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

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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 4

Suruchi Nepal, Giorgio Gabarrini, Florian Bonn, Stefano Grasso, Sigrid Rosema, Anne de Jong, Natacha Couto, Dörte Becher, John W.A. Rossen, Erik Bathoorn and Jan Maarten van Dijl

DĂŶƵƐĐƌŝƉƚƚŽďĞƐƵďŵŝƩĞĚ

DƵůƟͲŽŵŝĐƐĂŶĂůLJƐĞƐŽĨĐůŝŶŝĐĂůŝƐŽůĂƚĞƐďĞůŽŶŐŝŶŐƚŽ

ƚŚĞEnterobacter cloacaeĐŽŵƉůĞdžƵŶĐŽǀĞƌĚŝīĞƌĞŶĐĞƐŝŶ

ĞdžƉƌĞƐƐŝŽŶŽĨĚĞƚĞƌŵŝŶĂŶƚƐĨŽƌĂŶƟďŝŽƟĐƌĞƐŝƐƚĂŶĐĞĂŶĚ

ǀŝƌƵůĞŶĐĞ

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Abstract

Bacterial species belonging to the Enterobacter cloacae complex occupy many ĚŝīĞƌĞŶƚĞĐŽůŽŐŝĐĂůŶŝĐŚĞƐ͕ŝŶĐůƵĚŝŶŐƚŚĞŚƵŵĂŶďŽĚLJǁŚĞƌĞƚŚĞLJĐĂŶĐĂƵƐĞƐĞǀĞƌĞ ŝŶĨĞĐƟŽŶƐ͘dŚĞƉƌĞƐĞŶƚƐƚƵĚLJǁĂƐĂŝŵĞĚĂƚĚŝƐƟŶŐƵŝƐŚŝŶŐƉĂƌƟĐƵůĂƌĨĞĂƚƵƌĞƐŽĨ ‘E. cloacae͛ŝƐŽůĂƚĞƐĨƌŽŵƉĂƟĞŶƚƐĂƚƚŚĞhŶŝǀĞƌƐŝƚLJDĞĚŝĐĂůĞŶƚĞƌ'ƌŽŶŝŶŐĞŶ ƚŚĂƚ ǁĞƌĞ ƌĞŵĂƌŬĂďůĞ ŝŶ ƚĞƌŵƐ ŽĨ ƌĞƐŝƐƚĂŶĐĞ ƚŽ ĐĂƌďĂƉĞŶĞŵƐ Žƌ ĂƐƐŽĐŝĂƟŽŶ ǁŝƚŚďŽŶĞŝŶĨĞĐƟŽŶƐ͘dŽĚŝƐƟŶŐƵŝƐŚƚŚĞƐĞŝƐŽůĂƚĞƐ͕ĂĐŽŵďŝŶĞĚŐĞŶŽŵŝĐƐĂŶĚ ƉƌŽƚĞŽŵŝĐƐĂƉƉƌŽĂĐŚǁĂƐĂƉƉůŝĞĚ͘&ŝƌƐƚ͕ĐŽŵƉĂƌŝƐŽŶŽĨƚŚĞƌĞƐƉĞĐƟǀĞŐĞŶŽŵĞ ƐĞƋƵĞŶĐĞƐƐŚŽǁĞĚƚŚĂƚƚŚĞŝƐŽůĂƚĞƐǁŝƚŚĚŝīĞƌĞŶƚĐůŝŶŝĐĂůƉƌĞƐĞŶƚĂƟŽŶďĞůŽŶŐĞĚ ƚŽ ĚŝīĞƌĞŶƚ Enterobacter ƐƉĞĐŝĞƐ͘ dŚĞ ĐĂƌďĂƉĞŶĞŵ ƌĞƐŝƐƚĂŶƚ ŐƵƚ ŝƐŽůĂƚĞ ǁĂƐr ŝĚĞŶƟĮĞĚ ĂƐ E. roggenkampii͕ ǁŚĞƌĞĂƐ ĂŶŽƚŚĞƌ ŐƵƚ ŝƐŽůĂƚĞ ǁĂƐ ŝĚĞŶƟĮĞĚ ĂƐ E. hormaechei subsp͘ ŚŽīŵĂŶŶŝŝ͘ dŚĞ ďŽŶĞ ŝƐŽůĂƚĞƐ ďĞůŽŶŐ ƚŽ ĚŝīĞƌĞŶƚ E. hormaechei subspecies or i E. cloacae͘dŚŝƐŝŵƉůŝĞƐĂĚŝīĞƌĞŶƚĞǀŽůƵƟŽŶĂƌLJŚŝƐƚŽƌLJ

