University of Groningen A proteogenomic view on antibiotic resistance in pathogenic Enterobacter species Nepal, Suruchi

A proteogenomic view on antibiotic resistance in pathogenic Enterobacter species

Nepal, Suruchi

DOI:

10.33612/diss.103413360

IMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite from it. Please check the document version below.

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Publication date: 2019

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Citation for published version (APA):

Nepal, S. (2019). A proteogenomic view on antibiotic resistance in pathogenic Enterobacter species. University of Groningen. https://doi.org/10.33612/diss.103413360

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Chapter 6

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^ƵŵŵĂƌŝnjŝŶŐĚŝƐĐƵƐƐŝŽŶĂŶĚĨƵƚƵƌĞƉĞƌƐƉĞĐƟǀĞƐ

Species belonging to the Enterobacter cloacae complex are widespread in ŶĂƚƵƌĞ͕ǁŚŝĐŚƵŶĚĞƌƐĐŽƌĞƐƚŚĞŝƌŚŝŐŚƉŽƚĞŶƟĂůƚŽĂĚĂƉƚƚŽĚŝīĞƌĞŶƚĞĐŽůŽŐŝĐĂů

niches that range from the soil to the human body 1_{͘/ŶƚŚĞůĂƩĞƌĐŽŶƚĞdžƚƚŚĞLJ}

have emerged as nosocomial pathogens2 _{that increasingly display resistance}

ƚŽĂŶƟďŝŽƟĐƐ3_{. As shown by the studies on a highly drug resistant Enterobacter}

roggenkampii isolate with sequence type (ST) 232 that are documented in this i

ƚŚĞƐŝƐ͕ƚŚĞƌĞƐŝƐƚĂŶĐĞƉŚĞŶŽƚLJƉĞƐŽĨƌĞƉƌĞƐĞŶƚĂƟǀĞƐŽĨƚŚĞ E. cloacae complex ŶŽǁĞǀĞŶĞdžƚĞŶĚƚŽĐĂƌďĂƉĞŶĞŵƐ͕ǁŚŝĐŚĂƌĞŝŵƉŽƌƚĂŶƚ͚ůĂƐƚƌĞƐŽƌƚĂŶƟďŝŽƟĐƐ͛͘ To understand how such a resistance phenotype may have emerged, what is its present status, how it compares to other related isolates, and how it may evolve ŝŶLJĞĂƌƐƚŽĐŽŵĞǁĂƐƚŚĞƉƌŝŵĞŽďũĞĐƟǀĞŽĨƚŚĞŚĞƌĞĚĞƐĐƌŝďĞĚƌĞƐĞĂƌĐŚ͘ &ƌŽŵĂĐůŝŶŝĐĂůƉĞƌƐƉĞĐƟǀĞ͕ĂŶƐǁĞƌƐƚŽƚŚĞƋƵĞƐƟŽŶƐŚŽǁĂŶŝŶĨĞĐƟŽŶƉƌĞƐĞŶƚƐ ŝƚƐĞůĨ ĂŶĚ ŚŽǁ ŝƚ ĐĂŶ ďĞƐƚ ďĞ ƚƌĞĂƚĞĚ ŚĂǀĞ ƚŚĞ ĮƌƐƚ ƉƌŝŽƌŝƚLJ͘ dŽ ƚŚŝƐ ĞŶĚ͕ ŝƚ ŝƐ ƌĞůĞǀĂŶƚ ƚŽ ŝƐŽůĂƚĞ ĂŶĚ ŝĚĞŶƟĨLJ ƚŚĞ ĐĂƵƐĂƟǀĞ ĂŐĞŶƚ͕ ĂŶĚ ƚŽ ĚĞƚĞƌŵŝŶĞ ŝƚƐ ĂŶƟďŝŽƟĐƌĞƐŝƐƚĂŶĐĞƉƌŽĮůĞ͘dŚĞE. cloacae complex isolates that are described ŝŶƚŚŝƐƚŚĞƐŝƐǁĞƌĞŽďƚĂŝŶĞĚĨƌŽŵƉĂƟĞŶƚƐĂĚŵŝƩĞĚƚŽƚŚĞhŶŝǀĞƌƐŝƚLJDĞĚŝĐĂů ĞŶƚĞƌ'ƌŽŶŝŶŐĞŶ;hD'Ϳ͘ůĞĂƌůLJ͕ĨƌŽŵƚŚĞƚŚĞƌĂƉĞƵƟĐƉĞƌƐƉĞĐƟǀĞ͕ŬŶŽǁŝŶŐ ƚŚĞĂŶƟďŝŽƟĐƐƵƐĐĞƉƟďŝůŝƚLJƉƌŽĮůĞŽĨƚŚĞƐĞŝƐŽůĂƚĞƐǁĂƐŵŽƌĞŝŵƉŽƌƚĂŶƚƚŚĂŶ ŝĚĞŶƟĨLJŝŶŐƚŽǁŚŝĐŚƐƉĞĐŝĞƐǁŝƚŚŝŶƚŚĞE. cloacae complex they belonged exactly. dŚĞƐĂŵĞĂƉƉůŝĞĚƚŽƐƵĐŚŝƐŽůĂƚĞƐŽďƚĂŝŶĞĚĨƌŽŵƉĂƟĞŶƚƐŝŵƉůŝĐĂƚĞĚŝŶďŽŶĞ ŝŶĨĞĐƟŽŶƐ͕ǁŚŝĐŚǁĂƐĂŶƵŶƵƐƵĂůĐůŝŶŝĐĂůƉƌĞƐĞŶƚĂƟŽŶĨŽƌďĂĐƚĞƌŝĂŽĨƚŚĞE. cloacae ĐŽŵƉůĞdžĂƚƚŚĞƟŵĞŽĨŝƐŽůĂƟŽŶ͘ĐĐŽƌĚŝŶŐůLJ͕ƚŚĞƉƌĞƐĞŶƚƐƚƵĚLJŝƐŽůĂƚĞƐǁĞƌĞ ŝĚĞŶƟĮĞĚďLJƌŽƵƟŶĞĐƵůƚƵƌŝŶŐĂŶĚŵĂƚƌŝdžͲĂƐƐŝƐƚĞĚůĂƐĞƌĚĞƐŽƌƉƟŽŶŝŽŶŝnjĂƟŽŶ ƟŵĞͲŽĨͲŇŝŐŚƚ ŵĂƐƐ ƐƉĞĐƚƌŽŵĞƚƌLJ ;D>/ͲdK& D^Ϳ͕ ǁŚŝĐŚ ůĞĚ ƚŽ ƚŚĞŝƌ ƌĂƉŝĚ ŝĚĞŶƟĮĐĂƟŽŶ ĂƐ ͚E. cloacae͛͘ /ŵŵĞĚŝĂƚĞůLJ ƚŚĞƌĞĂŌĞƌ͕ ƚŚĞ ĂŶƟďŝŽƟĐ ƌĞƐŝƐƚĂŶĐĞ ƉƌŽĮůĞƐŽĨƚŚĞƐĞŝƐŽůĂƚĞƐǁĞƌĞĚĞƚĞƌŵŝŶĞĚ͕ƵŶĞdžƉĞĐƚĞĚůLJƌĞǀĞĂůŝŶŐĐĂƌďĂƉĞŶĞŵ ƌĞƐŝƐƚĂŶĐĞĨŽƌŽŶĞŝƐŽůĂƚĞĨƌŽŵĂŶĞŽŶĂƚĞ͕ǁŚŝůĞƐŚŽǁŝŶŐĂŶƟďŝŽƟĐƐƵƐĐĞƉƟďŝůŝƚLJ ĨŽƌƚŚĞŝƐŽůĂƚĞƐĨƌŽŵďŽŶĞŝŶĨĞĐƟŽŶƐ͘&ŽƌƚƌĞĂƚŵĞŶƚŽĨƚŚĞƌĞƐƉĞĐƟǀĞƉĂƟĞŶƚƐ ƚŚŝƐ ŝŶĨŽƌŵĂƟŽŶ ǁĂƐ ƐƵĸĐŝĞŶƚ͘ /Ŷ ĐŽŶƚƌĂƐƚ͕ ĨŽƌ ƵŶĚĞƌƐƚĂŶĚŝŶŐ ƚŚĞ ďŝŽůŽŐLJ ƵŶĚĞƌůLJŝŶŐƚŚĞĂŶƟďŝŽƟĐƌĞƐŝƐƚĂŶĐĞŵĞĐŚĂŶŝƐŵƐĂŶĚĐůŝŶŝĐĂůƉƌĞƐĞŶƚĂƟŽŶ͕ƚŚŝƐ was merely the beginning.

