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Elena Bülow

Tuesday 24 March 2015

The human gut microbiota as a reservoir for antimicrobial resistance genes

Promotor: Prof. dr. M.J.M. Bonten
Defence: 24 March 2015

In the last decades, the emergence and spread of resistant opportunistic pathogens is compromising the effectiveness of antimicrobial therapies. Understanding the emergence and global spread of drug-resistant microorganisms is thus crucial to combat antimicrobial resistance. The human gut harbors a densely populated and highly diverse microbial ecosystem - the gut microbiota - that can serve as a reservoir for antibiotic resistance genes ('the resistome') and for drug-resistant opportunistic pathogens. Critically ill patients that are hospitalized in Intensive Care Units (ICUs) are particularly susceptible to nosocomial infections caused by bacteria that are members of the normal human microbiota. In order to prevent or treat nosocomial infections, in these patients, antibiotic therapy is frequently used in patients that are hospitalized in ICUs. Other antimicrobial agents than antibiotics, such as disinfectants, are also important in limiting the prevalence of pathogenic microorganisms and nosocomial infections in clinical settings. However, their effectiveness is similarly compromised by the emergence of pathogens with reduced susceptibility to disinfectants. Previous studies have shown that the gut microbiota is significantly impacted by antimicrobial therapy. Yet, the dynamics of the gut resistome during such therapies and its contribution to the emergence and dissemination of antimicrobial resistance remain poorly understood.

In this thesis the dynamics of the gut microbiota and resistome of adult hospitalized patients in an ICU receiving prophylactic and curative antibiotic therapies were investigated using culture-independent techniques. Because gut-associated bacteria and their resistance genes may be shed into the environment through sewage, we also assessed the levels of antibiotic resistance genes in hospital sewage and in subsequent stages of wastewater treatment. The gut microbiota as a putative reservoir for disinfectant resistance genes is also studied.

The data described in chapter 2 show that prophylactic antibiotic therapy and ICU hospitalization can have a great impact on the resistome of ICU patients. The abundance of antibiotic resistance genes (ARGs), in particular aminoglycoside resistance genes, more than doubled in the gut resistom of a single ICU patient that received prophylactic antibiotic therapy throughout the period of hospitalization in the ICU. The resistance genes that were identified in drug-resistant fosmid clones ware carried by harmless gut commensals (Clostridium and Bacteroides) and located on mobile genetic elements, which indicated that these bacteria can form an important reservoir of antibiotic resistance genes. Resistance genes associated with mobile genetic elements could facilitate horizontal gene transfer of resistance genes from commensal bacteria to opportunistic pathogens. In chapter 3, we describe the dynamics of the microbiota and resistome of 13 ICU patients and 10 healthy adults. Our findings indicate that the microbiota of ICU patients is distinct from the microbiota of healthy adults and is characterized by changes in the abundance of the phyla Bacteroidetes and Firmicutes. Levels of ARGs increased during ICU hospitalization and were associated with changes in the composition of the microbiota. We also found that both the microbiota and the resistome was more varying in of ICU patients than in healthy adults. In chapter 4, we investigate the levels of ARGs in hospital sewage and their feit, during passage through the general sewage system. Our data show that hospital sewage is remarkably rich in antibiotic resistance genes, but the contribution of hospital sewage to the level of ARGs in urban sewage is limited. Levels of resistance genes and bacteria of the human microbiota further decreased during wastewater treatment, limiting the dispersal of antibiotic resistance genes from hospitals into the environment. In chapter 5, we studied whether the gut microbiota of hospitalized patients could act as a putative reservoir for genes conferring resistance to the disinfectant benzalkonium chloride (BC). We were able to identify three genes that conferred BC resistance in E. coli. Two of the identified genetic elements that carried the BC resistance genes also contributed to reduced susceptibility to antibiotics, suggesting that genes that confer resistance to disinfectants such as BC, may be genetically linked to genes that can confer resistance to antibiotics.

This thesis provides novel insights into the dynamics and diversity of the gut microbiota and the resistome of hospitalized patients, and highlights the significant effects of ICU hospitalization on the gut resistome and microbiota. The expansion of the gut resistome during ICU hospitalization and antimicrobial therapies shown here is a cause for concern. Follow up investigations combining gut microbiology and resistome studies may be be performed to assess the potential transfer of these resistance genes to nosocomial pathogens during and after hospitalization.