Frequent Use of Antibiotics Is Associated with Colorectal Cancer Risk: Results of a Nested Case–Control Study

Colorectal cancer (CRC) is the third most common cancer in men and second most common cancer in women worldwide [1]. Most cases of CRC develop according to the adenoma–carcinoma sequence, which is characterized by the accumulation of genetic and epigenetic mutations leading to benign premalignant lesions and eventually cancer [2]. Important risk factors associated with CRC include lifestyle factors, such as smoking, limited physical activity, and obesity, and a Western diet which is rich in animal fat, red and processed meat, and poor in fibers [3].

In the past few years, an increasing interest has emerged on the role of the gut microbiota in the development of CRC. The human microbiota consists of approximately 10¹⁴ bacterial cells of 500–1000 different species and is important for the defense against pathogens, the metabolization of polysaccharides, the production of certain vitamins and plays a key role in maintaining a healthy immune system [4]. Furthermore, the colonic microbiota ferments undigested carbohydrates from fibers into short-chain fatty acids including butyrate, acetate, and propionate [5, 6] which are the preferred energy source of the colon mucosa and possess anti-inflammatory, anti-proliferative, and anti-carcinogenic properties [7‐14]. High intakes of meat and animal fat, on the other hand, increase the bacterial production of genotoxic hydrogen sulfide and the secretion of bile acids which are metabolized into carcinogenic secondary bile acids by 7α-dehydroxylating bacteria [15, 16].

Only a few studies have investigated the association between the colonic microbiota and CRC development. These studies show important differences in the composition of the colon microbiota between low and high CRC risk populations based on their diet. For example, a reduced number of short-chain fatty acid-producing bacteria, such as the anti-inflammatory Faecalibacterium prausnitzii from the Clostridium cluster IV and Eubacterium/Roseburia species from cluster XIVa [17], and an increase in secondary bile acid-producing species are found in high CRC risk populations [15, 18]. Furthermore, in CRC patients compared to healthy controls, short-chain fatty acid-producing bacteria have been found to be depleted [19, 20] while the proinflammatory Fusobacterium and Porhyromonas genera were increased [19]. In another study in which stools of CRC patients and healthy subjects were investigated, an increase in Bacteroides‐Prevotella populations was demonstrated compared to healthy controls [21].

These findings suggest a potentially important role for the gut microbiota in the development of CRC. It may well be possible that other factors, besides diet, known to induce a disbalance of the gut microbiota are associated with an increased risk of developing CRC. In this regard, the use of antibiotics may be of interest since its use may seriously affect the diversity of the colonic microbiota. We therefore hypothesize that the (frequent) use of antibiotics is associated with an increased CRC risk. In the current study, we investigated whether the use of antibiotics and specific classes of antibiotics is associated with the risk of developing CRC in a population-based cohort. Secondly, we investigated potential effect modification by other factors that have an effect on the gut microbiota or have been found to be associated with CRC risk.

Between January 2006 and December 2011, 8141 subjects were identified with a DRG for CRC. After the exclusion of subjects with a DRG for inflammatory bowel disease (n = 88), prevalent CRC cases (n = 3518), or subjects with less than 6 years of follow-up (n = 506) before the first DRG, 4029 incident CRC cases remained available for the analysis. These cases were matched to 15,988 controls without inflammatory bowel disease or CRC.

The results of this nested case–control study indicate that the use of antibiotics is associated with a dose-dependent increased risk of developing CRC. The results were similar for models adjusted for sex and age and models additionally adjusted for comorbidities and comedication. These positive associations were found for both anti-aerobic and anti-anaerobic drugs; however, when stratified for different classes of antibiotics, we observed only statistically significant associations for penicillins and quinolones.

Previous studies investigating the association between the use of antibiotics and cancer risk demonstrated similar results. A Finnish cohort study including over three million subjects found relative risks of 1.37 (95 % CI 1.34–1.40) for developing any cancer and 1.15 (95 % CI 1.04–1.26) for developing colon cancer when comparing subjects with ≥6 prescriptions to those with 0–1 prescriptions over a 3-year period prior to cancer diagnosis [26]. In a recent nested case–control study in which Boursi et al. included over twenty thousand cases, a dose-dependent increased CRC risk for subjects who had used penicillins, cephalosporins, sulfonamides/trimethoprim, and nitroimidazoles in the 1–5 years prior to index date [27]. Their findings remained significant after adjusting for potential confounding lifestyle factors, comorbidities, comedication, and previous screening colonoscopy. Another nested case–control study by Wang et al. in almost 28,000 patients with type 2 diabetes also found a positive association between the use of anti-anaerobic antibiotics and both colon (OR 2.31, 95 % CI 2.12–2.52) and rectal cancer (OR 1.69, 95 % CI 1.50–1.90), but no association was found for anti-aerobic agents [28]. In contrast, in our study, we found a positive association between the use of anti-aerobic agents and developing CRC, although this was less pronounced when compared to anti-anaerobic agents. The gut microbiota is predominately composed of anaerobes, and the findings reported by Wang et al. and those of our study suggest that particularly the use of anti-anaerobic agents may promote colorectal tumor growth, although caution is required when drawing conclusions about possible explanatory mechanisms.