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dŽ ƌĞĂůůLJ ƵŶĚĞƌƐƚĂŶĚ ƚŚĞ ĚƌŝǀĞƌƐ ĨŽƌ ĂŶƟďŝŽƟĐ ƌĞƐŝƐƚĂŶĐĞ ĂŶĚ ĐůŝŶŝĐĂů ŶŝĐŚĞ preference of bacterial isolates it is necessary to dissect their molecular architecture, from the genome to the proteome and beyond. This is a very ĂŵďŝƟŽƵƐ ŐŽĂů ƚŚĂƚ͕ ƚŽĚĂLJ͕ ĐĂŶ ŽŶůLJ ďĞ ĂĐŚŝĞǀĞĚ ŝŶ ƉĂƌƚƐ͘  ŵĂũŽƌ ůŝŵŝƚĂƟŽŶ ŝŶ ƵŶĚĞƌƐƚĂŶĚŝŶŐ ƚŚĞ ďĞŚĂǀŝŽƌ ŽĨ ŝŶĨĞĐƟŽƵƐ ĂŐĞŶƚƐ ŝƐ ƚŚĂƚ ƚŚĞLJ ĐĂŶ ŽŶůLJ ďĞ ƐƚƵĚŝĞĚŝŶĚĞƚĂŝůĂŌĞƌƚŚĞŝƌŝƐŽůĂƟŽŶĨƌŽŵƚŚĞƉĂƟĞŶƚ͕ŝŶŽƚŚĞƌǁŽƌĚƐŽƵƚƐŝĚĞ ƚŚĞƉĂƟĞŶƚ͛ƐďŽĚLJ͘dŚĞƌĞĨŽƌĞ͕ƚŚĞŶĞdžƚďĞƐƚŽƉƟŽŶŝƐƚŽĂŶĂůLJnjĞŝŶĚĞƚĂŝůƚŚĞ ĐŽŵƉŽƐŝƟŽŶ ŽĨ ƚŚĞŝƌ ŐĞŶŽŵĞ ĨƌŽŵ ǁŚŝĐŚ ƉŽƐƐŝďůĞ ĐůŝŶŝĐĂů ďĞŚĂǀŝŽƌ ĐĂŶ ďĞ ŝŶĨĞƌƌĞĚ͘dŚĞƉŽƐƐŝďŝůŝƟĞƐĨŽƌƐƵĐŚŐĞŶŽŵŝĐĂŶĂůLJƐĞƐŚĂǀĞĞdžƉĂŶĚĞĚĂƚĂǀĞƌLJ high pace over the past four years, and this development was actually followed closely in the here presented studies. For example, when studies for Chapter 2 ǁĞƌĞ ƉĞƌĨŽƌŵĞĚ͕ ŶĞdžƚ ŐĞŶĞƌĂƟŽŶ ƐĞƋƵĞŶĐŝŶŐ ƚĞĐŚŶŽůŽŐLJ ŚĂĚ ũƵƐƚ ŽƉĞŶĞĚ ƚŚĞ ǁŝŶĚŽǁ ƚŽ ĨĂƐƚ ĂŶĚ ĂīŽƌĚĂďůĞ ŐĞŶŽŵĞĂŶĂůLJƐĞƐ͘ zĞƚ͕ƚŚĞƌĞǁĞƌĞůŝƚĞƌĂůůLJ ŵĂŶLJ ŐĂƉƐ ŝŶ ƚŚĞ ŽďƚĂŝŶĞĚ ƐĞƋƵĞŶĐĞ ŝŶĨŽƌŵĂƟŽŶ͕ ǁŚŝĐŚ ǁĞƌĞ ŝŶĐƌĞĂƐŝŶŐůLJ ĐůŽƐĞĚ ƚŽǁĂƌĚƐ ƚŚĞ ĞŶĚ ŽĨ ƚŚĞ ŚĞƌĞ ĚĞƐĐƌŝďĞĚ ƐƚƵĚŝĞƐ ƵƐŝŶŐ ƚŚŝƌĚͲŐĞŶĞƌĂƟŽŶ ŶĂŶŽƉŽƌĞͲďĂƐĞĚƐĞƋƵĞŶĐŝŶŐƚĞĐŚŶŽůŽŐLJ͘/ŶĨĂĐƚ͕ƚŚĞĐŽŵďŝŶĂƟŽŶŽĨŚŝŐŚͲƋƵĂůŝƚLJ ƐĞƋƵĞŶĐĞƐŽďƚĂŝŶĞĚďLJƐŚŽƌƚͲƌĞĂĚƐĞƋƵĞŶĐŝŶŐĂƉƉƌŽĂĐŚĞƐǁŝƚŚƚŚĞůŽŶŐƌĞĂĚƐŽĨ lower quality obtained by nanopore sequencing can now deliver highly accurate genome sequences using hybrid de novo assembly approaches. This hybrid ĂƐƐĞŵďůLJ ŝƐ ƉĂƌƟĐƵůĂƌůLJ ŝŵƉŽƌƚĂŶƚ ĨŽƌ ƚŚĞ ĂŶĂůLJƐŝƐ ŽĨ ŐĞŶŽŵĞƐ ƚŚĂƚ ĐŽŶƚĂŝŶ ŵĂŶLJ ƌĞƉĞĂƚĞĚ ƐĞƋƵĞŶĐĞƐ͕ ĂƐ ŝƚ ůĞĂĚƐ ƚŽ ůŽǁĞƌ ŶƵŵďĞƌƐ ŽĨ ĐŽŶƟŐƐ ǁŝƚŚ͕ ŽŶ average, higher lengths and with fewer sequencing errors than can be obtained

with either method by itself 4_{. Accordingly, the hybrid genome assemblies are}

ĐƵƌƌĞŶƚůLJƚŚĞďĞƐƚƐƚĂƌƟŶŐƉŽŝŶƚƐĨŽƌĨƵƌƚŚĞƌĚŽǁŶƐƚƌĞĂŵĂƉƉůŝĐĂƟŽŶƐ͕ƌĂŶŐŝŶŐ ĨƌŽŵŐĞŶĞĂŶŶŽƚĂƟŽŶƚŽĚĞƚĂŝůĞĚŐĞŶŽŵĞĐŽŵƉĂƌŝƐŽŶƐ͕ĂŶĚƚŚĞŝŶƚĞƌƉƌĞƚĂƟŽŶ ŽĨŵĂƐƐƐƉĞĐƚƌŽŵĞƚƌLJĚĂƚĂƚŽĂŶĂůLJnjĞƚŚĞƉƌŽƚĞŝŶĐŽŵƉŽƐŝƟŽŶŽĨƚŚĞƌĞƐƉĞĐƟǀĞ ŽƌŐĂŶŝƐŵƐ͕ĞŝƚŚĞƌƋƵĂůŝƚĂƟǀĞůLJŽƌƋƵĂŶƟƚĂƟǀĞůLJ͘

