Characterization and Antimicrobial Susceptibility Patterns of Enterococcus Species Isolated from Nosocomial Infections in a Saudi Tertiary Care Hospital over a Ten-Year Period (2012–2021)

Introduction: The Enterococcus genus is a common cause of nosocomial infections, with vancomycin-resistant enterococci (VRE) posing a significant treatment challenge. Method: This retrospective study, spanning ten years (2012 to 2021), analyzes antimicrobial susceptibility patterns of Enterococcus species from clinical samples in a Saudi Arabian tertiary care hospital. Result: A total of 1034 Enterococcus isolates were collected, 729 from general wards and 305 from intensive care unit (ICU) patients. VRE accounted for 15.9% of isolates. E. faecalis was the most common species (54.3% of isolates and 2.7% of VRE), followed by E. faecium (33.6% of isolates and 41.2% of VRE). E. faecium exhibited the highest resistance to ciprofloxacin (84.1%), ampicillin (81.6%), and rifampicin (80%), with daptomycin (0.6%) and linezolid (3.1%) showing the lowest resistance. In E. faecalis, ciprofloxacin resistance was highest (59.7%), followed by rifampicin (20.1%) and ampicillin (11.8%). Daptomycin (0%), linezolid (1.5%), and vancomycin (2.7%) had the lowest resistance. VRE cases had higher mortality rates compared to vancomycin-sensitive enterococci (VSE). Conclusion: Eight different strains of Enterocci were identified. E. faecalis was the most commonly identified strain, while E. faecium had the highest percentage of VRE. VRE cases had a significantly higher mortality rate than VSE cases.


Resistance Patterns against Selected Antimicrobial Agents
This study identified and reported eight different species of Enterococcus: E. avium, E. casseliflavus, E. durans, E. faecalis, E. faecium, E. gallinarum, E. hirae, and E. raffinosus.E. faecium showed high resistance to ampicillin (81.6%), whereas E. faecalis showed only 11.8% resistance.Linezolid resistance was observed only in E. faecium and E. faecalis cases, with prevalence rates of 3.1% and 1.5% in the total sample population, respectively.The highest resistance in E. faecium was observed against ciprofloxacin (84.1%), followed by ampicillin (81.6%), rifampicin (80%), and vancomycin (41.2%).The least resistant antibiotics for E. faecium were daptomycin (0.6%) and linezolid (3.1%).For E. faecalis, the highest resistance was observed against ciprofloxacin (59.7%), followed by rifampicin (20.1%) and ampicillin (11.8%).The least resistant antibiotics for E. faecalis were daptomycin (0%), linezolid (1.5%), and vancomycin (2.7%).(Table 2).Trends in ampicillin and vancomycin resistance in the whole study population, ICU, and general ward, as well as trends of E. faecalis and E. faecium over the study years, are shown in Figure 5. Ampicillin resistance increased in all groups between 2012 and 2021, particularly among ICU patients.No significant change in antibiotic resistance was observed in the last two years of the COVID-19 pandemic; however, if only ICU data is considered, there was a significant increase in ampicillin resistance in 2020 and 2021.Vancomycin resistance in ICU patients decreased slightly between 2020 and 2021, but it increased in the overall population and general ward patients.See Figure 5. nificant change in antibiotic resistance was observed in the last two years of the COVID-19 pandemic; however, if only ICU data is considered, there was a significant increase in ampicillin resistance in 2020 and 2021.Vancomycin resistance in ICU patients decreased slightly between 2020 and 2021, but it increased in the overall population and general ward patients.See Figure 5.  20) S NA-Resistance pattern is not available for the antimicrobial agent, R-All isolated samples are resistant to the antimicrobial agent, S-All isolated samples are sensitive to the antimicrobial agent.