ŽĨƚŚĞƌĞƐƉĞĐƟǀĞŝƐŽůĂƚĞƐ͕ǁŚŝĐŚŵŝŐŚƚĞdžƉůĂŝŶƚŚĞŝƌĚŝīĞƌĞŶƚŶŝĐŚĞƉƌĞĨĞƌĞŶĐĞ ŝŶƚŚĞŚƵŵĂŶďŽĚLJ͘dŚĞƐƵďƐĞƋƵĞŶƚƉƌŽƚĞŽŵĞĂŶĂůLJƐŝƐƐŚŽǁĞĚƚŚĂƚƚŚĞŝƐŽůĂƚĞƐ ĂƐƐŽĐŝĂƚĞĚǁŝƚŚďŽŶĞŝŶĨĞĐƟŽŶƐƉƌŽĚƵĐĞĚƉƌŽƚĞŝŶƐƌĞůĂƚĞĚƚŽƚLJƉĞs/ƐĞĐƌĞƟŽŶ ƐLJƐƚĞŵƐ͕ ǁŚŝĐŚ ĂƌĞ ŐĞŶĞƌĂůůLJ ŝŵƉůŝĐĂƚĞĚ ŝŶ ǀŝƌƵůĞŶĐĞ͕ ĂŶĚ ƉƌŽƚĞŝŶƐ ƌĞůĂƚĞĚ ƚŽ ŇĂŐĞůůĂƌĨƵŶĐƟŽŶ ĨŽƌďĂĐƚĞƌŝĂůŵŽƟůŝƚLJ͘ŽŶƐŝƐƚĞŶƚǁŝƚŚƚŚĞƐĞƉƌŽƚĞŽŵĞĚĂƚĂ͕ ƚŚĞďŽŶĞŝƐŽůĂƚĞƐĐŽŶƚĂŝŶĞĚƚǁŽƚŽƚŚƌĞĞƚLJƉĞ///ƐĞĐƌĞƟŽŶƐLJƐƚĞŵƐĨŽƌŇĂŐĞůůĂƌ ďŝŽŐĞŶĞƐŝƐ͕ ĂŶĚ ƚǁŽ ƚŽ ƚŚƌĞĞ ƚLJƉĞ s/ ƐĞĐƌĞƟŽŶ ƐLJƐƚĞŵƐ͘ dŚĞ ŐĞŶĞƐ ĨŽƌ ƚŚĞƐĞ ƐLJƐƚĞŵƐ ǁĞƌĞ Ăůů ŝŶƚĞŐƌĂƚĞĚ ďĞƚǁĞĞŶ ƚZEͲĞŶĐŽĚŝŶŐ ŐĞŶĞƐ͕ ƐƵŐŐĞƐƟǀĞ ŽĨ ĂĐƋƵŝƐŝƟŽŶ ďLJ ŚŽƌŝnjŽŶƚĂů ŐĞŶĞ ƚƌĂŶƐĨĞƌ͘ KŶ ƚŚĞ ŽƚŚĞƌ ŚĂŶĚ͕ ƚŚĞ ĐĂƌďĂƉĞŶĞŵͲ resistant E. roggenkampiiƉƌŽŵŝŶĞŶƚůLJĞdžƉƌĞƐƐĞĚƉƌŽƚĞŝŶƐƌĞůĂƚĞĚƚŽĂŶƟďŝŽƟĐi ƌĞƐŝƐƚĂŶĐĞĂŶĚƌĞƐŝƐƚĂŶĐĞƚŽĐŽƉƉĞƌ͘dŽŐĞƚŚĞƌ͕ƚŚĞƐĞŽďƐĞƌǀĂƟŽŶƐƐƵƉƉŽƌƚƚŚĞ ǀŝĞǁ ƚŚĂƚ ƚŚĞ ŝŶǀĞƐƟŐĂƚĞĚ ŝƐŽůĂƚĞƐ͕ Ăůů ďĞůŽŶŐŝŶŐ ƚŽ ƚŚĞ E. cloacae ĐŽŵƉůĞdž͕ ŚĂǀĞĞǀŽůǀĞĚŝŶĚŝīĞƌĞŶƚĚŝƌĞĐƟŽŶƐƚŽŽƉƟŵĂůůLJĂĚĂƉƚƚŽĚŝīĞƌĞŶƚŶŝĐŚĞƐǁŝƚŚ ĚŝīĞƌĞŶƚƐĞůĞĐƟǀĞƉƌĞƐƐƵƌĞƐ͘