Whether the observed associations between antibiotics and cancer risk reflect a causal relation is unclear. Antibiotics generally have no known genotoxic potential, and evidence of possible carcinogenic effects of antibiotics is limited [29]. However, some antibiotics up-regulate cyclooxygenase-2 and increase the production of prostaglandins [30], which are important in inflammatory responses and are known to promote the development of CRC [31]. Furthermore, it has been hypothesized that a depletion of anti-inflammatory and short-chain fatty acid-producing species, such as Faecalibacterium prausnitzii and Roseburia, and the abundance of pro-inflammatory microorganisms, such as Fusobacterium, Porhyromonas, Enterococcaceae, and Bacteroides‐Prevotella, and toxin-producing species including B. fragilis and some E. coli strains could change the gut microbiota in a more pro-carcinogenic environment [27]. This shift is thought to be dependent on subjects’ age, diet, and pathogen infections, but it can be hypothesized that the frequent use of antibiotics may alter short-chain fatty acid-producing species by causing a dysbiosis of the colonic microbiota, thereby increasing the risk of developing CRC [7‐14]. The observed association between the use of antibiotics and CRC risk in our study could also be caused by unmeasured confounding factors associated with CRC risk and a higher use of antibiotics, such as socioeconomic status, smoking, body mass index, and other lifestyle factors. This is supported by the relative modest risk estimates that we found in this study. Nonetheless, when we adjusted for potential confounding factors associated with lifestyle, including diabetes and the use of statins, acetylsalicylic acids, and nonsteroidal anti-inflammatory drugs, the results remained similar. Similarly, modest risk estimates were found in the study by Boursi et al. after adjusting for lifestyle factors, comorbidities, comedication, and previous screening colonoscopy. An alternative explanation for the findings of the present study could be that subjects with a weakened immune system are more susceptible for developing cancer and more frequently develop infections requiring antibiotics. In that case, the observed associations may only be an indicator for an increased cancer risk in general rather than an effector. This may be why in previous studies positive associations have been found between the use of antibiotics and tumors in other organs, such as breast, prostate, lung, and thyroid cancer and non-Hodgkin’s lymphoma, which, as far as we currently understand, do not have an association with the gut microbiota [26, 32‐36]. We observed no statistically significant interaction between the use of antibiotics and other factors that may affect the gut microbiota or that have previously been reported to be associated with CRC risk, which suggests that there is no synergetic effect of antibiotics with diabetes or other medications. We also did not find evidence for reversed causation since prolonging the lag time between the last time point of antibiotic use and CRC diagnosis from 1 to 2 or 3 years had no effect on the results. On the contrary, the risk estimates increased when a follow-up period further back in time was employed. This increases the likelihood of a causal effect since CRC has been estimated to develop over a period of 8–10 years [37].

The strengths of this study include the large number of CRC cases and matched controls that were identified in a health claim database providing high-quality data on the use of antibiotics and potential confounding medication and comorbidity. In the Netherlands, antibiotics can only be obtained with a prescription of a physician and therefore are registered in the health claim database. Both DRG and pharmacy registrations are complete and highly accurate because of the economic function of the database for the insurance company [22]. Furthermore, compared to most previous studies, we included a higher number of subjects from the general population [26, 28, 32].

A limitation of our study is that the diagnosis of CRC could not be histologically confirmed. Second, as previously mentioned, there were no data available on potential confounding lifestyle factors, which is a limitation of the present study. Third, antibiotics prescribed during hospitalizations are not registered in the database. Therefore, we could not include the antibiotics that are more frequently prescribed during hospitalization, such as cephalosporins, aminoglycosides, and imidazoles. There were also no data available on whether the dispensed antibiotics were actually used. Both may have diluted the true size of the association between antibiotics and CRC risk. Finally, we did not have data on the microbiota composition of the subjects included in our study. Combined data on the use of antibiotic agents and the composition of the gut microbiota could be of additive value to further detangle the association between antibiotic use and CRC development. Nevertheless, the dose-dependent increase and the similar risk estimates for antibiotic use up to 8 years before diagnosis suggest that a dysbiosis of the gut microbiota through the use of antibiotics may partially contribute to the development of CRC.

As of now, there is insufficient evidence to make a clinical recommendation regarding the use of antibiotics and CRC risk. The results of our study do, however, support the idea that a microbiotical disbalance in the colorectum may increase the risk of developing CRC. Two previous studies, of which one in a general population investigating a shorter exposure period and one only including subjects with type 2 diabetes, found a positive association between the use of antibiotics and CRC risk. These findings are now supported by our study in a general population. Additional epidemiological studies with long-term follow-up should focus on the mutual effects of antibiotic use and lifestyle factors to further elucidate the association between antibiotic use and CRC risk. Furthermore, observational studies including measurements of the (changes in) microbiota composition in relation to CRC risk may provide more insights in the role of the gut microbiota in the pathogenesis of CRC.

In conclusion, we observed a positive association between the use of antibiotics, especially when used frequently, and the risk of developing CRC. Whether this association resembles a causal relation must be investigated in future studies.