On speciation, 68 (51.51%) isolates were identified as Enterococcus faecalis, 61 (46.21%) as Enterococcus faecium and 2 (1.51%) isolates as Enterococcus gallinarum and 1 (0.75%) as Enterococcus raffinosus. Most of the studies in the different places of India show a lower proportion of E. faecium. The proportion of the E. faecium in the different studies in New Delhi, West Bengal, Sikkim have reported a proportion of E. faecium varying between 8.5% to 28.8%. (6–11) Similar reports are also available worldwide. (12,13) On the contrary, Yadav and Agarwal, in a study conducted at Lucknow reported higher proportion (47.5%) of E. faecium. (4) Interestingly, two studies from Mangalore show very different proportions of E. faecium. (14,15) This shows there is a varying proportion of E. faecium infection in the country and the city, at different time periods. The different studies have reported few uncommon species of enterococci like E. hirae, E. durans, E. avium, E. mundtii, E. gallinarum etc. (6,7,9,10) This is similar to our study where E. gallinarum and E. raffinosus were detected. Hence it is seen that majority of the enterococcal infections in the country are caused by E. faecalis and E. faecium.

Ninety-one (68.9%) of the enterococcal isolates were from urine samples, 34 (25.8%) of the isolates were from exudates and 7 (5.3%) of the isolates were from blood culture samples. This in contrast to the various studies in India, where about 80% of the isolates of enterococcal isolates were from the urine samples. (4,6) A higher proportion of the enterococci were reported from the exudate samples in our facility.

Highest number of the isolates in our study were resistant to fluoroquinolones (55% and 56%). This is similar to high resistance to fluoroquinolones, reported in the various studies in the country. (4,7) Most of the studies in the country report resistance of enterococci to high level gentamicin of 30%- 55%. (4,7,10,11,16) High level gentamicin resistance was detected in 45.45% of the enterococcal isolates in the present study. Ampicillin is a good antibiotic to treat the enterococcal infections, if found to be susceptible. Resistance to ampicillin was detected in 34.85% of the enterococcal isolates. Similar resistance rate to ampicillin was documented by few studies, (6) whereas other studies showed higher resistance rates. (4,7) Nitrofurantoin is a very good drug for susceptible enterococcal urinary tract infections. Nitrofurantoin resistance was detected in 22.73% of the isolates which is concordant with other studies in the country. (6,7)

With the increased use of vancomycin and teicoplanin in the hospital, there has been development of enterococcal vancomycin resistance. Vancomycin resistance was observed in 7.58% of the isolates and teicoplanin resistance in 4.55% of the isolates. This is in contrast to a meta-analysis, where pooled vancomycin resistance among the enterococcal isolates from India was 12.4%. (17) There are studies with various different rates of vancomycin resistance in the country. (4,7–11) Chakraborty et al, in a study in West Bengal in the year 2015,(9) reported no vancomycin resistance, whereas Meena et al, in a study published in 2017, reported a high 37% of vancomycin resistant enterococci in New Delhi. (8) The teicoplanin resistance in the present study is lesser than in other similar studies. (7,16)

There was no linezolid resistance among all the enterococcal isolates in the present study compared to a study at New Delhi, where in a span of 3 years, 202 linezolid resistant enterococcal strains were isolated. (18) Various studies in the country have reported various rates of linezolid resistance. (4,7,10)

The isolates of E. faecium did not differ from other isolates in terms of median age of the patients. The median age of the patients in both the groups was 59 years. There was no significant difference in terms of the gender of the patients. The E. faecium, were more frequently isolated from urine samples (77% vs 60.3 % p value = 0.041) and less from the exudate samples (18% vs 33.8%, p value = 0.042), when compared to E. faecalis. There was no significant difference among the E. faecium isolates in terms of the blood sample (4.9% vs 5.9%, p value = 1.000). It was observed in the study that E. faecium isolates were isolated significantly more from the samples of the inpatient departments when compared to E. faecalis isolates (82% vs 63.2% p value = 0.018)

It was observed that E. faecium isolates were significantly more resistant to ampicillin (57.4% vs 14.7% p value = 0.000). In their study at Etawah, Uttar Pradesh, Musaddiq et al reported much higher resistance to ampicillin in both E. fecium and E. fecalis isolates (90.32% vs 84.78%) (5). The E. faecium isolates were also significantly more resistant to the high-level gentamicin (54.1% vs 36.8% p value- 0.048), nitrofurantoin (37.7% vs 8.8%, p value =0.000), than E. faecalis isolates. E. faecium isolates were found to be more resistant, also to ciprofloxacin, levofloxacin, teicoplanin (8.2% vs 1.5%, p value = 0.100), albeit not significantly when compared to the E. faecalis isolates. Similarly, Das et al, documented higher resistance rates of E. faecium, compared to E. faecalis isolates at New Delhi, (7) Similar higher difference in resistance rates was observed in a study in Tabriz, Iran. (12)

The E. faecium isolates were significantly more resistant to vancomycin than E. faecalis (11.5% vs 1.5%, p value = 0.026) which is concordant with other studies. (4,7,12,16,19)

Limitations of the study is that it is a single centre study and the only the phenotypic characterisation of the enterococcal isolates was done. This study demonstrates a higher proportion of E. faecium causing clinical infections than in other parts of the country. E. faecium in our study has significantly higher resistance rates to ampicillin, high level gentamicin, nitrofurantoin and vancomycin. Factors leading to increased rates of E. faecium infections and its antibiotic resistance need to be looked for and addressed promptly.

Conclusion

In this study we found that the proportion of the E. faecium among the clinical isolates is higher compared to many other studies. This is of concern, as the resistance rates to various antibiotics are higher among the E. faecium. The E. faecium isolates were mostly from inpatients indicating antibiotic overuse may be a causal factor. There were significantly higher rates of resistance seen among the E. faecium isolates compared to E. faecalis isolates in our study. Fortunately, the overall rate of resistance to vancomycin is low and none of the isolates were resistant to linezolid. Strict infection control and antimicrobial stewardship measures need to be taken in the hospitals to decrease the prevalence of the E. faecium infections and the resistance among the enterococci to prevent any adverse outcome.

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