Discussion
In this study, we examined the prevalence and resistance patterns of Enterococcus species in the ICU and general ward of a tertiary care hospital in Saudi Arabia from 2012 to 2021.We identified and reported eight different Enterococcus species: E. avium, E. casseliflavus, E. durans, E. faecalis, E. faecium, E. gallinarum, E. hirae, and E. raffinosus.E. faecalis was the most frequently isolated Enterococcus species (54.3%), followed by E. faecium (33.5%).Our findings support the notion that E. faecalis is the most common cause of enterococcal infections [10,11].However, similar to other studies, our study showed an increasing proportion of infections caused by E. faecium.This suggests that E. faecium could become the dominant species causing enterococcal infections in the future.Therefore, it is important to monitor the prevalence of Enterococcus species over time.
Our findings revealed that VRE constituted 15.9% of the total sample, while VSE made up 84.1%.This VRE incidence was consistent with recent studies [12,13].The highest incidence of VRE was observed in E. casseliflavus (75%), followed by E. gallinarum (50%).However, these isolates were few, with only four and six samples respectively, indicating that more positive isolates are needed to represent the true population.These species are known to be intrinsically resistant to vancomycin due to the natural presence of resistant genes [14].E. faecalis represented 54.3% of the total population, with 2.7% of VRE cases, while E. faecium represented 33.6% of the total population, with 41.2% of VRE cases.This was similar to a previous report [4], in which 41.7% of ICU isolates exhibited resistance to vancomycin, with 60% of these strains being E. faecium and 40% E. faecalis.According to our findings, the resistance pattern (VRE) was more prevalent in the ICU population (24.9% of total ICU samples vs. 12.1% of total general ward samples).A high incidence of VRE in ICU samples has also variably reported in previous studies [4,7,12], with rates of 41.7%, 12.3%, and 17.3%, respectively.This could be because ICU patients are more susceptible to nosocomial infections due to weakened immunity, frequent exposure to antimicrobial agents, and severe illnesses.
In our study, VRE samples in males accounted for 53.7% of total VRE samples.However, the percentage of VRE among total female samples was 19.6%, while it was 13.6% of total male samples.Current literature suggests differences between sexes, with a male preference (59%) in the distribution of VRE [15].Previous research has found gender differences in infections caused by some pathogens such as Staphylococcus aureus (male predominance) [16] and Escherichia coli (female predominance) [17].Genetic [18] and hormonal factors [18,19] could contribute to this phenomenon.
Another significant difference in resistance was observed against rifampin, with rates of 20.1% and 80% against E. faecalis and E. faecium, respectively.Previous research on isolates from bovine milk supported our findings, with 13.6% of E. faecalis and 42.3% of E. faecium being rifampicin-resistant strains [25].Rifampicin resistance rates were also found to be 71.2% and 94.3% in E. faecalis and E. faecium isolates from various clinical infections, respectively [26].Previously, research [27] on urinary tract infection isolates found complete resistance to rifampicin in E. faecium (100%) and a significant proportion in E. faecalis (81.2%).Rifampicin is not commonly used in the treatment of enterococci as acquired resistance to rifampicin has been observed in both E. faecium and E. faecalis due to mutations in the gene encoding the RNA polymerase subunit (rpoB) [28,29].Recently, it was reported that HelD proteins from high G+C Actinobacteria, called HelR, were able to dissociate rifampicin-stalled RNA polymerase from DNA and provide rifampicin resistance [25].
Despite the fact that the COVID-19 pandemic was the primary cause of increased and inappropriate antimicrobial use [30], our study found no conclusive evidence of a rising trend of antimicrobial resistance as a result of the pandemic.Other studies have produced conflicting results, with some indicating that the pandemic was associated with an increase in VRE infection or colonization [31] while others discovered a decrease in VRE resistance during the COVID-19 pandemic [4,32].This disparity could be explained by the fact that some hospitals, possibly due to the heavy load they were exposed to during the pandemic, had decreased infection control activities, decreased adherence to blood culture and central line bundles, and antibiotic abuse in COVID-19 patients, as well as high rates of staff turnover and the presence of non-strictly qualified staff members in ICU settings during the first pandemic periods [33].
Our findings revealed that the mortality rate for VRE infections was higher (43.7%) than for VSE cases (23.5%).However, the effect of VRE infection on mortality is still debatable, with the associated comorbidities potentially skewing the estimates [34][35][36].
Due the study's retrospective design, one limitation was that no genomic analysis of resistant isolates, particularly VRE, was performed.Future research involving genomic analysis is warranted.Also, our study represents a regional-or, at most, a nationalobservation, which may limit its generalizability.

Conclusions
This study provides an overview of enterococcal infections and antibiotic susceptibility trends, guiding clinicians in the selection of appropriate empirical antibiotic therapy to improve clinical outcomes.From clinical specimens in a tertiary care hospital, we identified eight different Enterococcus species.E. faecalis was the most commonly identified strain, while E. faecium specimens had the highest percentage of VRE.VRE cases had a significantly higher mortality rate than VSE cases.Daptomycin, linezolid, and vancomycin had the least resistance among isolated strains.

Figure 1 .
Figure 1.Vancomycin resistance across the all the study's population.The overall vancomycin resistance was 15.9% while ICU patients had a 46.4% resistance burden and 53.6% for general ward patients.

Figure 3 .
Figure 3. Mortality status across the study population.Total study mortality was 25.6%, while ICU had 32%, and the general ward had a 13.5% mortality rate of VRE.

Figure 4 .
Figure 4. Trends of E. faecalis and E. faecium incidence across the years of study.

Figure 5 .
Figure 5. Trends of ampicillin and vancomycin resistance in the whole study population, ICU, and general ward as well as trends in E. faecalis and E. faecium over the study years.

Figure 5 .
Figure 5. Trends of ampicillin and vancomycin resistance in the whole study population, ICU, and general ward as well as trends in E. faecalis and E. faecium over the study years.

Table 2 .
Resistance patterns against selected antimicrobial agents of the whole study population.Resistance pattern is not available for the antimicrobial agent, R-All isolated samples are resistant to the antimicrobial agent, S-All isolated samples are sensitive to the antimicrobial agent.

Table 2 .
Resistance patterns against selected antimicrobial agents of the whole study population.