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

ĞƌϦ

/ŶƚƌŽĚƵĐƟŽŶ dŚĞŐĞŶƵƐEnterobacterŝŶĐůƵĚĞƐƌŽĚͲƐŚĂƉĞĚ͕ŶŽŶͲƐƉŽƌƵůĂƟŶŐ͕ŵŽƟůĞ͕ĨĂĐƵůƚĂƟǀĞr ĂŶĂĞƌŽďŝĐ͕'ƌĂŵͲŶĞŐĂƟǀĞďĂĐƚĞƌŝĂďĞůŽŶŐŝŶŐƚŽƚŚĞĨĂŵŝůLJŽĨEnterobacteriaceae͘ ZĞƉƌĞƐĞŶƚĂƟǀĞƐƉĞĐŝĞƐŽĨƚŚŝƐŐĞŶƵƐ͕ůŝŬĞ Enterobacter cloacae͕ĐĂŶďĞŝƐŽůĂƚĞĚ ĨƌŽŵĂǁŝĚĞǀĂƌŝĞƚLJŽĨĞŶǀŝƌŽŶŵĞŶƚƐ͕ƌĂŶŐŝŶŐĨƌŽŵƚŚĞƐŽŝůĂŶĚƐĞǁĂŐĞƚŽƚŚĞ ŚƵŵĂŶŐĂƐƚƌŽŝŶƚĞƐƟŶĂůƚƌĂĐƚ͘/ŶĨĂĐƚ͕ E. cloacae ŝƐĨƌĞƋƵĞŶƚůLJŝĚĞŶƟĮĞĚĂŵŽŶŐ ƚŚĞŚƵŵĂŶŐƵƚŵŝĐƌŽďŝŽƚĂ͘/ŶƌĞĐĞŶƚLJĞĂƌƐ͕E. cloacae is increasingly encountered ĂƐĂĐĂƵƐĂƟǀĞĂŐĞŶƚŽĨŽƉƉŽƌƚƵŶŝƐƟĐŝŶĨĞĐƟŽŶƐ͕ĞƐƉĞĐŝĂůůLJŝŶŝŶƚĞŶƐŝǀĞĐĂƌĞƵŶŝƚƐ 1_{͘&ŽƌŝŶƐƚĂŶĐĞ͕E. cloacaeŚĂƐďĞĞŶŝŵƉůŝĐĂƚĞĚŝŶƐŬŝŶĂŶĚƐŽŌƟƐƐƵĞŝŶĨĞĐƟŽŶƐ͕} ƌĞƐƉŝƌĂƚŽƌLJĂŶĚƵƌŝŶĂƌLJƚƌĂĐƚŝŶĨĞĐƟŽŶƐϮ͕ϯ͕_{ŝŶƚƌĂͲĂďĚŽŵŝŶĂůŝŶĨĞĐƟŽŶƐ͕ďĂĐƚĞƌĞŵŝĂ͕} ĞŶĚŽĐĂƌĚŝƟƐ͕ ƐĞƉƟĐ ĂƌƚŚƌŝƟƐ͕ ĂŶĚ ŽƐƚĞŽŵLJĞůŝƟƐ ŝŶ ŝŵŵƵŶŽĐŽŵƉƌŽŵŝƐĞĚ ƉĂƟĞŶƚƐϰ͕ϱ_{͘DŽƐƚ E. cloacae ŝƐŽůĂƚĞƐŝŵƉůŝĐĂƚĞĚŝŶŝŶĨĞĐƟŽŶƐĂƌĞƐƟůůƐĞŶƐŝƟǀĞƚŽ} ĂŶƟďŝŽƟĐƐ͕ďƵƚĂůƐŽĂŵŽŶŐƌĞĐĞŶƚE. cloacae ŝƐŽůĂƚĞƐĂƚƌĞŶĚƚŽǁĂƌĚƐŝŶĐƌĞĂƐĞĚ ĂŶƟďŝŽƟĐƌĞƐŝƐƚĂŶĐĞŝƐĞŵĞƌŐŝŶŐ6_͘

dŚĞ͚ƐƉĞĐŝĞƐ͛E. cloacaeǁĂƐŽƌŝŐŝŶĂůůLJƌĞĨĞƌƌĞĚƚŽĂƐ Bacillus cloacae in 1890 ďƵƚ͕ĂŌĞƌǀĂƌŝŽƵƐŶĂŵĞĐŚĂŶŐĞƐ͕ƚŚĞŶĂŵĞE. cloacaeǁĂƐĮŶĂůůLJĐŽŝŶĞĚŝŶϭϵϲϬ

7_{͘EŽŶĞƚŚĞůĞƐƐ͕ǁŚĞŶƐƚƵĚLJŝŶŐ͚E. cloacae͕͛ŝƚƐŚŽƵůĚďĞŶŽƚĞĚƚŚĂƚƚŚŝƐƐƉĞĐŝĞƐ}

ŝƐƋƵŝƚĞĚŝǀĞƌƐĞĂƐƵŶĚĞƌƐĐŽƌĞĚ ďLJDƵůƟ >ŽĐƵƐ ^ĞƋƵĞŶĐĞŶĂůLJƐŝƐ ĂŶĚ ,ƐƉϲϬ typing 8_{͘/ŶĐŽŶƚƌĂƐƚ͕ĚŝīĞƌĞŶƚE. cloacae ŝƐŽůĂƚĞƐĐĂŶŶŽƚďĞƌĞůŝĂďůLJĚŝƐƟŶŐƵŝƐŚĞĚ}

