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Prevalence and Antimicrobial Profile of Colonized ‎Enterococcus Species Isolated from Hospitalized and Non-hospitalized Patients, Khartoum, Sudan

Loai A Siddig*, Omnia M Hamid, Nasreldin Elhadi, Magdi A Bayoumi

Microbiology Department, Faculty of Medical Laboratory Sciences, University of Medical Sciences and ‎Technology (UMST), Khartoum, Sudan

Abstract: Objective: in this study, we evaluate the prevalence, antimicrobial profiles, and vancomycin resistance (van) genotype ‎of fecal Enterococcus isolates from hospitalized and non-hospitalized patients in Khartoum locality hospitals, Sudan. ‎Methodology: This is a cross-sectional study, conducted between Oct 2018 and March 2020 at four tertiary hospitals in the ‎Khartoum locality. A total number of 588 fecal samples were collected and processed using microbiological culture media ‎‎ (Bile Esculin agar), gram stain, and gram-positive biochemical set tests to identify Enterococci species. Antibiogram of ‎Enterococci strains was performed, and the disk diffusion method of Kirby-Bauer has been used with the broth microdilution‎ method for vancomycin ‎minimum inhibitory concentration. A multiplex polymerase chain reaction assay was used to provide simultaneous identification at ‎the species level and detection of vancomycin resistance (van) genotypes characterization.‎ Results: All tested enterococci were confirmed to the species level, van genes were detected, and the MIC values determined vancomycin. Overall, Enterococcal species were isolated from 170/588 (28.9%) of the study subjects. Among the ‎Enterococcus isolates, 70 (41.2%) were isolated from hospitalized patients, and 100 (58.8%) were isolated from non-hospitalized patients. The isolates were E. faecium 108 (63.5%), followed by E. faecalis 43 (25.3%) ‎and 19 (11.2%) other Enterococcus spp. Enterococcus isolates show overall high resistance to ceftazidime (80.0%), ‎followed by amoxicillin-clavulanic acid (70%), gentamycin (69.4%), and erythromycin (52.4%). A higher ‎prevalence of resistance to ampicillin, rifampin, and teicoplanin was detected in E. faecium than that in E. faecalis and ‎other Enterococcus spp., while a greater prevalence of resistance to ceftazidime, and ciprofloxacin was ‎found in E. faecalis. ‎Twenty-five (14.7%) strains of fecal Enterococci were found to be vancomycin resistant with vanA 19 (11.2%), vanB 5 ‎‎(2.9%), and 1 (0.6%) vanC1 genotypes. The most predominant van producer strains were E. faecium [18, 16.7%; vanA (n= ‎‎17) & vanB (n= 1)] followed by E. faecalis [6, 14.0%; vanA (n= 2), and vanB (n= 4)] and Other Enterococcus spp. [1, 5.3%; ‎vanC1 (n= 1)].‎ Conclusion: The present study provides the first comprehensive report ‎of the ‎antimicrobial ‎resistance pattern ‎ and shows Khartoum localities are repositories for the vancomycin resistance ‎Enterococcus with vanA, vanB, and vanC1 genotypes in human feces of both hospitalized and non-hospitalized patients. ‎It is imperative to track and implement infection control measures in both settings to prevent the spread of these strains.‎

Keywords: fecal enterococci, vancomycin-resistant enterococci, van genotypes, Khartoum, Sudan

Received September 01, 2022; Revised October 02, 2022; Accepted October 10, 2022

Citation: Loai A Siddig, Omnia M Hamid, Nasreldin Elhadi, Magdi A Bayoumi, Prevalence and Antimicrobial Profile of Colonized ‎Enterococcus Species Isolated from Hospitalized and Non-hospitalized Patients, Khartoum, Sudan. American Journal of Infectious Diseases and Microbiology, 10 (4). 119-125. January 2022. doi:10.12691/ajidm-10-4-1

1. Introduction

The genus Enterococcus is a natural human microbiota colonized in the oral cavity as well as vagina in humans., where they have adapted to nutrient-rich, oxygen-depleted, and ecologically complex environments [1], includes more than 17 species, distinguished based on pigment production, motility, and ability to produce acids from various carbohydrates [2]. Enterococcus faecium and Enterococcus faecalis make up about 90% of infections caused by members of this genus [3], besides they are the most abundant species of this Enterococcus genus found in fecal content comprising up to 1% of the adult intestinal microbiota [4]. Furthermore, E. faecium and E. faecalis emerged as serious nosocomial infections such as surgical wound infections, bacteremia, endocarditis, meningitis, and urinary tract infections [5]. Other members of this genus do not often cause human infections [6].

