Clostridioides difficile and Enterococci’s Interplay in the Human Gut: Bacterial Alliance or Competition? A Systematic Literature Review

Clostridioides difficile and Enterococcus spp. are two common bacterial pathogens populating the human microbiota. We possess scant data on how Clostridioides difficile interacts with Enterococcus spp. in the gut microbiota in subjects colonized with Clostridioides difficile or during a Clostridioides difficile infection. We carried out a systematic review of studies on Enterococcus spp. and Clostridioides difficile’s interaction in the gut microbiota and on the effect of Enterococcus spp. gut colonization on CDI development. Studies on Enterococcus spp. and Clostridioides difficile’s interaction in the gut microbiota and on the effect of Enterococcus spp. gut colonization on CDI were searched using the search terms “clostridium”, “clostridioides”, “difficile” and “enterococcus” on the MEDLINE and SCOPUS databases. PubMed was searched until 1 May 2023. An English language restriction was applied. The risk of bias in the included studies was not assessed. Quantitative and qualitative information was summarized in textual descriptions. Fourteen studies, published from August 2012 to November 2022, on Clostridioides difficile and Enterococcus spp.’s interaction in the gut microbiota met the inclusion criteria. The studies included in our systematic review reported evidence that the Enterococcus spp. intestinal burden represents a risk factor for the occurrence of CDI. There is supporting evidence that Enterococcus spp. play a role in CDI development and clinical outcomes.


Introduction
In the last decade, our knowledge on the microbial ecosystem populating the human gut has considerably increased. The gut microbiota is an extremely heterogeneous environment populated by different bacteria, viruses and fungi continuously interacting with each other and with the human immune system.
In the case of gut dysbiosis, Clostridioides difficile represents a potential pathogen causing diarrhea.
However, we possess scant data on how Clostridioides difficile interacts with the variety of microorganisms populating the microbiota in subjects colonized with Clostridioides difficile or in Clostridioides difficile infection (CDI) patients.
There is a lack of knowledge on the interaction between two common bacterial pathogens populating the human microbiota, i.e., Clostridioides difficile and Enterococcus spp.
Interestingly, Enterococcus spp. and Clostridioides difficile share several similarities. Both of these two bacteria belong to the Firmicutes phylum and possess intrinsic resistance to common antibiotics [1][2][3][4][5]. Moreover, they may compose part of the gut microbiota in healthy subjects but are also among the main causes of opportunistic infections [1][2][3][4][5]. 2 of 11 Moreover, Enterococcus spp. and Clostridioides difficile share the ability to proliferate following microbiota dysbiosis, i.e., after antibiotic administration [6,7]. Adding complexity to this picture, Enterococcus spp. possess the ability to acquire resistance to several antibiotics, including the first-line CDI antimicrobial treatment, vancomycin [8].
Recently, some studies investigated the potential interplay between Enterococcus spp. and Clostridioides difficile in the gut microbiota and the impact of Enterococcus spp. on CDI occurrence, development and clinical outcomes [8,9]. However, the effect of Enterococcus spp. gut colonization on susceptibility to CDI and on CDI patients' outcomes remains largely unknown.
We performed a systematic literature review to assess the available data on Enterococcus spp. and Clostridioides difficile's interaction in the gut microbiota and on the effect of Enterococcus spp. gut colonization on CDI development.

Search Strategy and Article Identification
Published articles on Enterococcus spp. and Clostridioides difficile's interaction in the gut microbiota and on the effect of Enterococcus spp. gut colonization on CDI were searched on the MEDLINE and SCOPUS databases.
Searching the MEDLINE database, the following search terms were used: [(clostridium) OR (clostridioides) AND (difficile) AND (enterococcus)]. The filters clinical study, clinical trial, meta-analysis, multicenter study, observational study and randomized controlled trial were applied with respect to article type, and the filter from 1 January 2020 to 1 May 2023 was applied with respect to publication date.
Searching the SCOPUS database, the following search terms were used: [(clostridioides) AND (difficile) AND (enterococcus)]. The filter article was applied with respect to document type; the filter English was applied with respect to language; the filter enterococcus was applied with respect to keywords; and the filter from 2019 to 2023 was applied with respect to publication date.
Supplementary Figure S1 describes the specifications of the query details used on the MEDLINE and SCOPUS databases, respectively ( Figure S1).
No attempt was made to obtain information about unpublished studies. Review articles and meta-analyses, correction articles, case reports, editorials and clinical trial protocols were excluded from further assessment.