ďLJDĂƚƌŝdžͲƐƐŝƐƚĞĚ>ĂƐĞƌĞƐŽƌƉƟŽŶ/ŽŶŝnjĂƟŽŶDĂƐƐ^ƉĞĐƚƌŽŵĞƚƌLJ;D>/ͲdK& D^ͿŽƌϭϲƐƌZEƚLJƉŝŶŐϵ͕ϭϬ_{͘dŚŝƐŵĂŬĞƐĂƉƌĞĐŝƐĞĚŝƐƟŶĐƟŽŶŽĨE. cloacae isolates}

ĚŝĸĐƵůƚĨŽƌƚŚĞƌŽƵƟŶĞĚŝĂŐŶŽƐƟĐƉƌŽĐĞĚƵƌĞ͘/ŶĨĂĐƚ͕ƚŚŝƐĚŝĸĐƵůƚLJŚĂƐůĞĚƚŽ ŵƵůƟƉůĞƚĂdžŽŶŽŵLJĐŚĂŶŐĞƐƚŚĂƚƚŽŽŬƉůĂĐĞƐŝŶĐĞƚŚĞĚĞĮŶŝƟŽŶŽĨE. cloacae as a species in 19607_{ĂŶĚ͕ƚŽĚĂLJ͕ŝƚŝƐƉƌŽďĂďůLJŵŽƌĞĂƉƉƌŽƉƌŝĂƚĞƚŽƌĞĨĞƌƚŽƚŚĞ͚E.} cloacaeĐŽŵƉůĞdž͛͘ ZĞĐĞŶƚůLJ͕ ǁĞ ŝĚĞŶƟĮĞĚ Ă ĐĂƌďĂƉĞŶĞŵ ƌĞƐŝƐƚĂŶƚ ŝƐŽůĂƚĞ ďĞůŽŶŐŝŶŐ ƚŽ ƚŚĞ E. cloacaeĐŽŵƉůĞdž͕ǁŚŝĐŚŚĂĚƚŚĞƐĞƋƵĞŶĐĞƚLJƉĞ;^dͿϮϯϮĂŶĚǁŚŝĐŚĞdžƉƌĞƐƐĞĚ Ă ĐĞƉŚĂůŽƐƉŽƌŝŶĂƐĞ ǁŝƚŚ ĐĂƌďĂƉĞŶĞŵĂƐĞ ĂĐƟǀŝƚLJ 11_{͘ ĂƐĞĚ ŽŶ ŝƚƐ ǁŚŽůĞͲ} ŐĞŶŽŵĞƐĞƋƵĞŶĐĞ͕ǁĞƐƵďƐĞƋƵĞŶƚůLJƐƉĞĐŝĮĞĚƚŚŝƐŝƐŽůĂƚĞŵŽƌĞƉƌĞĐŝƐĞůLJĂƐE.

roggenkampii͘ůƚŚŽƵŐŚĐĂƌďĂƉĞŶĞŵƌĞƐŝƐƚĂŶĐĞŝƐƐƟůůƌĂƌĞŝŶE. cloacae and its

ĐůŽƐĞƌĞůĂƟǀĞƐ͕ƚŚĞŝĚĞŶƟĮĐĂƟŽŶŽĨƚŚŝƐƌĞƐŝƐƚĂŶĐĞƉŚĞŶŽƚLJƉĞŝŶE. roggenkampii ŝƐŚŝŐŚůLJǁŽƌƌŝƐŽŵĞŝŶǀŝĞǁŽĨƚŚĞĨĂĐƚƚŚĂƚďĂĐƚĞƌŝĂďĞůŽŶŐŝŶŐƚŽƚŚĞE. cloacae ĐŽŵƉůĞdžĐĂŶĐĂƵƐĞƐĞƌŝŽƵƐŝŶǀĂƐŝǀĞĚŝƐĞĂƐĞ͘dŚĞƌĞĨŽƌĞ͕ǁĞǁĂŶƚĞĚƚŽƉŝŶƉŽŝŶƚƚŽ

