Nonalcoholic fatty liver disease (NAFLD) has become the leading cause of chronic liver disease with increasing prevalence worldwide. Clostridioides difficile infection (CDI) remains the most common cause of nosocomial diarrhea in developed countries.

To assess the impact of NAFLD on the outcomes of hospitalized patients with CDI.

This study was a retrospective cohort study. The Nationwide Inpatient Sample database was used to identify a total of 7239 adults admitted as inpatients with a primary diagnosis of CDI and coexisting NAFLD diagnosis from 2010 to 2014 using ICD-9 codes. Patients with CDI and coexisting NAFLD were compared to those with CDI and coexisting alcoholic liver disease (ALD) and viral liver disease (VLD), individually. Primary outcomes included mortality, length of stay, and total hospitalization charges. Secondary outcomes were in-hospital complications. Multivariate regression was used for outcome analysis after adjusting for possible confounders.

CDI with NAFLD was independently associated with lower rates of acute respiratory failure (2.7% vs 4.2%, P < 0.01; 2.7% vs 4.2%, P < 0.05), shorter length of stay (days) (5.75 ± 0.16 vs 6.77 ± 0.15, P < 0.001; 5.75 ± 0.16 vs 6.84 ± 0.23, P <0.001), and lower hospitalization charges (dollars) (38150.34 ± 1757.01 vs 46326.72 ± 1809.82, P < 0.001; 38150.34 ± 1757.01 vs 44641.74 ± 1660.66, P < 0.001) when compared to CDI with VLD and CDI with ALD, respectively. CDI with NAFLD was associated with a lower rate of acute kidney injury (13.0% vs 17.2%, P < 0.01), but a higher rate of intestinal perforation (P < 0.01) when compared to VLD. A lower rate of mortality (0.8% vs 2.7%, P < 0.05) but a higher rate of intestinal obstruction (4.6% vs 2.2%, P = 0.001) was also observed when comparing CDI with NAFLD to ALD.

Hospitalized CDI patients with NAFLD had more intestinal complications compared to CDI patients with VLD and ALD. Gut microbiota dysbiosis may contribute to the pathogenesis of intestinal complications.

Nonalcoholic fatty liver disease (NAFLD) is a heterogeneous disease with a spectrum from simple steatosis to nonalcoholic steatohepatitis (NASH), cirrhosis, and hepatocellular carcinoma[1,2]. With a prevalence of 10 to 46 percent in the United States and 6% to 35% worldwide[3,4], NAFLD has become the leading cause of chronic liver disease, and its prevalence continues to increase, paralleled by the increase of obesity and type 2 diabetes[5].

Clostridioides difficile (C. difficile) is a gram-positive, spore-forming bacterium, known as the most common pathogen causing nosocomial diarrhea in developed countries[6]. Symptoms of C. difficile infection (CDI) range from mild to severe diarrhea, which can progress to sepsis, fulminant colitis, and bowel perforation[7]. Severe colitis may also present as ileus and megacolon, which are characterized by symptoms of intestinal obstruction[8,9]. Gut microbiota dysbiosis due to the administration of antibiotics is the most prominent risk factor for the development of CDI. Advanced age, prolonged hospitalization and gastric acid suppression are some common additional risk factors for CDI[10,11].

Recently, a number of animal and human studies have revealed the role of the gut microbiota in the pathophysiology of NAFLD. It is proposed that dysbiosis-induced dysregulation of the gut barrier function and translocation of the bacteria link the gut microbiome to NAFLD[12,13]. In addition, it has been well documented that patients with chronic liver disease are more susceptible to CDI due to frequent hospitalization and antibiotics use. Specifically, recent studies have observed that NAFLD is an independent risk factor for CDI by single-centered retrospective design[14,15].

Although a strong association between NAFLD and CDI has been observed, gut microbiota dysbiosis likely plays a vital role in the pathogenesis of both aforementioned diseases. However, the inpatient outcomes of CDI in the NAFLD population, have not been well studied in large populations. The aim of this nationwide study was to assess the impact of NAFLD on the outcomes of hospitalized patients with CDI.

This nationwide retrospective cohort study investigated the inpatient clinical characteristics and outcomes of CDI in hospitalized patients with coexisting liver diseases, with comparisons between NAFLD, VLD and ALD. We demonstrated that patients hospitalized with CDI and coexisting NAFLD had overall more favorable outcomes including a lower rate of RF, lower hospitalization charges and a shorter LOS when compared to those with ALD and VLD individually. Interestingly, higher rates of intestinal complications were observed in the CDI with NAFLD group when compared to the CDI with ALD or VLD groups. Specifically, a significantly higher rate of intestinal obstruction was seen in the CDI with NAFLD group when compared to the CDI with ALD group, and a higher rate of intestinal perforation was seen when compared to CDI patients with concomitant VLD.