Ɛ ĞdžĞŵƉůŝĮĞĚ ďLJ ƚŚĞ ƐƚƵĚLJ ĚĞƐĐƌŝďĞĚ ŝŶ ŚĂƉƚĞƌ Ϯ͕ ƚŚĞ ŚŝŐŚ ƋƵĂůŝƚLJ ŚLJďƌŝĚ genome assembly of a carbapenem resistant isolate belonging to the E. cloacae ĐŽŵƉůĞdžĂůůŽǁĞĚĂĚĞƚĂŝůĞĚƌĞĐŽŶƐƚƌƵĐƟŽŶŽĨĞǀŽůƵƟŽŶĂƌLJĞǀĞŶƚƐƚŚĂƚŵĂLJŚĂǀĞ ƐĞƚ ƚŚĞ ƐƚĂŐĞ ĨŽƌ ŝƚƐ ŚŽƌŝnjŽŶƚĂůůLJ ĂĐƋƵŝƌĞĚ ƌĞƐŝƐƚĂŶĐĞ͘ /Ŷ ƉĂƌƟĐƵůĂƌ͕ ŝƚ ĂůůŽǁĞĚ ƚŚĞŝĚĞŶƟĮĐĂƟŽŶŽĨĂŵŽďŝůĞŐĞŶŽŵŝĐŝƐůĂŶĚ;D'/ͿƚŚĂƚǁĂƐƐƉƌĞĂĚĂŵŽŶŐƚŚĞ ĂŶĐĞƐƚƌLJŽĨƚŚĞŝŶǀĞƐƟŐĂƚĞĚŝƐŽůĂƚĞ͕ůŽŶŐďĞĨŽƌĞƚŚĞƐƉĞĐŝĞƐďĞůŽŶŐŝŶŐƚŽƚŚĞE.

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ŚĂƉƚ

ĞƌϨ

ďĞĐĂŵĞĂĚŽĐŬŝŶŐƉůĂƞŽƌŵĨŽƌĂƌĂŶŐĞŽĨĚŝīĞƌĞŶƚƌĞƐŝƐƚĂŶĐĞŐĞŶĞƐ͕ĞƐƉĞĐŝĂůůLJƚŚĞ clinically relevant genes specifying AmpC type cephalosporinases, one of which ƚƵƌŶĞĚŽƵƚƚŽƉŽƐƐĞƐƐĂůƐŽĐĂƌďĂƉĞŶĞŵĂƐĞĂĐƟǀŝƚLJ;ŝ͘Ğ͘D/ZϭϳͿ͘dŚŝƐŽďƐĞƌǀĂƟŽŶ ŚĂĚƐĞǀĞƌĂůŝŵƉŽƌƚĂŶƚŝŵƉůŝĐĂƟŽŶƐ͘/ŶƚŚĞĮƌƐƚƉůĂĐĞ͕ŝƚƐŚŽǁĞĚƚŚĂƚĞůĞŵĞŶƚƐ that are generally regarded as belonging to the core genome of a bacterial ƐƉĞĐŝĞƐŵĂLJĂĐƚƵĂůůLJƐƟůůďĞŵŽďŝůĞŐĞŶŽŵŝĐĞůĞŵĞŶƚƐ͘dŚŝƐŝƐĂĨĂĐƚƚŚĂƚŝƐŽŌĞŶ ŽǀĞƌůŽŽŬĞĚŝŶďĂĐƚĞƌŝĂůŐĞŶŽŵĞŝŶǀĞƐƟŐĂƟŽŶƐ͘zĞƚ͕ƚŚĞŽďƐĞƌǀĂƟŽŶƐĚĞƐĐƌŝďĞĚ ŝŶŚĂƉƚĞƌϮƐŚŽǁƚŚĂƚŝƚŝƐŝŵƉŽƌƚĂŶƚƚŽůŽŽŬďĞLJŽŶĚƚŚĞƐƉĞĐŝĞƐďĂƌƌŝĞƌƐǁŚĞŶ ĚŝƐĐĞƌŶŝŶŐĐŽƌĞĂŶĚĂĐĐĞƐƐŽƌLJŐĞŶŽŵĞƐ͘^ĞĐŽŶĚ͕ƚŚĞƌĞƐƵůƚƐĨŽĐƵƐĂƩĞŶƟŽŶŽŶ ƚŚĞĨĂĐƚƚŚĂƚƐƵĐŚ͚ĂŶĐŝĞŶƚD'/Ɛ͛ŵĂLJƌĞƉƌĞƐĞŶƚŚŽƚƐƉŽƚƐĨŽƌƚŚĞĂĐĐƵŵƵůĂƟŽŶ ŽĨ ĐůŝŶŝĐĂůůLJ ƌĞůĞǀĂŶƚ ƌĞƐŝƐƚĂŶĐĞ ƉƌŽƉĞƌƟĞƐ͕ ĂƐ ĞdžĞŵƉůŝĮĞĚ ďLJ ƚŚĞ ampC type C

cephalosporinase genes. Third, the results show that not all cephalosporinases ĂƌĞĞƋƵĂůĂƐƐŽŵĞŽĨƚŚĞŵ͕ůŝŬĞD/Zϭϳ͕ŵĂLJƉŽƐƐĞƐƐĐĂƌďĂƉĞŶĞŵĂƐĞĂĐƟǀŝƚLJ͘KĨ ŶŽƚĞ͕ƚŚŝƐĮŶĚŝŶŐŚĂƐďĞĞŶŝŵƉůĞŵĞŶƚĞĚŝŶƌŽƵƟŶĞĚŝĂŐŶŽƐƟĐƐĂƚhD'͕ǁŚĞƌĞ ƉůĂƚĞƐǁŝƚŚĐůŽdžĂĐŝůůŝŶĂƌĞŶŽǁƵƐĞĚƚŽĂƐƐĞƐƐƉŽƐƐŝďůĞĐĂƌďĂƉĞŶĞŵĂƐĞĂĐƟǀŝƚLJ ŽĨ ĐĞƉŚĂůŽƐƉŽƌŝŶĂƐĞƐ͘ >ĂƐƚůLJ͕ ƚŚĞ ŚLJďƌŝĚ ŐĞŶŽŵĞ ĂƐƐĞŵďůLJ ŚĂƐ ƐĞƚ ƚŚĞ ƐƚĂŐĞ ĨŽƌĂƉƌŽƚĞŽŵŝĐĚŝƐƐĞĐƟŽŶŽĨƚŚĞĐĂƌďĂƉĞŶĞŵƌĞƐŝƐƚĂŶƚE.roggenkampii study ŝƐŽůĂƚĞĚĞƐĐƌŝďĞĚŝŶŚĂƉƚĞƌϮ͕ŚŝŐŚůŝŐŚƟŶŐƚŚĞŚŝŐŚĂďƵŶĚĂŶĐĞŽĨD/Zϭϳ͘dŚĞ ƐĐŝĞŶƟĮĐƉĂƚŚĨƌŽŵĐůŝŶŝĐĂůŝƐŽůĂƚĞƚŚƌŽƵŐŚŐĞŶŽŵĞƐĞƋƵĞŶĐŝŶŐƚŽĂŶĂůLJƐŝƐŽĨƚŚĞ ƉƌŽƚĞŽŵĞŝƐƐŬĞƚĐŚĞĚŝŶ&ŝŐƵƌĞϭ͘