Eligibility Criteria
Original articles reporting data on Clostridioides difficile and Enterococcus spp.'s interaction in the gut microbiota were eligible for inclusion.

Study Selection and Data Extraction
Eligibility assessment and extraction of data were performed independently by two investigators. Each investigator was blinded to the other investigator's data extraction. In the case of disagreement between the two reviewers, a third reviewer was consulted. The risk of bias in the included studies was not assessed.
For each included study, we collected data regarding the study design, the study population and the study setting, the sample size and the patients' outcome if applicable.

Data Synthesis
Quantitative and qualitative information was summarized by means of textual descriptions.   Two studies were excluded because they were review articles. One hundred studies were excluded for not reporting data on Clostridioides difficile and Enteroc spp.'s interaction.
A summary description of the included studies is reported in Tables 1-4. Two studies were excluded because they were review articles. One hundred forty studies were excluded for not reporting data on Clostridioides difficile and Enterococcus spp.'s interaction.
A summary description of the included studies is reported in Tables 1-4.

Enterococcus spp. and C. difficile's Interplay during Clostridioides Difficile Infection
An in vivo study used the mouse model to test the hypothesis that specific bacterial gut communities determine a variation in CDI severity. Different gut communities were derived by colonizing germfree mice with human fecal communities. The mice were then infected with a Clostridioides difficile clinical isolate, resulting in morbidity and histopathologic differences. Fecal communities rich in Enterococcus spp. were associated with more severe CDI outcomes [12]. Agar well-diffusion assay was used to test the inhibitory activity of isolated colonies against toxigenic C. difficile strains. The cytopathic effects of C.
difficile on colon adenocarcinoma cells were evaluated through immunofluorescence assay.
In total, 85 distinct bacterial colonies were isolated from the feces of 38 breast-fed infants. Of these, six Enterococcus faecalis isolates showed anti-C. difficile activity. The six strains inhibited spore germination (100 − 98.20 ± 2.17%) and sporulation. The cell-free supernatant of these strains reduced the cytopathic effects of C. difficile on colon adenocarcinoma cells (HT-29 cells).
VRE represents vancomycin-resistant Enterococci, CDI represents Clostridioides difficile infection, IQR represents interquartile range and CI represents confidence interval.    Moreover, a study aimed to define the interaction between Enterococcus spp. and Clostridioides difficile during CDI. The study comprised two parts; the first one was performed using the mouse model, and the second one was on pediatric human patients to evaluate the role of Enterococcus spp. in determining CDI severity [13].
In this study, mice were infected with Clostridioides difficile following the antibioticmediated depletion of endogenous enterococci. The enterococcal depletion resulted in a delay in Clostridioides difficile colonization [13]. Afterwards, to test whether this effect was directly attributable to the enterococci in the mice gut, Enterococcus faecalis was introduced immediately preceding CDI. The introduction of Enterococcus faecalis recovered early Clostridioides difficile colonization [13].
Moreover, to test the reciprocal effect of Clostridioides difficile on enterococcal fitness in the gut, mice were infected with toxigenic and nontoxigenic Clostridioides difficile strains. Enterococcal burdens significantly increased in the presence of toxigenic Clostridioides difficile, demonstrating that Clostridioides difficile toxin-mediated damage provides a fitness advantage to Enterococcus spp. in the gut.
In addition, the authors performed fluorescent in situ hybridization during CDI in mice, showing that Enterococcus spp. colocalize with Clostridioides difficile in the lumen and in biofilm-like aggregates on the host epithelium. Clostridioides difficile readily formed biofilms with Enterococcus faecalis, and this markedly enhanced Clostridioides difficile survival during vancomycin treatment.
Moreover, to examine the effect of Enterococcus spp. on CDI pathogenesis, the authors compared fecal Clostridioides difficile toxin titers from mice infected with Clostridioides difficile alone or Clostridioides difficile plus Enterococcus faecalis. Clostridioides difficile toxin fecal titers were higher in the mice infected with both Clostridioides difficile and Enterococcus faecalis (p: 0.003) [13].
Finally, the second part of the study was on pediatric human patients. During this part of the study, the authors aimed to quantify the Enterococcus spp. burden in pediatric patients with CDI. The authors reported a positive correlation between Enterococcus spp. and Clostridioides difficile gut burdens (Spearman's ρ correlation: 0.551) [13].
Some authors proposed that Enterococcus spp. strains might have a protective effect towards Clostridioides difficile infections. Romyasamit et al. aimed to identify potential Enterococcus spp. strains exerting a protective effect against Clostridioides difficile. With respect to this aim, 38 fecal samples were collected from healthy breastfed infants. The authors isolated six Enterococcus faecalis strains inhibiting Clostridioides difficile spore germination and sporulation. The cytopathic effects of Clostridioides difficile on colon adenocarcinoma cells were reduced through a pretreatment with the cell-free supernatant of these Enterococcus faecalis strains [14].