Enterococci can acquire antibiotic resistance by being exposed to antibiotics or by acquiring genetic resistance factors from one bacterium to another [7]. The emergence of vancomycin-resistant Enterococci species (VRE) has concerned great public health due to the lack of options for therapeutic. Furthermore, it may be possible for resistance genes to transfer horizontally and for virulence factors to be increased [8]. E. faecium has become a more important nosocomial pathogen in the last two decades due to its ability to acquire resistance to a greater number of drugs than E faecalis [9,10].

In many countries and areas, rising proportions of vancomycin-resistant E. faecium were more commonly observed [11,12]. In 2017, WHO designated VRE as one of the most significant antibiotic-resistant bacteria on their “Global Priority List of Antibiotic-Resistant Bacteria”. The prevalence of VRE has been documented globally in various parts of the world, but there are no comprehensive data on its prevalence in Africa, including Sudan.

2. Methodology

2.1. Study Design and Area

A descriptive cross-sectional study was conducted at four tertiary hospitals in Khartoum Locality-Sudan from October 2018 to March 2020.

2.2. Specimen Collection

Data was collected based on the participant's permission in this study. The stool specimens were collected from hospitalized patients in different wards and non-hospitalized patients at selected hospitals. Immediately, stool specimens were transported to the Research Laboratory of Graduate College at the University of Medical Sciences and Technology-UMST-Sudan and analyzed.

2.3. Isolation and Identification of Enterococcus Species

The collected stool specimens were directly streaked on Bile Esculin Agar (BEA) medium (Hi-Media, India), and incubated at 37°C for 24 hours; plates were observed for the appearance of characteristic colonies of grey color and surrounded with a black halo (hydrolysis of esculin). The target strains were identified using different biochemical tests including Gram stain, catalase and oxidase production, growth at 10°C and 45°C, growth in the presence of 6.5 % Sodium Chloride, growth at pH 9.6, and reduced litmus milk [13]. Before analysis, all isolates were preserved on 20% glycerol stock at -80°C, until further analysis.

2.4. PCR Identification of Enterococcus Species Strains

The use of species-specific primers of PCR, which allows for simultaneous identification of enterococci to species level [14], and glycopeptide resistance genotypes (vanA, vanB, vanC1, and vanC2/3) was performed by Multiplex PCR [15], illustrated in (Table 1). Genomic DNA was extracted using the G-spin Genomic DNA Extraction Kit (Intron, Korea), according to the manufacturer's instructions. DNA concentrations were quantified using a NanoDrop spectrophotometer. DNA purity was calculated from the ratio of A₂₆₀ and A₂₈₀, and DNA quality was evaluated using agarose gel electrophoresis. All PCR reaction mixtures were analyzed by electrophoresis through a 1% agarose gel in 1 × TBE buffer pH 8.3. The sizes of the PCR products were compared with a 100bp molecular size ladder (MOLEQULE-ON, Auckland, New Zealand). The gels were stained with ethidium bromide and visualized under ultraviolet light using a Gene Genius Bioimaging System (SynGene, Maryland, USA).

Table 1. List of primers used in Multiplex PCR assays for species identification and genes encoding vancomycin resistance amplification with sequence and amplicon size (bp)

2.5. Antimicrobial Susceptibility Testing (AST)

Antimicrobial susceptibility testing was done using disk diffusion technique according to Kirby–Bauer method [16] on Muller-Hinton agar (Hi-Media, India). Fifteen antimicrobial agents, include, Amoxicillin-clavulanic acid (30 μg), Ampicillin (30 µg), Ceftriaxone (30 µg), Ceftazidime (30 µg), Ciprofloxacin (5 µg), Daptomycin (30 µg), Erythromycin (15 µg), Gentamicin (10 µg), Levofloxacin (15 µg), Linezolid (30 µg), Penicillin-G (10 IU), Rifampin (15 µg), Teicoplanin (30 µg), Tetracycline (10 µg), and Vancomycin (30 µg). The diameter of zone inhibition was measured and reported based on Hi-Media Antimicrobial Susceptibility Systems guidelines, as susceptible (S), or resistant (R). For quality control, the disks were checked by using reference strain E. faecalis ATCC29212 strain was used. Minimum inhibitory concentrations (MICs) of vancomycin were determined by the broth microdilution techniques and interpreted according to the CLSI guidelines [17], the VRE isolates were included based on showing MIC ≤4 mg/mL is susceptible, 8–16 mg/mL is intermediate, and ≥32 mg/ml indicates resistance for vancomycin, and MIC = 256 mg/ml was considered as highly resistant to vancomycin.