537819-L-bw-Nepal 537819-L-bw-Nepal 537819-L-bw-Nepal 537819-L-bw-Nepal Processed on: 13-11-2019 Processed on: 13-11-2019 Processed on: 13-11-2019

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&ŝŐƵƌĞ ϭ͘ KǀĞƌǀŝĞǁ ŽĨ ĞdžƉĞƌŝŵĞŶƚƐ ĂŶĚ ĚĂƚĂ ĂŶĂůLJƐĞƐ ƵŶĚĞƌƚĂŬĞŶ ĨŽƌ ƚŚĞ ƉƌĞƐĞŶƚWŚƌĞƐĞĂƌĐŚ͘;ͿůŝŶŝĐĂůďĂĐƚĞƌŝĂůŝƐŽůĂƚĞƐŽĨŝŶƚĞƌĞƐƚǁĞƌĞĐŽůůĞĐƚĞĚ͕ ĐƵůƚƵƌĞĚĂŶĚƚĞƐƚĞĚĨŽƌĂŶƟďŝŽƟĐƌĞƐŝƐƚĂŶĐĞ͘;ͿdŚĞŝƐŽůĂƚĞƐǁĞƌĞƐƵďƐĞƋƵĞŶƚůLJ ĐŚĂƌĂĐƚĞƌŝnjĞĚ ďLJ E ƐĞƋƵĞŶĐŝŶŐ ƵƐŝŶŐ ƚǁŽ ĚŝƐƟŶĐƚ ŵĞƚŚŽĚƐ͕ ŝ͘Ğ͕͘ /ůůƵŵŝŶĂ ƐŚŽƌƚͲƌĞĂĚƐĞƋƵĞŶĐŝŶŐĂŶĚKdžĨŽƌĚEĂŶŽƉŽƌĞdĞĐŚŶŽůŽŐLJůŽŶŐͲƌĞĂĚƐĞƋƵĞŶĐŝŶŐ͘ hƉŽŶƐƵƐďƐĞƋƵĞŶƚŚLJďƌŝĚEƐĞƋƵĞŶĐĞĂƐƐĞŵďůLJƵƐŝŶŐƚŚĞ^WĚĞƐŽƌhŶŝĐLJĐůĞƌ ĂƉƉƌŽĂĐŚĞƐ͕ ĨƵƌƚŚĞƌ ĂŶŶŽƚĂƟŽŶ ǁĂƐ ƉĞƌĨŽƌŵĞĚ ƚŽ ŝĚĞŶƟĨLJ ŐĞŶŽŵŝĐ ŝƐůĂŶĚƐ͕ ƉůĂƐŵŝĚƐ ĂŶĚ ŝŶƚĞŐƌŽŶƐ͕ ƌĞƐŝƐƚĂŶĐĞ ŐĞŶĞƐ ĂŶĚ ƌĞƐŝƐƚĂŶĐĞͲƌĞůĂƚĞĚ ŐĞŶĞƐ ƵƐŝŶŐ ƐƵŝƚĂďůĞďŝŽͲŝŶĨŽƌŵĂƟĐƐƚŽŽůƐ͘EĞdžƚ͕ĂƚĂďůĞǁŝƚŚŝŶĨŽƌŵĂƟŽŶŽŶƚŚĞŐĞŶĞŶĂŵĞ͕ ƉƌŽƚĞŝŶŶĂŵĞ͕ƉƌŽƚĞŝŶƐĞƋƵĞŶĐĞ͕ŐĞŶĞŽŶƚŽůŽŐLJ;'KͿĂŶĚ'KƚĞƌŵƐ͕ďŝŽůŽŐŝĐĂů ƉƌŽĐĞƐƐĞƐĂŶĚĐůƵƐƚĞƌƐŽĨŽƌƚŚŽůŽŐŽƵƐ;K'ͿĨƵŶĐƟŽŶĂůĐĂƚĞŐŽƌŝĞƐǁĂƐƉƌĞƉĂƌĞĚ͘ dŚĞƉĞƉƟĚĞƐĞƋƵĞŶĐĞƐĚĞĚƵĐĞĚĨƌŽŵƚŚĞǁŚŽůĞͲŐĞŶŽŵĞƐĞƋƵĞŶĐĞƐǁĞƌĞƚŚĞŶ ƵƐĞĚ ĂƐ Ă ƌĞƐŽƵƌĐĞ ĨŽƌ ƉĞƉƟĚĞ ŝĚĞŶƟĮĐĂƟŽŶ ďLJ ŵĂƐƐ ƐƉĞĐƚƌŽŵĞƚƌLJ͘ ;Ϳ ĂƚĂ from the proteome and genome analyses were compared and interpreted, ƚĂŬŝŶŐĂĚǀĂŶƚĂŐĞŽĨƚŚĞƐŽͲĐĂůůĞĚ<''ƉĂƚŚǁĂLJƐ͕ƚŽƵŶĚĞƌƐƚĂŶĚƚŚĞďĂĐƚĞƌŝĂů responses to an imipenem challenge, and to predict the possible future of ĂŶƟďŝŽƟĐƌĞƐŝƐƚĂŶĐĞĚĞǀĞůŽƉŵĞŶƚ͘

537819-L-bw-Nepal 537819-L-bw-Nepal 537819-L-bw-Nepal 537819-L-bw-Nepal Processed on: 13-11-2019 Processed on: 13-11-2019 Processed on: 13-11-2019

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ŚĂƉƚ

ĞƌϨ

ƉĂƌƟĐƵůĂƌĂĚǀĂŶƚĂŐĞŽĨŚĂǀŝŶŐĂŚŝŐŚƋƵĂůŝƚLJŐĞŶŽŵĞƐĞƋƵĞŶĐĞĂƐŽďƚĂŝŶĞĚ by hybrid genome assembly is that it allows detailed genome comparisons of

related Enterobacter species belonging to the r E. cloacae complex. Indeed, such

ĂƉŚLJůŽŐĞŶĞƟĐĂŶĂůLJƐŝƐǁĂƐƉĞƌĨŽƌŵĞĚ͕ƌĞǀĞĂůŝŶŐƚŚĂƚƚŚĞĐĂƌďĂƉĞŶĞŵƌĞƐŝƐƚĂŶƚ

study isolate of Chapter 2 was de facto an E. roggenkampii isolate. i

Importantly, the more classical proteome approach applied for the studies ĚĞƐĐƌŝďĞĚ ŝŶ ŚĂƉƚĞƌ Ϯ ƉƌŽǀŝĚĞƐ Ă ŐŽŽĚ ŽǀĞƌǀŝĞǁ ŽĨ ƚŚĞ ͚ƉƌŽƚĞŝŶ ůĂŶĚƐĐĂƉĞ͛ ŽĨƚŚĞďĂĐƚĞƌŝĂůĐĞůů͕ďƵƚŝƚƉƌŽǀŝĚĞƐŶŽĚLJŶĂŵŝĐŝŶĨŽƌŵĂƟŽŶŽŶŚŽǁƚŚĞĐĞůůƐ ƌĞƐƉŽŶĚ ƚŽ ĞŶǀŝƌŽŶŵĞŶƚĂů ŝŶƐƵůƚƐ͕ ƐƵĐŚ ĂƐ ƚŚĞ ĂĚŵŝŶŝƐƚƌĂƟŽŶ ŽĨ ĂŶƟďŝŽƟĐƐ͘ ,ŽǁĞǀĞƌ͕ ŬŶŽǁŝŶŐ ŚŽǁ ƚŚĞ ĐĞůůƐ ƌĞƐƉŽŶĚ ƚŽ ƚŚĞ ƉƌĞƐĞŶĐĞ ŽĨ ĂŶ ĂŶƟďŝŽƟĐ ƉƌŽǀŝĚĞƐ ǀĂůƵĂďůĞ ŝŶĨŽƌŵĂƟŽŶ ŽŶ ĂĚĂƉƟǀĞ ďĞŚĂǀŝŽƌ ƚŚĂƚ͕ ƵƉŽŶ ůŽŶŐͲƚĞƌŵ ĂŶƟďŝŽƟĐĞdžƉŽƐƵƌĞ͕ŵĂLJďĞĐŽŵĞůŽĐŬĞĚŝŶƚŽĂŶŝŶƚƌŝŶƐŝĐƌĞƐŝƐƚĂŶĐĞŐĞŶŽƚLJƉĞ͘ Therefore, the studies documented in Chapter 3 assessed how the carbapenem