Enterococci Intestinal Burden as a Risk Factor for CDI
The ANTICIPATE study was an international multicenter prospective observational cohort study performed to estimate CDI incidence and to assess clinical characteristics and biomarkers to predict CDI in patients receiving newly initiated antibiotic treatments [15]. The study enrolled 1007 patients receiving antibiotic treatment with penicillins, cephalosporins, carbapenems, fluoroquinolones or clindamycin. The enrolled patients were followed up for 90 days. The estimated cumulative incidence of CDI was 1.1% (95% confidence interval (CI): 0.6-2.1) and 1.9% (95% CI: 1.1-3.0) within 28 and 90 days, respectively. The study found that the high intestinal abundance of Enterococcus spp. relative to Ruminococcus spp. predicted an increased CDI risk (hazard ratio (95% CI): 5.4 (2.1-18.7)) [15].
The ANTICIPATE study group also investigated the intestinal microbiota of hospitalized patients to identify the microbial markers predictive of CDI. Among the 1007 patients included in the study, 135 had antibiotic-associated diarrhea, and 15 were diagnosed with CDI. Stool samples from 33 of the 135 patients with diarrhea, including 6 CDI patients were collected at the occurrence of the first diarrheal episode and were analyzed through 16S rRNA gene profiling and sequence typing. The patients developing CDI exhibited significantly lower microbial diversity prior to antibiotic treatment and a distinct microbiota phenotype enriched in Enterococcus spp. and depleted of Ruminococcus, Blautia, Prevotella and Bifidobacterium spp. compared to the non-CDI patients. Alpha diversity was lower in the patients developing CDI compared to the patients developing antibiotic-associated diarrhea or not developing diarrhea (p ≤ 0.049) [16].
Moreover, a case-control study was performed to evaluate the composition of the gut microbiota dominant bacterial groups in CDI patients compared to healthy controls. The study enrolled a total of 50 CDI inpatients and 50 healthy controls. The abundances of Enterococcus spp. were higher in the CDI group compared with the healthy control group (p < 0.05) [17].
Finally, one study used the gene sequencing approach to better understand the clinical and microbiome-based factors associated with CDI. In the study, 16S rRNA gene sequenc-ing was used to characterize the gut microbiomes of 94 CDI patients, 89 diarrheal and 155 nondiarrheal controls. The clinical and microbiome data were merged to generate models to differentiate between the three groups of subjects. The study found that the CDI patients had a significantly higher abundance of Enterococcus spp. in their gut microbiota compared to the healthy controls [1].