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ǁŚĂƚĞdžƚĞŶƚƚŚĞE. roggenkampii^dϮϯϮŝƐŽůĂƚĞĚŝīĞƌƐĨƌŽŵŽƚŚĞƌĐůŝŶŝĐĂůŝƐŽůĂƚĞƐi

belonging to the E. cloacae ĐŽŵƉůĞdž͘ dŽ ƚŚŝƐ ĞŶĚ͕ ǁĞ ĨŽůůŽǁĞĚ Ă ŵƵůƟͲŽŵŝĐƐ ĂƉƉƌŽĂĐŚ͕ ǁŚŝĐŚ ŚŝŐŚůŝŐŚƚƐ ƉĂƌƟĐƵůĂƌ ĚŝīĞƌĞŶĐĞƐ ďĞƚǁĞĞŶ ƚŚĞ ŝŶǀĞƐƟŐĂƚĞĚ E.

roggenkampii ^dϮϯϮŝƐŽůĂƚĞ͕ƚŚĞi E. cloacaeƚLJƉĞƐƚƌĂŝŶdϭϯϬϰϳ͕ĂŶĚŝƐŽůĂƚĞƐ

of the E. cloacae ĐŽŵƉůĞdž ƚŚĂƚ ǁĞƌĞ ĂƐƐŽĐŝĂƚĞĚ ǁŝƚŚ ŚƵŵĂŶ ďŽŶĞ ŝŶĨĞĐƟŽŶƐ͘ EŽƚĂďůLJ͕ƚŚĞůĂƩĞƌĐůŝŶŝĐĂůĂƐƐŽĐŝĂƟŽŶǁĂƐƵŶĞdžƉĞĐƚĞĚĂƚƚŚĞƟŵĞƚŚĞƐĞŝƐŽůĂƚĞƐ ǁĞƌĞŝĚĞŶƟĮĞĚďƵƚ͕ƚŽĚĂLJ͕ŝƐŽůĂƚĞƐďĞůŽŶŐŝŶŐƚŽƚŚĞE. cloacae complex are in ĨĂĐƚŝŶĐƌĞĂƐŝŶŐůLJŝŵƉůŝĐĂƚĞĚŝŶďŽŶĞŝŶĨĞĐƟŽŶƐ5_͘