resistant E. roggenkampii isolate responded to the presence of the carbapenem i

imipenem and, again, the availability of the hybrid genome assembly turned ŽƵƚ ĂĚǀĂŶƚĂŐĞŽƵƐ ĂƐ ŝƚ ĂůůŽǁĞĚ ƚŚĞ ŝĚĞŶƟĮĐĂƟŽŶ ŽĨ ĂŶ ĂĚĚŝƟŽŶĂů ƐĞƚ ŽĨ ϰϬϬ ƉƌŽƚĞŝŶƐ ƚŚĂƚ ǁĞƌĞ ŝŶŝƟĂůůLJ ŽǀĞƌůŽŽŬĞĚ͘ KĨ ŶŽƚĞ͕ ŝƚ ǁĂƐ ĂŶƟĐŝƉĂƚĞĚ ƚŚĂƚ Ă ƋƵĂŶƟƚĂƟǀĞ ƉƌŽƚĞŽŵĞ ĂŶĂůLJƐŝƐ ƚŽ ĨŽůůŽǁ ƚŚĞ ƌĞƐƉŽŶƐĞƐ ŽĨ Ă ŚŝŐŚůLJ ĂŶƟďŝŽƟĐ ƌĞƐŝƐƚĂŶƚďĂĐƚĞƌŝĂůŝƐŽůĂƚĞƚŽƚŚĞƉƌĞƐĞŶĐĞŽĨĂŶĂŶƟďŝŽƟĐƚŽǁŚŝĐŚŝƚŝƐƌĞƐŝƐƚĂŶƚ ŵŝŐŚƚƉƌŽǀŝĚĞĂŐůĂŶĐĞŽĨƉŽƚĞŶƟĂůĨƵƚƵƌĞĂĚĂƉƟǀĞŽƌĞǀĞŶŝŶƚƌŝŶƐŝĐƌĞƐŝƐƚĂŶĐĞ͘ /ŶĚĞĞĚ͕ƚŚĞƌĞƐƵůƚƐƐŚŽǁĞĚƚŚĂƚƚŚĞƉƌĞƐĞŶĐĞŽĨŝŵŝƉĞŶĞŵĂƚĂĐŽŶĐĞŶƚƌĂƟŽŶ ďĞůŽǁ ƚŚĞ ŵŝŶŝŵĂů ŝŶŚŝďŝƚŽƌLJ ĐŽŶĐĞŶƚƌĂƟŽŶ ƚƌŝŐŐĞƌĞĚ ǀĂƌŝŽƵƐ ƌĞƐƉŽŶƐĞƐ ƚŚĂƚ ĂůůŽǁƚŚĞďĂĐƚĞƌŝĂƚŽŵŝŶŝŵŝnjĞ;ŝͿƚŚĞǁĂƐƚĞŽĨĐĞůůƵůĂƌƌĞƐŽƵƌĐĞƐĨŽƌĐĞůůǁĂůů ďŝŽŐĞŶĞƐŝƐ ƵƉŽŶ ŝŵƉĂŝƌĞĚ ĨƵŶĐƟŽŶ ŽĨ ƉĞŶŝĐŝůůŝŶ ďŝŶĚŝŶŐ ƉƌŽƚĞŝŶƐ͕ ĂŶĚ ;ŝŝͿ ƚŚĞ ƉŽƚĞŶƟĂůůLJ ĚĞƚƌŝŵĞŶƚĂů ŐĞŶĞƌĂƟŽŶ ŽĨ ƌĞĂĐƟǀĞ ŽdžLJŐĞŶ ƐƉĞĐŝĞƐ ;ZK^Ϳ ĚƵĞ ƚŽ ŝŶĐƌĞĂƐĞĚŇƵdžƚŚƌŽƵŐŚƚŚĞŐůLJĐŽůLJƟĐĂŶĚƚƌŝĐĂƌďŽdžLJůŝĐĂĐŝĚ;dͿƉĂƚŚǁĂLJƐƚŚĂƚ ŵĂLJŽĐĐƵƌǁŚĞŶĐĞůůǁĂůůďŝŽŐĞŶĞƐŝƐŝƐŝŵƉĂŝƌĞĚ͘ĚĚŝƟŽŶĂůůLJ͕ƚŚĞŐĞŶĞƌĂƟŽŶŽĨ ZK^ďLJƌĞƐƉŝƌĂƟŽŶǁĂƐƉŽƚĞŶƟĂůůLJĚĞĐƌĞĂƐĞĚďLJ;ŝͿƌĞĚƵĐĞĚĞdžƉƌĞƐƐŝŽŶŽĨƚŚĞ ďŝŽƐLJŶƚŚĞƟĐƉĂƚŚǁĂLJĨŽƌŝƌŽŶͲƐƵůƉŚƵƌĐůƵƐƚĞƌĨŽƌŵĂƟŽŶ͕ĂŶĚ;ŝŝͿďLJƵƉƌĞŐƵůĂƟŽŶ ŽĨĂŬĞLJĞŶnjLJŵĞŝŶƚŚĞƉĂƚŚǁĂLJĨŽƌĨŽƌŵĂƟŽŶŽĨƚŚĞĂŶƟŽdžŝĚĂŶƚǀŝƚĂŵŝŶ<͘dŚĞƐĞ ŽďƐĞƌǀĂƟŽŶƐƐŚŽǁƚŚĂƚƚŚĞďĂĐƚĞƌŝĂƐƟůůŚĂǀĞŵĂŶLJŽƉƟŽŶƐƚŽŽǀĞƌĐŽŵĞƚŚĞ ĚĞƚƌŝŵĞŶƚĂůĞīĞĐƚƐŽĨĂŶƟďŝŽƟĐƐůŝŬĞŝŵŝƉĞŶĞŵ͕ŝŶĂĚĚŝƟŽŶƚŽƚŚĞƉƌŽĚƵĐƟŽŶ ŽĨD/ZϭϳĂŶĚƚŚĞŵƵƚĂƟŽŶŽĨƉŽƌŝŶƐŝŶƚŚĞŽƵƚĞƌŵĞŵďƌĂŶĞĂƐĚŽĐƵŵĞŶƚĞĚŝŶ ŚĂƉƚĞƌϮ͘/ŶƚĞƌĞƐƟŶŐůLJ͕ƚŚĞĮŶĚŝŶŐƐƉƌĞƐĞŶƚĞĚŝŶŚĂƉƚĞƌϯĂƌĞĐŽŶƐŝƐƚĞŶƚǁŝƚŚ

537819-L-bw-Nepal 537819-L-bw-Nepal 537819-L-bw-Nepal 537819-L-bw-Nepal Processed on: 13-11-2019 Processed on: 13-11-2019 Processed on: 13-11-2019