Vancomycin-Resistant Enterococci and Clostridioides difficile 3.4.1. Vancomycin-Resistant Enterococci Colonization as a Risk Factor for CDI
A retrospective cohort study was performed among nine intensive care units to evaluate the risk and pathogenic distribution of enteric infection in patients colonized with vancomycin-resistant Enterococcus spp. (VRE). The study included 131 VRE-colonized patients. The authors reported a trend towards an increased risk of CDI in the VRE-colonized patients. In the study, Clostridioides difficile was the most common pathogen detected in the VRE-colonized patients [18].
In a study performed to investigate the presence of multidrug-resistant (MDR) bacteria among hospitalized patients, stool samples from both CDI and non-CDI patients were analyzed through culture, matrix-assisted laser desorption/ionization (MALDI-TOF) mass spectrometry, polymerase chain reaction (PCR) and whole-genome sequencing for bacterial identification and characterization. The study did not find a significant difference in the prevalence of VRE between the CDI and non-CDI patients [19].
Recently, a microbiology surveillance study was performed to identify patients colonized with VRE among CDI patients admitted at a Slovakian military hospital. The authors reported that VRE was identified in 44 out of 113 (38.9%) stool samples that were positive for CD toxins [20].

Effect of the CDI Antimicrobial Treatment on the Rate of Vancomycin-Resistant Enterococci Colonization
Oral vancomycin is one of the most frequently used antibiotics for the treatment of CDI. There are concerns that this might increase the risk of selecting vancomycin-resistant enterococci [21].
Recently, a double-blind randomized controlled trial was performed to evaluate the effect of oral vancomycin on the gut microbiota of hospitalized patients. The trial included 15 patients with at least one diarrheal stool sample that tested positive for C. difficile via nucleic acid amplification tests but negative via toxin enzyme immunoassay. Patients were randomized 1:1 to receive 10 days of oral vancomycin, 125 milligrams four times per day, or a matching placebo. Stool specimens were collected at 8 weeks from randomization. The authors reported an increase in microbiota beta diversity (p: 0.0059) in the vancomycintreated group. Overall, VRE colonization was found in five (26%) patients, three of them in the vancomycin group. No significant difference in the prevalence of VRE was observed between the two study arms [21].
A multicenter retrospective study was performed to evaluate the risk of VRE among 15,780 CDI patients. The CDI patients were included if they were treated with metronidazole or oral vancomycin and had no history of VRE colonization. The authors reported no differences between the patients treated with oral vancomycin or metronidazole, developing VRE at 3 months (adjusted relative risk of 0.96; 95% CI: 0.77 to 1.20), with an absolute risk difference of −0.11% (95% CI: −0.68% to 0.47%). Similar findings were observed within 6 months [22].
A 2-year retrospective cohort study compared the VRE colonization rates between previously VRE-negative patients receiving either metronidazole or oral vancomycin as a CDI-specific treatment. In the study, 170 hospitalized CDI patients treated with metronidazole or oral vancomycin were monitored for VRE acquisition. The VRE status was assessed at the beginning of the CDI treatment in order to differentiate between the patients with and without preexisting VRE colonization. In total, 14 patients acquired VRE colonization after the first CDI antibiotic treatment. The study did not find significant differences between the VRE acquisition rates of the metronidazole-or vancomycin-treated patients (p: 0.98) [23].
Moreover, a large double-blind randomized multicenter phase III controlled trial was performed to test the hypothesis that fidaxomicin promotes less VRE colonization than vancomycin. In this trial, a total of 548 CDI patients were randomized to receive treatment with 10 days of fidaxomicin versus 10 days of vancomycin; of them, 301 (55%) had stool samples available both prior to and at the completion of CDI therapy: 160 vancomycintreated patients and 141 fidaxomicin-treated patients. Pre-and posttreatment stool samples were collected and assessed for VRE colonization. In comparison with the vancomycintreated patients, the fidaxomicin-treated patients had less frequent acquisition of VRE (8/114 patients, 7%, versus 41/133 patients, 31%; p < 0.001) [24].