Results and Discussion

WŚLJůŽŐĞŶĞƟĐĚŝīĞƌĞŶĐĞƐŝŶEnterobacter cloacae isolates

ůƚŽŐĞƚŚĞƌ͕ƐĞǀĞŶ͚E. cloacae͛ŝƐŽůĂƚĞƐĐŽůůĞĐƚĞĚĨƌŽŵƉĂƟĞŶƚƐĂƚƚŚĞhŶŝǀĞƌƐŝƚLJ DĞĚŝĐĂůĞŶƚĞƌ'ƌŽŶŝŶŐĞŶ͕ƚŚĞEĞƚŚĞƌůĂŶĚƐ͕ǁĞƌĞŝŶĐůƵĚĞĚŝŶƚŚĞƉƌĞƐĞŶƚƐƚƵĚLJ͕ ĂůŽŶŐ ǁŝƚŚ ƚŚĞ d ϭϯϬϰϳ E. cloacae ƚLJƉĞ ƐƚƌĂŝŶ͘ &ŝǀĞ ŽĨ ƚŚĞƐĞ ƐƚƌĂŝŶƐ ǁĞƌĞ ŝƐŽůĂƚĞĚ ĨƌŽŵ ďŽŶĞ ŝŶĨĞĐƟŽŶƐ͕ ǁŚŝůĞ ƚǁŽ ŽƚŚĞƌ ŝƐŽůĂƚĞƐ ǁĞƌĞ ŽďƚĂŝŶĞĚ ĨƌŽŵ ŶĞŽŶĂƚĂůƌĞĐƚĂůƐǁĂďƐ͘KŶĞŽĨƚŚĞůĂƩĞƌƚǁŽŝƐŽůĂƚĞƐǁĂƐƉƌĞǀŝŽƵƐůLJĐŚĂƌĂĐƚĞƌŝnjĞĚ as E͘ roggenkampii͕ ĂŶĚ ĨŽƵŶĚ ƚŽ ďĞ ƌĞƐŝƐƚĂŶƚ ƚŽ ĐĂƌďĂƉĞŶĞŵƐ͘ dŚĞ ŽƚŚĞƌ ĐůŝŶŝĐĂůŝƐŽůĂƚĞƐƐŚŽǁĞĚŝŶƐŝŐŶŝĮĐĂŶƚĂŶƟďŝŽƟĐƌĞƐŝƐƚĂŶĐĞůĞǀĞůƐ;^ƵƉƉůĞŵĞŶƚĂƌLJ dĂďůĞ^ϭͿ͘^ŝŶĐĞƚŚĞĚŝƐƟŶĐƟŽŶŽĨƐƉĞĐŝĞƐďĞůŽŶŐŝŶŐƚŽƚŚĞE. cloacae complex ŝƐĚŝĸĐƵůƚ͕ǁĞĚĞĐŝĚĞĚƚŽĐŚĂƌĂĐƚĞƌŝnjĞƚŚĞƐĞůĞĐƚĞĚĐůŝŶŝĐĂůŝƐŽůĂƚĞƐďLJǁŚŽůĞͲ ŐĞŶŽŵĞƐĞƋƵĞŶĐŝŶŐ͕ƵƐŝŶŐĂŚLJďƌŝĚĂƐƐĞŵďůLJĂƉƉƌŽĂĐŚƚŚĂƚĐŽŵďŝŶĞĚƐŚŽƌƚͲ ĂŶĚ ůŽŶŐͲƌĞĂĚ ƐĞƋƵĞŶĐŝŶŐ ĚĂƚĂ͘ EĞdžƚ͕ ƚŚĞ ƚĂdžŽŶŽŵŝĐĂů ĚŝƐƚĂŶĐĞ ďĞƚǁĞĞŶ ƚŚĞ ƐĞůĞĐƚĞĚ ŝƐŽůĂƚĞƐ ǁĂƐ ĂƐƐĞƐƐĞĚ ďLJ ƉŚLJůŽŐĞŶĞƟĐ ĂŶĂůLJƐĞƐ͕ ǁŚŝĐŚ ŝŶĐůƵĚĞĚ ϯϲ ƉƵďůŝĐůLJ ĂǀĂŝůĂďůĞ ŐĞŶŽŵĞ ƐĞƋƵĞŶĐĞƐ ŽĨ ĚŝīĞƌĞŶƚ ƐƉĞĐŝĞƐ ďĞůŽŶŐŝŶŐ ƚŽ ƚŚĞ E. cloacae ĐŽŵƉůĞdž;^ƵƉƉůĞŵĞŶƚĂƌLJĂƚĂ^ϭͿ͘dŚĞŶĞŝŐŚďŽƌũŽŝŶŝŶŐƚƌĞĞďĂƐĞĚŽŶ ƚŚĞǁŚŽůĞͲŐĞŶŽŵĞƐĞƋƵĞŶĐĞƐŝƐƉƌĞƐĞŶƚĞĚŝŶ&ŝŐƵƌĞϭ͘/ŵƉŽƌƚĂŶƚůLJ͕ƚŚŝƐĂŶĂůLJƐŝƐ ƐŚŽǁĞĚƚŚĂƚƚŚĞƐĞůĞĐƚĞĚ͚E. cloacaeDzƐƚƵĚLJŝƐŽůĂƚĞƐďĞůŽŶŐƚŽĚŝīĞƌĞŶƚƐƉĞĐŝĞƐ ĂŶĚ ƐƵďͲƐƉĞĐŝĞƐ͘ /Ŷ ƉĂƌƟĐƵůĂƌ͕ ƚŚĞ ŝƐŽůĂƚĞƐ ϲϭϮϬϮ ĂŶĚ ϱϱϲϬϮ͕ ƌĞƐƉĞĐƟǀĞůLJ ĂƐƐŽĐŝĂƚĞĚ ǁŝƚŚ ƵƉƉĞƌ Ăƌŵ ĂŶĚ ŚĞĞů ďŽŶĞ ŝŶĨĞĐƟŽŶƐ͕ ĂƌĞ Enterobacter hormaechei subsp͘ƐƚĞŝŐĞƌǁĂůƟŝ ĂŶĚƌĞƉƌĞƐĞŶƚĂƌĞůĂƟǀĞůLJƌĞĐĞŶƚůLJĚŝƐĐŽǀĞƌĞĚŝ

subspecies of Enterobacter hormaechei͘dŚĞŝƐŽůĂƚĞϰϰϭϬϭ͕ǁŚŝĐŚǁĂƐĂƐƐŽĐŝĂƚĞĚ ǁŝƚŚĂŚĞĞůďŽŶĞŝŶĨĞĐƟŽŶ͕ĐůƵƐƚĞƌĞĚƚŽŐĞƚŚĞƌǁŝƚŚƚŚĞE. cloacae type strain

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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97

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dϭϯϬϰϳ͕ĂŶĚŝƚǁĂƐƚŚĞƌĞĨŽƌĞŝĚĞŶƟĮĞĚĂƐE. cloacae subsp. cloacae͘dŚĞ ŝƐŽůĂƚĞϵϵϭϬϭ͕ĂƐƐŽĐŝĂƚĞĚǁŝƚŚĂƟďŝĂŝŶĨĞĐƟŽŶ͕ĐůƵƐƚĞƌĞĚǁŝƚŚŵĞŵďĞƌƐŽĨƚŚĞ E.