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ƌĞƐƵůƚƐĨƌŽŵƉƌĞǀŝŽƵƐƐƚƵĚŝĞƐďLJŽƚŚĞƌƐ͕ǁŚŽŝŶǀĞƐƟŐĂƚĞĚƌĞƐƉŽŶƐĞƐƚŽƘͲůĂĐƚĂŵ ĂŶƟďŝŽƟĐƐĂŶĚƐŚŽǁĞĚƚŚĂƚƚŚĞƐĞĂŶƟďŝŽƟĐƐĚŽŶŽƚŽŶůLJĐĂƵƐĞĂďĂĐƚĞƌŝĐŝĚĂů ǁĞĂŬĞŶŝŶŐŽĨƚŚĞďĂĐƚĞƌŝĂůĐĞůůǁĂůů͕ďƵƚĂůƐŽĞůŝĐŝƚĞdžĐĞƐƐŝǀĞĨŽƌŵĂƟŽŶŽĨZK^ ǁŚŝĐŚŵĂLJĐŽŶƚƌŝďƵƚĞƚŽƚŚĞďĂĐƚĞƌŝĂůŬŝůůŝŶŐ567_{͘>ŝŬĞǁŝƐĞ͕ŽƚŚĞƌƐƚƵĚŝĞƐƐŚŽǁĞĚ} ƚŚĂƚ ŽǀĞƌĐŽŵŝŶŐ ZK^ ĨŽƌŵĂƟŽŶ ďLJ ƐůŽǁŝŶŐ ĚŽǁŶ ƚŚĞ ĐĂƌďŽŶ ŇƵdž ƚŚƌŽƵŐŚ ŐůLJĐŽůLJƐŝƐĂŶĚƚŚĞdĐLJĐůĞĂůůŽǁĞĚƚŚĞĨŽƌŵĂƟŽŶŽĨƐŽͲĐĂůůĞĚďĂĐƚĞƌŝĂů>ͲĨŽƌŵƐ͕ ǁŚŝĐŚůĂĐŬĂĐĞůůǁĂůů8_{͘dŚĞƐĞŽďƐĞƌǀĂƟŽŶƐĨŽĐƵƐĂƩĞŶƟŽŶŽŶƚŚĞĐĞŶƚƌĂůƌŽůĞŽĨ}

metabolic pathways in the bacterial life cycle and the possibility to target central ĐĂƌďŽŶŵĞƚĂďŽůŝƐŵǁŝƚŚŶŽǀĞů͕LJĞƚƚŽďĞĚĞǀĞůŽƉĞĚ͕ĂŶƟďŝŽƟĐƐ͘&ŽƌŝŶƐƚĂŶĐĞ͕ŝĨ ŝƚǁŽƵůĚďĞƉŽƐƐŝďůĞƚŽŝŶĐƌĞĂƐĞŵĞƚĂďŽůŝĐĂĐƟǀŝƚLJŝŶƚŚĞƉƌĞƐĞŶĐĞŽĨĂŶƟďŝŽƟĐƐ ůŝŬĞŝŵŝƉĞŶĞŵ͕ƌĂƚŚĞƌƚŚĂŶůĞƫŶŐƚŚĞďĂĐƚĞƌŝĂƐůŽǁĚŽǁŶƚŚĞŝƌŵĞƚĂďŽůŝƐŵ͕ŝƚ ƐŚŽƵůĚďĞƉŽƐƐŝďůĞƚŽĞůĞǀĂƚĞZK^ƉƌŽĚƵĐƟŽŶƚŽůĞǀĞůƐƚŚĂƚƐƚƌŽŶŐůLJĞŶŚĂŶĐĞƚŚĞ ďĂĐƚĞƌŝĂůŬŝůůŝŶŐ͘ůƚĞƌŶĂƟǀĞůLJ͕ƐƵĐŚĂŶĞīĞĐƚĐŽƵůĚƉŽƚĞŶƟĂůůLJďĞĂĐŚŝĞǀĞĚďLJ ŝŶƚĞƌĨĞƌŝŶŐǁŝƚŚƚŚĞĚŽǁŶͲƌĞŐƵůĂƟŽŶŽĨƚŚĞďŝŽƐLJŶƚŚĞƟĐƉĂƚŚǁĂLJĨŽƌŝƌŽŶͲƐƵůƉŚƵƌ ĐůƵƐƚĞƌƐ͘/ŶƚŚĞůĂƩĞƌĐĂƐĞ͕ƚŚĞ&ƵƌƌĞŐƵůĂƚŽƌŵŝŐŚƚďĞĂŶŝŶƚĞƌĞƐƟŶŐƚĂƌŐĞƚ͕ĂƐ ƚŚĞĮŶĚŝŶŐƐĚĞƐĐƌŝďĞĚŝŶŚĂƉƚĞƌϯŝŶĚŝĐĂƚĞƚŚĂƚƚŚĞƉƌĞƐƵŵĞĚĚŽǁŶƌĞŐƵůĂƟŽŶ ŽĨŝƌŽŶͲƐƵůƉŚƵƌĐůƵƐƚĞƌďŝŽŐĞŶĞƐŝƐŝƐŵĞĚŝĂƚĞĚďLJƵƉƌĞŐƵůĂƟŽŶŽĨ&Ƶƌ͘

tŚŝůĞƚŚĞŚŝŐŚͲƋƵĂůŝƚLJŐĞŶŽŵĞƐĞƋƵĞŶĐĞŽĨƚŚĞE. roggenkampii isolate unveiled i

ƚŚĞƉƌĞƐĞŶĐĞĂŶĚƐĞƋƵĞŶĐĞŝĚĞŶƟƚLJŽĨŝƚƐampC gene, more classical microbiological C

approaches were necessary to show that the encoded cephalosporinase also had ĐĂƌďĂƉĞŶĞŵĂƐĞĂĐƟǀŝƚLJ͘dŚŝƐǁĂƐĂĮŶĚŝŶŐƚŚĂƚĐŽƵůĚŶŽƚŚĂǀĞďĞĞŶƉƌĞĚŝĐƚĞĚ from the genome sequence. In turn, this underpins the fact that the genome sequence is only the blueprint of what a bacterium is actually capable of. Also, ƚŚĞŐĞŶŽŵĞƐĞƋƵĞŶĐĞǁŽƵůĚŶŽƚĂůůŽǁƚŚĞƉƌĞĚŝĐƟŽŶŽĨƚŚĞĂĚĂƉƟǀĞďĞŚĂǀŝŽƌ

ŽĨƚŚĞŝŶǀĞƐƟŐĂƚĞĚE. roggenkampii isolate in the presence of imipenem. Another i

ĨĞĂƚƵƌĞŝŶƚĞƌŵƐŽĨĂŶƟďŝŽƟĐƌĞƐŝƐƚĂŶĐĞďĞŚĂǀŝŽƌƚŚĂƚĐŽƵůĚŶŽƚďĞƉƌĞĚŝĐƚĞĚ from the genome sequence alone, at least not yet, was the fact that the studied

E. roggenkampii isolate carried an i aadA gene for aminoglycoside resistance on

an In127 integron but, nonetheless, showed no resistance to aminoglycosides. tŚŝůĞƚŚŝƐŽďƐĞƌǀĂƟŽŶƚĞůůƐƵƐƐŽŵĞƚŚŝŶŐĂďŽƵƚƚŚĞĞǀŽůƵƟŽŶĂƌLJŚŝƐƚŽƌLJŽĨƚŚĞ