2.6. Detection of Vancomycin Resistance Genes

A Multiplex PCR was used for the detection of genes encoding van A, B, C1, and C2/3 [15]. The PCR reaction mix contained absolute Master mix (MOLEQULE-ON, Auckland, New Zealand) ready mix, 0.4 µM of each primer pair (Table 1), following thermal cycling conditions were used: cycles of initial denaturing (95°C, 3 minutes), and 30 cycles of amplification consisting of denaturation (95°C, 30 seconds), annealing (55°C, 30 seconds), extension (72°C, 1 minute), with (72°C, 5 minutes) for the final extension. PCR products were assessed by electrophoresis in 1% agarose gel using 100 V for 45 min. The gels were stained with ethidium bromide and visualized under ultraviolet light using a Gene Genius Bioimaging System (SynGene, Maryland, USA). A 100bp ladder (MOLEQULE-ON, Auckland, New Zealand) was used as a reference to determine the expected size of fragments. The primers used for PCR amplification of the genes encoding vancomycin resistance are listed in (Table 1).

2.7. Statistical Analysis

Data were entered into Microsoft Excel 2016, and Statistical Package for the Social Sciences software version 24.0 (IMB SPSS Inc., Chicago, IL) was used. Results were presented using frequency and percentages for quantitative Variables.

2.8. Ethical Considerations

Ethical approval for this study was obtained from the Graduate College-UMST and the Federal Ministry of Health, Sudan-Research Ethics Committee, Besides; the patients were informed about the study and the informed consent form was signed. Confidentiality was assured. No names were in the format used. The data were to be used for research only.

3. Results

3.1. Prevalence and Distribution of Fecal Enterococcus Species

Overall, Enterococci strains were isolated from 170/588 (28.9%) of the study subjects, during October 2018-March 2020, from four Khartoum localities hospitals. 70/170 (41.2%) were from hospitalized patients, and 100/170 (58.8%) were from non-hospitalized patients. The most dominant species among the isolated strains were E. faecium, with 108 (63.5%), followed by E. faecalis, with 43 (25.3%), while other Enterococcus spp. were 19 (11.2%) shown in (Table 2).

Table 2. Distribution of Fecal Enterococcus Species among hospitalized and non-hospitalized patients at Khartoum locality hospitals, Sudan, 2018-2020

3.2. Antimicrobial Susceptibility Profile of Enterococcus Species

The resistance profile rates of antimicrobial resistance in isolates of E. faecium, E. faecalis, and other Enterococcus spp. were presented in (Table 3). A significantly higher prevalence of resistance to Enterococcus spp. involved five antibiotics, ceftazidime, amoxicillin-clavulanic acid, gentamycin, erythromycin, and ceftriaxone.

Table 3. Antimicrobial resistance profile of fecal Enterococcus strains among hospitalized and non-hospitalized patients at Khartoum locality hospitals, Sudan, 2018-2020

Based on the results, resistance to the beta-lactam group showed (75%, 81/108) E. faecium, (60.5%, 26/43) E. faecalis, and (63.2%, 12/19) for other Enterococcus spp. were resistant to amoxicillin-clavulanic acid, overall (70.0%, 119/170). Low resistance to ampicillin overall (18.8%, 32/170), and penicillin overall (15.9%, 27/170).

Among the cephalosporins group, high resistance to ceftazidime was shown in E. faecalis (83.7%, 36/43), followed by E. faecium (79.6%, 86/108), and other Enterococcus spp. (73.7%, 14/19). Ceftazidime was the most resistant antibiotic in Enterococcus strains (80%, 136/170). Notably, overall resistance to ceftriaxone when compared to ceftazidime was low (37%, 63/170). For aminoglycoside resistance, gentamycin resistance was observed in (71.3%, 77/108) of E. faecium, (65.1%, 28/43) of E. faecalis, and (68.4%, 13/19) of other Enterococcus spp. with overall (69.4%, 118/170).

For the glycopeptide group, a total of (14.7%, 25/170), of Enterococcus spp. were resistant to vancomycin, (16.7%, 18/108) in E. faecium, (14%, 6/43) in E. faecalis, (5.3%, 1/19) in other Enterococcus spp. Teicoplanin, on the other hand, was resistant to (17.6%, 19/108) of E. faecium and (2.3%, 1/43) of E. faecalis. There was no resistance to teicoplanin among the other isolates of Enterococcus spp. tested.