Our findings of worse intestinal complications in patients hospitalized with CDI and coexisting NAFLD compared to CDI patients with VLD and ALD, linked the gut pathology to the liver. The crosstalk between the gut and liver is increasingly recognized as the gut-liver axis[18]. Receiving more than 70% of the blood supply from the intestinal venous outflow, the liver represents the first line of defense against gut derived antigens with a broad array of immune cells[19]. The liver also releases many bioactive mediators into the systemic circulation, allowing for communication with the intestine. In the intestine, the endogenous and exogenous products from host and microbial metabolism translocate to the liver through the portal venous system, ultimately influencing liver function[20].

How does NAFLD influence the intestinal complications of CDI through the gut-liver axis? Convincing evidence has shown that NAFLD is associated with significantly increased gut permeability and inflammation in both animal[21] and human models. Miele et al[22] found that NAFLD patients had significantly increased gut permeability measured by urine radiolabeled markers and immunohistochemical analysis of zona occludens -1 expression in intestinal biopsy specimens, compared with healthy volunteers. They also discovered that both gut permeability and the prevalence of small intestinal bacterial overgrowth are correlated with the severity of steatosis. Verdam et al[23] found that plasma immunoglobulin G levels against endotoxin were increased in NASH patients, which positively correlated with the severity of inflammation. Furthermore, transmission electron microscopy observed irregular microvilli and widened tight junctions in the gut mucosa of the NAFLD patients[24]. In addition, decreased numbers of CD4+ and CD8+ T lymphocytes and increased levels of TNF-α, IL-6 and IFN-γ were detected in the NAFLD patient group compared to healthy control. All of these results suggested impaired gut permeability and increased levels of inflammation at both the tissue and cellular levels in NAFLD disease models.

The gut microbiota-mediated inflammation, the related disturbance of the intestinal integrity and the impairment in mucosal immune function have been reported to play important roles, not only in the pathophysiology of CDI[25] but also in the pathogenesis of NAFLD[13,24,26]. The gut microbiota normally exerts significant influence on intestinal epithelial cell health, nutrient metabolism and mucosal defense[19,27]. Early evidence in animal studies demonstrated that altered gut microbiota composition[28] independently contributed to the development of NAFLD in mice. In addition, altered interaction between the gut and the host (produced by defective inflammasome sensing in inflammasome-deficient mouse models) may govern the rate of progression of multiple metabolic syndrome-associated abnormalities[29]. With the recent developments in genome sequencing technologies, bioinformatics, and culturomics; it has been recognized that NAFLD and NASH are associated with decreased richness of the gut flora and increased risk of pathogenic flora in pediatric and adult patients[30-34], which are both well known risk factors for CDI. Although it is still unclear which specific microorganisms are harmful given conflicting results in human and animal studies[35], it is believed that gut microbiota-derived signatures extracted by whole-genome shotgun sequencing of DNA can be used for diagnosis of advanced fibrosis in NAFLD[36], and modification of gut microbiota analyzed by 16S ribosomal RNA pyrosequencing can be used for therapeutic purposes in NASH patients[37]. Additionally, increased pathogenic flora in NAFLD and NASH further disturb the immune balance and cause worsened dysbiosis through various mechanisms involving short-chain fatty acids[38], lipopolysaccharide[21], choline metabolism[39], bile acid metabolism[40] and bacteria-derived ethanol[41]. Collectively, NAFLD and NASH related alterations of gut microbiota and its downstream dysbiosis pathways may contribute to CDI risk and worse intestinal complications.

On the other end, we sought to identify the characteristics of gut microbiota changes in ALD and VLD. Compared to NAFLD, ALD is remarkably similar histologically[42] and initiated directly from the gut by alcohol intake or binges. It has been well documented that alcohol intake can lead to changes in gut microbiota composition[43] and gut permeability[44] early on, even before the development of liver disease. These alterations involve multiple physical and biochemical layers of defense in the intestinal barrier[19]. In VLD, the gut microbiome works as an effective tool early on for immunity against the hepatitis virus, and helps with viral clearance[45]. In chronic VLD, large translocations of intestinal microbiota were observed and thought to contribute to not only dysregulation of immune cells and dysfunction of the intestinal barrier, but also viral replication[27]. Comparison analysis revealed that, compared to other cirrhosis etiologies, alcoholic cirrhosis is associated with worse gut dysbiosis after adjusting for Model For End-Stage Liver Disease score and body mass index[46]. In two other studies[47,48], which primarily compared the gut microbiota composition in HBV/HCV related and alcoholic cirrhosis, no difference was observed at the phylum and class level.