ŝŶǀĞƐƟŐĂƚĞĚE. roggenkampii isolate, it also reinforces the need for combining i

ŐĞŶŽŵĞĂŶĂůLJƐĞƐǁŝƚŚĨƵŶĐƟŽŶĂůĂƐƐĂLJƐŽƌƉƌŽƚĞŽŵŝĐƐĂƉƉƌŽĂĐŚĞƐƚŽŽďƚĂŝŶĂ ĐůĞĂƌĞƌǀŝĞǁŽĨǁŚĂƚƚŚĞŝŶǀĞƐƟŐĂƚĞĚďĂĐƚĞƌŝĂĂƌĞƌĞĂůůLJƵƉƚŽ͘/ŶĂĚĚŝƟŽŶ͕ĨƵƚƵƌĞ ƵƐĞŽĨŵĂĐŚŝŶĞůĞĂƌŶŝŶŐĂŶĚĂƌƟĮĐŝĂůŝŶƚĞůůŝŐĞŶĐĞĂƉƉƌŽĂĐŚĞƐŵĂLJŽǀĞƌĐŽŵĞƚŚĞ

537819-L-bw-Nepal 537819-L-bw-Nepal 537819-L-bw-Nepal 537819-L-bw-Nepal Processed on: 13-11-2019 Processed on: 13-11-2019 Processed on: 13-11-2019

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ŚĂƉƚ

ĞƌϨ

ĐƵƌƌĞŶƚůŝŵŝƚĂƟŽŶƐŽĨŐĞŶŽŵŝĐĂŶĂůLJƐĞƐ͘

Chapter 4 presents the results of a combined genomics and proteomics ĂƉƉƌŽĂĐŚ ƚŽ ĮŶĚ ŽƵƚ ǁŚŝĐŚ ĨĞĂƚƵƌĞƐ ĐŽƵůĚ ĚŝƐƟŶŐƵŝƐŚ ŚƵŵĂŶ ŐƵƚͲƌĞƐŝĚĞŶƚ isolates belonging to the E. cloacae complex from related isolates implicated in ďŽŶĞŝŶĨĞĐƟŽŶƐ͘&ŝƌƐƚ͕ĂĐŽŵƉĂƌŝƐŽŶŽĨƚŚĞŐĞŶŽŵĞƐĞƋƵĞŶĐĞƐƐŚŽǁĞĚƚŚĂƚƚŚĞ ŝŶǀĞƐƟŐĂƚĞĚŝƐŽůĂƚĞƐǁŝƚŚĚŝīĞƌĞŶƚĐůŝŶŝĐĂůƉƌĞƐĞŶƚĂƟŽŶƐďĞůŽŶŐĞĚƚŽĚŝīĞƌĞŶƚ

Enterobacter ƐƉĞĐŝĞƐ͘ dŚĞ ŐƵƚ ƌĞƐŝĚĞŶƚ ŝƐŽůĂƚĞƐ ǁĞƌĞ ŝŶ ĨĂĐƚ ŝĚĞŶƟĮĞĚ ĂƐr E. roggenkampii and E. hormaechei subsp͘ŚŽīŵĂŶŶŝŝ, whereas the bone isolates

ďĞůŽŶŐĞĚ ƚŽ ĚŝīĞƌĞŶƚ E. hormaechei subspecies or i E. cloacae. This provided

ĂůƌĞĂĚLJ Ă ƐƚƌŽŶŐ ĐůƵĞ ĨŽƌ Ă ĚŝīĞƌĞŶƚ ĞǀŽůƵƟŽŶĂƌLJ ŚŝƐƚŽƌLJ ŽĨ ƚŚĞ ƌĞƐƉĞĐƟǀĞ ŝƐŽůĂƚĞƐ͕ǁŚŝĐŚŵŝŐŚƚĞdžƉůĂŝŶĂĚŝīĞƌĞŶƚŶŝĐŚĞƉƌĞĨĞƌĞŶĐĞŝŶƚŚĞŚƵŵĂŶďŽĚLJ͘ Intriguingly, the subsequent proteome analysis showed that the bone isolates ƉƌŽĚƵĐĞĚŵĂŶLJƉƌŽƚĞŝŶƐƌĞůĂƚĞĚƚLJƉĞs/ƐĞĐƌĞƟŽŶƐLJƐƚĞŵƐ͕ǁŚŝĐŚĂƌĞŐĞŶĞƌĂůůLJ ŝŵƉůŝĐĂƚĞĚŝŶǀŝƌƵůĞŶĐĞ͕ĂŶĚƉƌŽƚĞŝŶƐƌĞůĂƚĞĚƚŽŇĂŐĞůůĂƌĨƵŶĐƟŽŶĨŽƌďĂĐƚĞƌŝĂů ŵŽƟůŝƚLJ͘ŽŶƐŝƐƚĞŶƚǁŝƚŚƚŚĞƐĞƉƌŽƚĞŽŵĞĚĂƚĂ͕ƚŚĞďŽŶĞŝƐŽůĂƚĞƐĐŽŶƚĂŝŶĞĚƚǁŽ ƚŽƚŚƌĞĞƚLJƉĞ///ƐĞĐƌĞƟŽŶƐLJƐƚĞŵƐĨŽƌŇĂŐĞůůĂƌďŝŽŐĞŶĞƐŝƐ͕ĂŶĚƚǁŽƚŽƚŚƌĞĞƚLJƉĞ s/ƐĞĐƌĞƟŽŶƐLJƐƚĞŵƐ͘dŚĞŐĞŶĞƐĨŽƌƚŚĞƐĞƐLJƐƚĞŵƐǁĞƌĞĂůůŝŶƚĞŐƌĂƚĞĚďĞƚǁĞĞŶ ƚZEͲĞŶĐŽĚŝŶŐ ŐĞŶĞƐ͕ ƐƵŐŐĞƐƟǀĞ ŽĨ ĂĐƋƵŝƐŝƟŽŶ ďLJ ŚŽƌŝnjŽŶƚĂů ŐĞŶĞ ƚƌĂŶƐĨĞƌ͘

KŶ ƚŚĞ ŽƚŚĞƌ ŚĂŶĚ͕ ƚŚĞ ĐĂƌďĂƉĞŶĞŵͲƌĞƐŝƐƚĂŶƚ E. roggenkampii isolate morei

ƉƌŽŵŝŶĞŶƚůLJĞdžƉƌĞƐƐĞĚƉƌŽƚĞŝŶƐƌĞůĂƚĞĚƚŽĂŶƟďŝŽƟĐƌĞƐŝƐƚĂŶĐĞĂŶĚƌĞƐŝƐƚĂŶĐĞƚŽ ŚĞĂǀLJŵĞƚĂůƐ͘dŽŐĞƚŚĞƌ͕ƚŚĞƐĞŽďƐĞƌǀĂƟŽŶƐĂƚƚŚĞŐĞŶŽŵĞĂŶĚƉƌŽƚĞŽŵĞůĞǀĞůƐ ƐƵƉƉŽƌƚƚŚĞǀŝĞǁƚŚĂƚƚŚĞŝŶǀĞƐƟŐĂƚĞĚŝƐŽůĂƚĞƐ͕ĂůůďĞůŽŶŐŝŶŐƚŽƚŚĞE. cloacae ĐŽŵƉůĞdž͕ ŚĂǀĞ ĞǀŽůǀĞĚ ŝŶ ĚŝīĞƌĞŶƚ ĚŝƌĞĐƟŽŶƐ ƚŽ ŽƉƟŵĂůůLJ ĂĚĂƉƚ ƚŽ ĚŝīĞƌĞŶƚ ŶŝĐŚĞƐǁŝƚŚĚŝīĞƌĞŶƚƐĞůĞĐƟǀĞƉƌĞƐƐƵƌĞƐ͘/ŶƚŚĞĐŽŶƚĞdžƚŽĨƚŚŝƐĞǀŽůƵƟŽŶ͕ƚŚĞLJ ŚĂǀĞĂĐƋƵŝƌĞĚĚŝīĞƌĞŶƚŵŽůĞĐƵůĂƌĨƵŶĐƟŽŶƐƚŚĂƚŚĂǀĞŝŶĐƌĞĂƐĞĚƚŚĞŝƌĮƚŶĞƐƐŝŶ ƚŚĞƌĞƐƉĞĐƟǀĞŶŝĐŚĞƐ͘ ZĞůĂƚĞĚ ƚŽ ďĂĐƚĞƌŝĂů ĞǀŽůƵƟŽŶ ĂŶĚ ƚŚĞ ŚŽƌŝnjŽŶƚĂů ƚƌĂŶƐĨĞƌ ŽĨ ƌĞƐŝƐƚĂŶĐĞ genes, Chapter 5 ĨŽĐƵƐĞƐ ĂƩĞŶƟŽŶ ŽŶ ƚǁŽ ƉůĂƐŵŝĚƐ ĞŶĐŽĚŝŶŐ ƚŚĞ KyͲϰϮϳ carbapenemase, which were isolated from a Klebsiella pneumoniae and an E.