Other alternative antibiotics to treat infection by Enterococcus strains also showed different rates of resistance. A low resistance rate was observed in both; ciprofloxacin 27 (15.9%), and levofloxacin 12 (7.1%), among all Enterococcus spp. Resistance to rifampicin was observed in (21.8%) of the isolates, (25.3%) to tetracycline, (52.4%) to erythromycin, (8.2%) to linezolid, and (11.8 %) to teicoplanin shown in (Table 3). Overall, E. faecium showed higher resistance rates than those of E. faecalis and other Enterococcus species.

3.3. PCR Analysis of Vancomycin Resistance Genes

Among the study subjects, 25/170 (14.7%) isolates were resistant to vancomycin. Overall vanA gene was detected in 19 (11.2%) of Enterococci spp. consisting of 17 E. faecium and 2 E. faecalis isolates, whereas vanB was detected in 5 (2.9%) isolates, consisting of 1 E. faecium and 4 E. faecalis isolate, Whereas the vanC1 gene was detected in only 1 of the other Enterococcus spp. while vanC2/C3 genes were not detected in any of the fecal Enterococcus spp. isolated strains (Table 4).

Table 4. Distribution of van genotypes of fecal Enterococci strains among hospitalized and non-hospitalized patients at Khartoum locality hospitals, Sudan, 2018-2020

3.4. Detection Rate of Vancomycin MICs

In 25-van resistance gene producing fecal Enterococci isolates, Enterococci strains possessing the vanA gene had the highest vancomycin resistance with these strains having MICs of ≥128µg/mL, while vanB strains showed MIC of 64 and 32 µg/mL in E. faecium and E. faecalis respectively The strain carrying vanC1 has vancomycin MICs of 32µg/ml. The other resistance regarding the MICs of vancomycin resistance among fecal Enterococci strains is represented in (Table 5).

Table 5. Vancomycin MICs profile to fecal Enterococci strains among 170 enterococci at Khartoum locality hospitals, Sudan, 2018-2020

4. Discussion

Until the 1980s, Enterococcus spp. were merely intestinal microbes of little clinical significance. Now, they are among the most common nosocomial pathogen, so physicians are becoming more worried, as resistance in enterococci rises and the incidence of VRE colonization increases where vancomycin is one of the antibiotics used to treat infections caused by Gram-positive multidrug-resistant organisms (MDRO), such as Enterococci.

The present study provides the first comprehensive report of the antimicrobial resistance pattern and shows Khartoum localities are repositories for the vancomycin resistance Enterococcus with vanA, vanB, and vanC1 genotypes in human feces of both hospitalized and non-hospitalized patients.

In our study, the overall prevalence of fecal Enterococci was 170/588 (28.9%), where the prevalence of Enterococci was higher among non-hospitalized patients (56.8%; 100/170) than in hospitalized patients (41.2%; 70/170). This was similar to the previous report from the Netherlands [18], and in contrast to our finding a study from Palestine [19] reported that fecal Enterococci was higher among hospitalized patients than in non-hospitalized patients. The difference may be explained due to study period variance and the area covered.

E. faecium was the most dominating species having 108 (63.5%) Isolates, followed by 43 (25.3%) E. faecalis, while other Enterococcus spp. were 19 (11.2%). This species distribution is comparable to the distribution of enterococcal species in other studies from China [20,21].

In our study, all enterococcus species Isolates showed resistance against all types of antibiotics used except one that showed sensitivity to all antibiotics. The high resistance rates to a wide range of antimicrobial agents may be due to excessive overuse and misuse of antibiotics in both human and animal medicine [22], and easy access to an antibiotic from a pharmacy without a prescription especially in low economic countries including Sudan [23].

The current study showed that (80.0%) of fecal Enterococcus isolates were resistant to ceftazidime followed by (70.0%) to amoxicillin-clavulanic acid, (69.4%) to gentamycin, (52.4%) to erythromycin and (37.1%) to ceftriaxone. This is consistent with those published in other countries Palestine [19], Ethiopia [24], Boston [25], and Argentina [26]. The reporting of high rates of resistance to aminoglycosides and cephalosporin’s in these strains will limit the options available for treating patients in our region. Although, CLSI indicates that, for Enterococcus spp., cephalosporins, and aminoglycosides (except for high level resistance screening), may appear active in vitro but they are not effective clinically, and isolates should not be reported as susceptible [27], here we report them as we are not treating patients.

Enterococci exhibit decreased susceptibility to penicillins and ampicillins as a result of the expression of low-level Penicillin Binding Proteins (PBPs). In the present study (15.9%), and (18.8%) of isolates were resistant to penicillin and ampicillin respectively. Based on our result, E.faecium were highly resistant to ampicillin than E. faecalis and this was in agreement with the previous study from Argentina showed that ampicillin resistance, which is rare in Enterococcus faecalis, occurs in most hospital-associated Enterococcus faecium isolates [26].

