A proteogenomic view on antibiotic resistance in pathogenic Enterobacter species

Bacteria belonging to the family of Enterobacteriaceae are renowned for causing bloodstream infections, hospital- and healthcare-associated pneumonia, intra-abdominal infections, and urinary tract infections. These infections are usually effectively fought with antibiotics. However, certain Enterobacteriaceae produce enzymes that make them invulnerable, even to the so-called carbapenems, which are antibiotics with an exceptionally broad spectrum of bactericidal activity. Accordingly, carbapenems are considered as “last resort” antibiotics. Today, carbapenem resistant bacteria are a worldwide threat for healthcare settings, especially since infections caused by these highly antibiotic resistant pathogens are nearly untreatable and associated with high mortality rates. The present thesis describes studies on Enterobacteriaceae, belonging to the Enterobacter cloacae complex, which were remarkable either because they were highly carbapenem resistant, or because they were associated with unusual bone infections. The results show that carbapenem resistance in the investigated bacteria was acquired by horizontal transfer of carbapenemase genes, involving a thus far overlooked ancient mobile genomic element or a novel megaplasmid with many additional antibiotic resistance genes. Moreover, by applying integrated in-depth genomics and proteomics analyses, other factors were found to contribute to carbapenem resistance, including restricted cell envelope permeability, reduced metabolism, limited formation of reactive oxygen species, and increased antioxidant production. On the other hand, isolates associated with bone infections were sensitive to most antibiotics, but expressed a large array of virulence factors. Altogether, the present studies have greatly increased our understanding of antibiotic resistance mechanisms and virulence in Enterobacteriacea that belong to the E. cloacae complex

An ancient family of mobile genomic islands introducing cephalosporinase and carbapenemase genes in Enterobacteriaceae

The exchange of mobile genomic islands (MGIs) between microorganisms is often mediated by phages, which may provide benefits to the phage's host. The present study started with the identification of Enterobacter cloacae, Klebsiella pneumoniae and Escherichia coli isolates with exceptional cephalosporin and carbapenem resistance phenotypes from patients in a neonatal ward. To identify possible molecular connections between these isolates and their β-lactam resistance phenotypes, the respective bacterial genome sequences were compared. This unveiled the existence of a family of ancient MGIs that were probably exchanged before the species E. cloacae, K. pneumoniae and E. coli emerged from their common ancestry. A representative MGI from E. cloacae was named MIR17-GI, because it harbors the novel β-lactamase gene variant blaMIR17. Importantly, our observations show that the MIR17-GI-like MGIs harbor genes associated with high-level resistance to cephalosporins. Among them, MIR17-GI stands out because MIR17 also displays carbapenemase activity. As shown by mass spectrometry, the MIR17 carbapenemase is among the most abundantly expressed proteins of the respective E. cloacae isolate. Further, we show that MIR17-GI-like islands are associated with integrated P4-like prophages. This implicates phages in the spread of cephalosporin and carbapenem resistance amongst Enterobacteriaceae. The discovery of an ancient family of MGIs, mediating the spread of cephalosporinase and carbapenemase genes, is of high clinical relevance, because high-level cephalosporin and carbapenem resistance have serious implications for the treatment of patients with enterobacteriaceal infections

Antibiotic resistance plasmids cointegrated into a megaplasmid harbouring the blaOXA-427 carbapenemase gene

OXA-427 is a new class-D carbapenemase encountered in different species of Enterobacteriaceae in a Belgian hospital. To study the dispersal of this gene, we describe the comparative analysis of two plasmids containing the blaOXA-427 gene isolated from a Klebsiella pneumoniae and an Enterobacter cloacae complex strain. The two IncA/C2 plasmids containing blaOXA-427 share the same backbone, however, in the K. pneumoniae this plasmid is cointegrated into an IncFIB plasmid forming a 321 kb megaplasmid with multiple multi-resistance regions

Proteomic Charting of Imipenem Adaptive Responses in a Highly Carbapenem Resistant Clinical Enterobacter roggenkampii Isolate

Gram-negative bacteria belonging to the Enterobacter cloacae complex are increasingly implicated in difficult-to-treat nosocomial infections, as exemplified by a recently characterized highly carbapenem-resistant clinical Enterobacter roggenkampii isolate with sequence type (ST) 232. While mechanisms of carbapenem resistance are well-understood, little is known about the responses of highly drug-resistant bacteria to these antibiotics. Our present study was therefore aimed at charting the responses of the E. roggenkampii ST232 isolate to the carbapenem imipenem, using a 'stable isotope labeling of amino acids in cell culture' approach for quantitative mass spectrometry. This unveiled diverse responses of E. roggenkampii ST232 to imipenem, especially altered levels of proteins for cell wall biogenesis, central carbon metabolism, respiration, iron-sulfur cluster synthesis, and metal homeostasis. These observations suggest a scenario where imipenem-challenged bacteria reduce metabolic activity to save resources otherwise used for cell wall biogenesis, and to limit formation of detrimental reactive oxygen species at the cytoplasmic membrane due to respiration and Fenton chemistry. We consider these observations important, because knowing the adaptive responses of a highly resistant bacterium of the E. cloacae complex to last-resort antibiotics, such as imipenem, provides a 'sneak preview' into the future development of antibiotic resistance in this emerging group of pathogens

Antibiotic resistance plasmids cointegrated into a megaplasmid harbouring the blaOXA-427 carbapenemase gene

OXA-427 is a new class-D carbapenemase encountered in different species of Enterobacteriaceae in a Belgian hospital. To study the dispersal of this gene, we describe the comparative analysis of two plasmids containing the blaOXA-427 gene isolated from a Klebsiella pneumoniae and an Enterobacter cloacae complex strain. The two IncA/C2 plasmids containing blaOXA-427 share the same backbone, however, in the K. pneumoniae this plasmid is cointegrated into an IncFIB plasmid forming a 321 kb megaplasmid with multiple multi-resistance regions.status: publishe

An ancient family of mobile genomic islands introducing cephalosporinase and carbapenemase genes in Enterobacteriaceae

The exchange of mobile genomic islands (MGIs) between microorganisms is often mediated by phages, which may provide benefits to the phage’s host. The present study started with the identification of Enterobacter cloacae, Klebsiella pneumoniae and Escherichia coli isolates with exceptional cephalosporin and carbapenem resistance phenotypes from patients in a neonatal ward. To identify possible molecular connections between these isolates and their β-lactam resistance phenotypes, the respective bacterial genome sequences were compared. This unveiled the existence of a family of ancient MGIs that were probably exchanged before the species E. cloacae, K. pneumoniae and E. coli emerged from their common ancestry. A representative MGI from E. cloacae was named MIR17-GI, because it harbors the novel β-lactamase gene variant blaMIR17. Importantly, our observations show that the MIR17-GI-like MGIs harbor genes associated with high-level resistance to cephalosporins. Among them, MIR17-GI stands out because MIR17 also displays carbapenemase activity. As shown by mass spectrometry, the MIR17 carbapenemase is among the most abundantly expressed proteins of the respective E. cloacae isolate. Further, we show that MIR17-GI-like islands are associated with integrated P4-like prophages. This implicates phages in the spread of cephalosporin and carbapenem resistance amongst Enterobacteriaceae. The discovery of an ancient family of MGIs, mediating the spread of cephalosporinase and carbapenemase genes, is of high clinical relevance, because high-level cephalosporin and carbapenem resistance have serious implications for the treatment of patients with enterobacteriaceal infections