Discussion
Currently, the exact relationship and the interplay between Enterococcus spp. and Clostridioides difficile in the human gut remain largely unknown.
In our systematic review, we included 14 studies dealing with Clostridioides difficile and Enterococcus spp.'s interaction in the gut microbiota.
According to our results, when we looked at studies evaluating the possible role of the Enterococci intestinal burden as a risk factor for CDI onset, we found initial evidence that a high intestinal burden of Enterococcus spp. may represent a risk factor for the occurrence of CDI. On this issue, the large "ANTICIPATE" study reported that, when compared to non-CDI patients, CDI patients possess a significantly lower gut microbial diversity and a microbiota enriched in Enterococcus spp. [16].
Overall, regarding the link between VRE and Clostridioides difficile, the included studies did not find significant differences in the prevalence of VRE colonization between CDI and non-CDI patients. Moreover, no significant differences were reported among the included studies on the risk of developing VRE colonization after a CDI treatment course with oral vancomycin or metronidazole.
Finally, regarding the possible effect of the Enterococci intestinal burden in increasing CDI morbidity, studies performed in the animal model reported increased CDI severity with fecal communities enriched in Enterococcus spp. [12,13]. This indicated that Enterococci in the gut microbiota may alter the gastrointestinal environment following antibiotic treatment and support early Clostridioides difficile colonization and CDI occurrence.
It should be emphasized that the included studies were heterogeneous, being performed in different epidemiological settings with different designs and aims. Moreover, it is of note that, among the included studies, there are studies enrolling small study populations and therefore not reaching statistically significant results. Certainly, a major limitation of our review is the low number of included studies dealing with the specific issue regarding Enterococcus spp. and C. difficile's interaction.
Importantly, Enterococcus spp. may play a more complex role in affecting CDI pathogenesis and the outcome of CDI patients; the studies performed so far may be unable to evaluate these effects. As an example, Enterococci have been shown to have the potential to acquire resistance to vancomycin, which is a first-line antimicrobial agent used to treat CDI [5]. This could potentially reduce the effectiveness of vancomycin in treating Enterococci coinfections in CDI patients, determining worse patient outcomes.
In addition, the modulation of bacterial metabolism and the nutritional landscape in the gut microbiota may alter CDI development. A recent study suggests that Enterococci and Clostridioides difficile may interact through metabolic cross talk during CDI, enhancing reciprocal colonization, persistence and pathogenesis in the gut [13]. Again, further studies are needed to better evaluate these mechanisms.
Smith et al. recently suggested that Enterococci may increase Clostridioides difficile pathogenesis by enhancing toxin production [13]. These observations should be confirmed in studies on human subjects.
Moreover, recent studies demonstrated that Enterococcus faecalis's biofilm structure is important for enhancing Clostridioides difficile survival following antibiotic exposure and suggested that dual-species biofilms may promote persistence during infection [13]. Importantly, biofilms provide ideal conditions for horizontal gene transfer between bacterial species.
This preliminary evidence that Enterococci and Clostridioides difficile may share mobile genetic elements in the gut is alarming, but no studies have evaluated the potential clinical impact of the transmission of vancomycin resistance genes between VRE and Clostridioides difficile so far.
Interestingly, a study by Romyasamit C. et al. identified select Enterococci strains as potential probiotics for preventing or controlling CDI. The strains reduced the TdcA and TdcB toxic effects on human cells and prevented Clostridioides difficile spore production and germination [14]. This intuition may pave the road towards an innovative treatment approach against CDI, but further in vivo studies on the inhibition of Clostridioides difficile using ad hoc Enterococcus strains are required.
To conclude, the studies included in our systematic review reported evidence that the Enterococcus spp. intestinal burden represents a risk factor for the occurrence of CDI.
Regarding the possible link between VRE and Clostridioides difficile, the included studies did not report VRE colonization as a risk factor for CDI, and there is no clear evidence on the CDI treatment with oral vancomycin increasing the rate of VRE colonization.
Overall, while the potential interplay between Enterococcus spp. and Clostridioides difficile in the human gut microbiota is still not fully understood, there is supporting evidence that Enterococcus spp. play a role in CDI development and clinical outcomes. Further research is needed to fully elucidate the nature of this interplay and its potential implications for CDI prevention and treatment.
Supplementary Materials: The following supporting information can be downloaded at https://www. mdpi.com/article/10.3390/jcm12154997/s1, Figure S1: the complete specifications of the query details used on the MEDLINE and SCOPUS databases.
Funding: This research received no external funding.
Institutional Review Board Statement: Ethical review and approval were waived for this study due to the nature of this article that is a systematic literature review.
Informed Consent Statement: Informed consent was obtained from all subjects involved in the studies cited.