Intriguingly, in our study, the majority (94.5%) of patients in CDI with NAFLD group were non-cirrhotic; the percentage of cirrhotic patients in CDI with NAFLD group was significantly less than those in CDI with ALD or VLD group. CDI with NAFLD group was associated with a higher rate of intestinal complications after adjusting for cirrhosis and its complications. These results suggested that NAFLD is associated with altered gut microbiota that is predisposed to CDI and its complications, likely independent from the liver disease severity. In fact, NAFLD has been reported as an independent risk factor for CDI[14]. Although ALD and VLD cirrhosis was previously found to be associated with worse gut dysbiosis than NAFLD cirrhosis, this finding should be treated cautiously for non-cirrhotic patients, because the alteration of the gut microbiome is associated with the severity of liver disease, as significant differences in gut microbiota have been found between non-cirrhotic, compensated and decompensated cirrhotic patients[49,50]. Importantly, the standard of care therapies in cirrhotic patients such as lactulose, rifaximin, antibiotics and acid-suppressants that can affect the gut microbiota, may be playing a critical role[51]. In summary, our study suggested that NAFLD may be associated with worse dysbiosis in early liver disease stages and therefore a higher risk for CDI and its complications compared to ALD and VLD.

Aside from aforementioned gut microbiota changes that directly link NAFLD to CDI and intestinal complications, NAFLD related metabolic syndrome and systemic inflammation also play crucial roles in intestinal pathology. Recently, metabolic dysfunction-associated fatty liver disease has been proposed as a more appropriate name to replace NAFLD by an international panel of experts, with emphasis on the underlying metabolic dysfunction[52,53]. Clinical evidence has demonstrated that NAFLD, along with other components of metabolic syndrome, such as diabetes and obesity, are associated with an increased prevalence of small intestinal bacterial overgrowth (SIBO)[54,55] by insulin resistance, oxidative stress and chronic low grade inflammation[56]. Subsequently, the dysmotility induced by SIBO can further promote SIBO in NAFLD patients, causing a vicious cycle[57]. In fact, dysmotility itself is associated with NAFLD and may be a potential therapeutic target for NAFLD from a Japanese study[58,59]. Moreover, diabetes, a component of metabolic syndrome which may cause vasculopathies and neuropathies in the intestines, also contributes to dysmotility[60]. Additionally, diverticular disease, irritable bowel disease[61] and inflammatory bowel disease[62], together with SIBO and dysmotility have all been shown to have increased prevalence in NAFLD patients. Not surprisingly, the structural and functional abnormalities in the gut associated with NAFLD and metabolic dysfunction further increase the risk of CDI and its complications.

The strengths of this study include the utilization of the NIS database to provide a unique opportunity to investigate a nationwide population hospitalized for CDI. To the best of our knowledge, this study is a leading clinical research analysis that provided a comprehensive nationwide comparison of outcomes between NAFLD and other common chronic liver diseases, ALD and VLD, in hospitalized CDI patients. There are also limitations in this study. Particularly, NIS data acquisition relies on the accuracy ICD-9-CM codes for medical diagnoses and no lab results, biopsy or image studies were available for NAFLD diagnosis and severity stratification. It is also difficult to determine which cases of CDI were hospital acquired or community acquired because ICD-9 codes are assigned at discharge. To strengthen the validity of ICD-9 codes for NAFLD, VLD and ALD, we used not only diagnostic codes but also excluded the codes for all other chronic liver diseases (Supplementary Table 1)[63]. The ICD-9 codes for CDI were validated previously with good diagnostic accuracy[64,65].

In conclusion, this study found more favorable overall outcomes but higher rates of intestinal complications in patients hospitalized with CDI and coexisting NAFLD, compared to CDI with coexisting ALD and VLD, individually. These results suggested that NAFLD may be associated with a higher risk of CDI associated intestinal complications through alteration of gut microbiota. Our study also suggested that NAFLD associated metabolic syndrome may contribute significantly to the gut dysbiosis even in the early liver disease stages and cause increased risk for CDI and its complications. During the last few years, the novel and rapidly evolving research technologies for the gut microbiome have been opening up an exciting era in the microbiota therapeutics for different disease models[66]. Tremendous progress has been observed in the treatment of NAFLD and CDI through gut microbiome manipulation. Our study may help increase awareness and diagnose intestinal complications in patients with two common diseases: CDI and NAFLD. Unraveling the significance of interactions between gut microbiota, gut immunity and systemic metabolic impact of NAFLD with prospective studies will provide more insights into the future microbiota therapeutics for CDI and NAFLD.

The authors thank Dr. Chunyi Wu, PhD of Epidemiology from University of Michigan Medical School for her assistance with the statistical analysis.