cloacaeĐŽŵƉůĞdžĐůĂĚĞ^ͺEϮϮ͘dŚĞŝƐŽůĂƚĞϴϴϳϬϭ͕ǁŚŝĐŚǁĂƐĚĞƌŝǀĞĚĨƌŽŵŇƵŝĚ

ĐŽůůĞĐƚĞĚĨƌŽŵĂŚŝƉƌĞŐŝŽŶ͕ĐůƵƐƚĞƌĞĚƚŽŐĞƚŚĞƌǁŝƚŚŵĞŵďĞƌƐŽĨƚŚĞE. cloacae ĐŽŵƉůĞdžĐůĂĚĞ^ͺ'EϬϲϮϯϮ͘>ĂƐƚůLJ͕ƚŚĞŐƵƚŝƐŽůĂƚĞϭϰϭϬϮϰ<ǁĂƐŝĚĞŶƟĮĞĚĂƐ E.

hormaechei ƐƵďƐƉ͘ ŚŽīŵĂŶŶŝŝ͘ƐƉƌĞǀŝŽƵƐůLJƐŚŽǁŶ͕ƚŚĞĂůƌĞĂĚLJĐŚĂƌĂĐƚĞƌŝnjĞĚ

carbapenem resistant E. roggenkampiiŝƐŽůĂƚĞ;ϯϯϵϯϴϵ>ͿǁĂƐŐƌŽƵƉĞĚƚŽŐĞƚŚĞƌi

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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98 Z  Ă ƌďĂƉĞŶĞŵ su scep tib ilit y ^ ^ ^ ^ ^ ^ ^ Z = ZĞ Ɛ ŝƐ ƚĂ Ŷƚ ^ = ^ Ƶ Ɛ Đ Ğ Ɖ ƚŝ ď ůĞ d LJ Ɖ Ğ s tr a in 'Ƶ ƚ 'Ƶ ƚ Bo n e Bo n e Bo n e Bo n e Bo n e be lo ng in g t o t he E. cloac ae ĐŽ ŵ ƉůĞ dž͕ ĂŶĚ ƚŚ Ğ E. cloac ae ƚLJƉĞ Ɛƚƌ ĂŝŶ d  ϭϯϬϰϳ͘  ŶĞŝ ŐŚď Žƌ ũŽŝŶŝŶ Őƚƌ ĞĞ ǁ ĂƐ ĐŽŶƐ ƚƌƵĐƚ ĞĚ ďĂƐĞĚ ŽŶ ƚŚ Ğ ŐĞŶŽŵĞ ƐĞƋƵĞŶĐĞƐ ŽĨ ƚŚĞ ƐƚƵĚLJ ŝƐŽůĂ ƚĞƐ͕ ƚŚĞ E. cloac ae ƚLJƉĞ Ɛƚƌ ĂŝŶ ĂŶĚ ƌĞƉƌ ĞƐĞŶ ƚĂƟǀ Ğ ƉƵďůŝĐůLJ Ăǀ ĂŝůĂďůĞ ŐĞŶŽŵĞ ƐĞƋƵĞŶĐĞƐ ŽĨ ďĂĐƚ ĞƌŝĂ be lo ng in g t o t he E. cloac ae ĐŽ ŵ ƉůĞ džƵ ƐŝŶ ŐZŝĚŽŵ ^ĞƋ^ƉŚĞƌ Ğн ǀĞƌ ƐŝŽŶ ϱ͘ϭ͘Ϭ͘ dŚĞ ƚƌ ĞĞ Śŝ ŐŚůŝ ŐŚ ƚƐ ƚŚĞ Ěŝ īĞƌ ĞŶ ƚĐůĂĚĞƐ ďĞůŽŶ ŐŝŶ Őƚ Žƚ ŚĞ E. cloac ae ĐŽŵƉůĞ dž͘ dŚĞ ƌĞƐŝƐ ƚĂŶĐĞ ;ZͿ Žƌ ƐƵƐĐĞƉƟďŝůŝƚLJ ;^Ϳ ƚŽ ĐĂƌďĂƉĞŶĞŵ ƐŽ Ĩƚ ŚĞ Ɛƚ ƵĚ LJŝƐ ŽůĂ ƚĞ ƐŝƐ ŝŶ ĚŝĐ ĂƚĞ Ě͘ dŚ ĞƐ ĐĂ ůĞ ďĂ ƌŝŶ ĚŝĐ ĂƚĞ Ɛƚ ŚĞ ŐĞŶĞ ƟĐĚŝǀ Ğƌ ŐĞŶĐĞƌ ĞůĞǀ ĂŶ ƚƚ Žďƌ ĂŶĐŚůĞŶ ŐƚŚĂŶĚŝƐďĂƐĞĚŽŶƵŶŝƚƐŽĨƚŚĞŶƵŵďĞƌŽĨŶƵĐůĞŽƟĚĞĚŝ īĞƌ ĞŶĐĞƐƉĞƌƐŝƚ ĞĂĐƌ ŽƐƐϳϵϰůŽĐŝ͘