The remaining antimicrobial drugs showed low resistance impact toward linezolid (8.2%), daptomycin (4.7%), and levofloxacin (8.2%), therefore they can be prescribed. The emergence of VRE has prompted the use of novel and modified therapeutic agents including linezolid and daptomycin, although resistance to those agents has already been reported in clinical settings. However, none of these newly licensed agents (linezolid, and daptomycin), has been entirely free of resistance [25,28,29].

Linezolid, as one of the last resorts for the treatment of enterococcal infection among humans, was included in the WHO list of critically important antibiotics [30,31], However, linezolid-resistant enterococci (LRE) are quite predominant among E.faecalis in Asia, this finding is similar to our study, that showed a high proportion of LRE among E. faecalis (11.6%) compared to other species. This resistance rate was considered higher than that in a study involving clinical samples conducted in China (3.34%) [32].

VRE prevalence worldwide ranges from 9.8% to 26.4% [33,34,35,36], and it tends to rise over time. Our study also shows that out of 170 Enterococcus isolated, 25 (14.7%), were vancomycin-resistant (VRE). This prevalence result is in agreement with similar studies done in Iraq (14%) [37], Europe (13%) [38], South Korea (16%) [39], India (14%) [40], and Ethiopia (14.8%) [41].

The most common species of Enterococcus isolated during the study period is E. faecium. Out of 108 E. faecium isolates detected, (VREFm), constituted (16.7%, 18/108), while E. faecalis (VREFs), constituted (14%, 6/43) of the isolates, and only one (5.3%, 1/19) among the other Enterococcus species. This is in concordance with the Indian study conducted by Khandelwal et al. in 2020, where E. faecium is the predominant species isolated [40].

The prevalence of VRE in the gastrointestinal tract was higher among non-hospitalized patients (64%; 16/25) than in hospitalized patients (36%; 9/25), this was similar to the previous report from Palestine [42], and in contrast to finding a study from Greece [43], and Korea [44].

Phenotypic resistance against glycopeptides (e.g., vancomycin, teicoplanin), is genetically encoded by resistance determinants designated as van-operons. In the context of human infection, vanA and vanB are the most relevant resistance determinants, encoding a combined resistance against vancomycin and teicoplanin. Our findings indicate that among all 25 vancomycin-resistant strains, 11.2% were vanA, 2.9% vanB, and 0.6% vanC1, however, the vanC2/3 genotype was not found. In Sudan, vanA was found to be the predominant glycopeptide resistance determinant, which is in line with findings from Germany [45]. Among these, a recent study conducted by L. Matthew et al. in 2016, shows that resistance mediated by vanA is most common followed by vanB and vanC1 [46].

According to our data, The MICs of vancomycin for fecal VRE strains were shifted towards the upper limits of tested ranges, with MICs for the majority of vanA gene producers exceeding that limit; 128 g/mL in E. faecium (70.6%; 12/17) and E. faecalis (50.0%; 1/2) isolates. For vanB gene producers, 64 g/mL in E. faecium (100.0%; 1/1) and vancomycin MICs 32 μg/mL in E. faecalis (100.0%;4/4) isolates. Whereas in other Enterococcus spp vanC1producer; the vancomycin MICs were 32 μg/mL. This is similar with previous report from Australia [47], China [48], India [49], and Texas [50].

5. Conclusion

This study reveals E. faecium and E. faecalis constituted the predominant strains among colonized Enterococcus spp. isolated from human feces of hospitalized and non-hospitalized patients in Khartoum, Sudan. There has been a shift in antibiotic resistance patterns among fecal Enterococci strains toward aminoglycosides, β-lactams with inhibiter, cephalosporins, and macrolides. All these data show a very high prevalence of drug resistance in Enterococcal strains isolated in our settings.

The emerging strains of VRE in hospitalized and non-hospitalized patients at Khartoum in the current study was (14.7%). The most predominant van genotypes strains vanA (11.2%), vanB 5(2.9%) and vanC1 (0.6%). Thus, to limit VRE infections and development, focused infection prevention measures should be implemented, and periodic surveillance of antibacterial susceptibilities is recommended to detect emerging antimicrobial resistance and prevent the spread of antibacterial-resistant strains.

Conflicts of Interest

The authors declare no conflict of interest.

Funding

Not funded (corresponding author fund all work as part of PhD degree).

Data Availability

All data generated or analyzed during this study are Included in this published article (are available from the Corresponding author on reasonable request as Excel file.

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