cloacae ĐŽŵƉůĞdž ƐƚƌĂŝŶ͕ ƌĞƐƉĞĐƟǀĞůLJ͘ dŚĞƐĞ ƚǁŽ /ŶĐͬϮ ƉůĂƐŵŝĚƐ ƐŚĂƌĞ ƚŚĞ

ƐĂŵĞďĂĐŬďŽŶĞ͕ďƵƚŝŶƚŚĞ K. pneumoniae isolate this plasmid is cointegrated ŝŶƚŽĂŶ/ŶĐ&/ƉůĂƐŵŝĚ͕ǁŚŝĐŚŚĂƐƌĞƐƵůƚĞĚŝŶĂϯϮϭͲŬďŵĞŐĂƉůĂƐŵŝĚ͘KĨŶŽƚĞ͕ ƚŚŝƐŵĞŐĂƉůĂƐŵŝĚĐĂƌƌŝĞƐŵƵůƟƉůĞŵƵůƟͲƌĞƐŝƐƚĂŶĐĞƌĞŐŝŽŶƐ͘dŚĞƉƌĞƐĞŶĐĞŽĨƚŚĞ ĚŝīĞƌĞŶƚ ƌĞƐŝƐƚĂŶĐĞ ŐĞŶĞƐ ǁŝƚŚŝŶ ƚŚĞ ŵĞŐĂƉůĂƐŵŝĚ ŝƐ ĚĞĮŶŝƚĞůLJ Ă ǁŽƌƌŝƐŽŵĞ

537819-L-bw-Nepal 537819-L-bw-Nepal 537819-L-bw-Nepal 537819-L-bw-Nepal Processed on: 13-11-2019 Processed on: 13-11-2019 Processed on: 13-11-2019

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ŽďƐĞƌǀĂƟŽŶ͕ďĞĐĂƵƐĞƚŚŝƐŵĂLJƐŝŐŶŝĮĐĂŶƚůLJĨĂĐŝůŝƚĂƚĞƚŚĞŝƌĚŝƐƐĞŵŝŶĂƟŽŶƚŽŽƚŚĞƌ ďĂĐƚĞƌŝĂ ƚŚĂƚ ŵĂLJ ĂůƌĞĂĚLJ ƉŽƐƐĞƐƐ ŽƚŚĞƌ ĂŶƟďŝŽƟĐ ƌĞƐŝƐƚĂŶĐĞƐ͘ dŚĞ ŝĚĞŶƟĮĞĚ ŵĞŐĂƉůĂƐŵŝĚĐŽƵůĚ͕ƚŚƵƐ͕ƚƵƌŶŝŶƚŽĂǀĞŚŝĐůĞĨŽƌƚŚĞĞŵĞƌŐĞŶĐĞŽĨƉĂŶͲƌĞƐŝƐƚĂŶƚ Enterobacteriaceae. /Ŷ ĐŽŶĐůƵƐŝŽŶ͕ ƚŚĞ ƉƌĞƐĞŶƚ WŚ ƚŚĞƐŝƐ ƌĞƉŽƌƚƐ ŽŶ ƚŚĞ ŝĚĞŶƟĮĐĂƟŽŶ ĂŶĚ ĐŚĂƌĂĐƚĞƌŝnjĂƟŽŶ ŽĨ ďĂĐƚĞƌŝĂů ŝƐŽůĂƚĞƐ ďĞůŽŶŐŝŶŐ ƚŽ ƚŚĞ E. cloacae complex, ǁŚŝĐŚǁĞƌĞƌĞŵĂƌŬĂďůĞŝŶƚĞƌŵƐŽĨƚŚĞŝƌĂŶƟďŝŽƟĐƌĞƐŝƐƚĂŶĐĞƉƌŽĮůĞŽƌĐůŝŶŝĐĂů ƉƌĞƐĞŶƚĂƟŽŶ͘ /Ŷ ƚŚĞ ǀĂƐƚ ŵĂũŽƌŝƚLJ ŽĨ ĐĂƐĞƐ͕ ƐƵĐŚ ŽďƐĞƌǀĂƟŽŶƐ ĂƌĞ ĞƐƐĞŶƟĂůůLJ ĂƉƉůŝĞĚƚŽƚƌĞĂƚƚŚĞƉĂƟĞŶƚƐŝŶĨĞĐƚĞĚďLJƚŚĞƐĞďĂĐƚĞƌŝĂ͕ǁŚĞƌĞĂƐƚŚĞĐŽŵƉůĞdž underlying mechanisms that have led to their drug resistance or niche preference ƌĞŵĂŝŶĐŽǀĞƌĞĚ͘KŶůLJǁŝƚŚƚŚĞĂĚǀĞŶƚŽĨƌĂƉŝĚŵƵůƟͲŽŵŝĐƐƚĞĐŚŶŽůŽŐLJĂŶĚŝƚƐ ŝŵƉůĞŵĞŶƚĂƟŽŶĂƚƚŚĞŝŶƚĞƌĨĂĐĞŽĨĐůŝŶŝĐĂůĂŶĚĨƵŶĚĂŵĞŶƚĂůƐĐŝĞŶƟĮĐƌĞƐĞĂƌĐŚ͕ŝƚ has become possible to open the molecular window that permits unprecedented views into the past, presence and future of the isolated pathogens. This is an ĞdžĐŝƟŶŐĚĞǀĞůŽƉŵĞŶƚĂƐƚŚĞĐŽůůĞĐƚĞĚŝŶƐŝŐŚƚƐŵĂLJƵůƟŵĂƚĞůLJŚĞůƉƵƐƚŽĚĞǀĞůŽƉ ďĞƩĞƌƐƚƌĂƚĞŐŝĞƐĂŶĚĚƌƵŐƐƚŽƉƌĞǀĞŶƚŽƌƚƌĞĂƚĚĂŶŐĞƌŽƵƐŶŽƐŽĐŽŵŝĂůŝŶĨĞĐƟŽŶƐ͘

537819-L-bw-Nepal 537819-L-bw-Nepal 537819-L-bw-Nepal 537819-L-bw-Nepal Processed on: 13-11-2019 Processed on: 13-11-2019 Processed on: 13-11-2019

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References

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ϴ͘ <ĂǁĂŝ͕z͘et al. Crucial role for central carbon metabolism in the bacterial