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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99

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ŽŵƉĂƌĂƟǀĞ ƉƌŽƚĞŽŵĞ ĂŶĂůLJƐŝƐ ĚŝīĞƌĞŶƟĂƚĞƐ ƚŚĞ ĐĂƌďĂƉĞŶĞŵ ƌĞƐŝƐƚĂŶƚ E. roggenkampiiŐƵƚŝƐŽůĂƚĞĨƌŽŵŝƐŽůĂƚĞƐŝŵƉůŝĐĂƚĞĚŝŶďŽŶĞŝŶĨĞĐƟŽŶƐi ŽƚŚƚŚĞĐĂƌďĂƉĞŶĞŵƌĞƐŝƐƚĂŶĐĞĂŶĚƚŚĞĂƐƐŽĐŝĂƟŽŶǁŝƚŚďŽŶĞŝŶĨĞĐƟŽŶƐǁĞƌĞ ƵŶĞdžƉĞĐƚĞĚŽďƐĞƌǀĂƟŽŶƐĨŽƌŝƐŽůĂƚĞƐĨƌŽŵƚŚĞ E. cloacaeĐŽŵƉůĞdž͘tĞƚŚĞƌĞĨŽƌĞ ĂƐŬĞĚƚŚĞƋƵĞƐƟŽŶǁŚĞƚŚĞƌƚŚĞƌĞƐƉĞĐƟǀĞŝƐŽůĂƚĞƐŵŝŐŚƚƉƌĞƐĞŶƚĚŝīĞƌĞŶĐĞƐ ŝŶƚŚĞŝƌƉƌŽƚĞŝŶĞdžƉƌĞƐƐŝŽŶƉƌŽĮůĞƐƚŚĂƚĐĂŶŶŽƚďĞŝŶĨĞƌƌĞĚĨƌŽŵƚŚĞŐĞŶŽŵĞ ƐĞƋƵĞŶĐĞ͘dŽƚŚŝƐĞŶĚ͕ƚŚĞƐĞǀĞŶĐůŝŶŝĐĂůŝƐŽůĂƚĞƐĂƐǁĞůůĂƐƚŚĞE. cloacae type ƐƚƌĂŝŶdϭϯϬϰϳǁĞƌĞŐƌŽǁŶŝŶ,/ďƌŽƚŚ͕ŚĂƌǀĞƐƚĞĚĂƚƚŚĞƐĂŵĞŐƌŽǁƚŚƐƚĂŐĞ͕ ĂŶĚƐƵďũĞĐƚĞĚƚŽƉƌŽƚĞŽŵĞĂŶĂůLJƐŝƐ͘dŚŝƐůĞĚƚŽƚŚĞŝĚĞŶƟĮĐĂƟŽŶŽĨϵϳϵƉƌŽƚĞŝŶƐ ŝŶƚŽƚĂů;^ƵƉƉůĞŵĞŶƚĂƌLJdĂďůĞ^ϮͿ͘ZŽƵŐŚůLJϮϱйŽĨƚŚĞŝĚĞŶƟĮĞĚƉƌŽƚĞŝŶƐǁĞƌĞ ĚĞƚĞĐƚĞĚ ŝŶ Ăůů ŝŶǀĞƐƟŐĂƚĞĚ ŝƐŽůĂƚĞƐ͕ ƚŚƵƐ ƌĞƉƌĞƐĞŶƟŶŐ ƚŚĞŝƌ ĐŽƌĞ ƉƌŽƚĞŽŵĞ ;&ŝŐƵƌĞ ϮͿ͘ LJ ĂŶĂůLJnjŝŶŐ ůƵƐƚĞƌƐ ŽĨ KƌƚŚŽůŽŐŽƵƐ 'ĞŶĞƐ ;K'Ϳ ĐĂƚĞŐŽƌŝĞƐ ĂŶĚ ĨƵŶĐƟŽŶƐďĂƐĞĚŽŶ'ĞŶĞKŶƚŽůŽŐLJ;'KͿ͕ŝƚǁĂƐĨŽƵŶĚƚŚĂƚƚŚĞůĂƌŐĞƐƚĐĂƚĞŐŽƌŝĞƐ ŽĨŝĚĞŶƟĮĞĚƉƌŽƚĞŝŶƐŽĨƚŚĞĐŽƌĞƉƌŽƚĞŽŵĞĂƌĞƌĞůĂƚĞĚƚŽƚƌĂŶƐůĂƟŽŶ͕ƌŝďŽƐŽŵĂů ƐƚƌƵĐƚƵƌĞ ĂŶĚ ďŝŽŐĞŶĞƐŝƐ͕ ĞŶĞƌŐLJ ƉƌŽĚƵĐƟŽŶ ĂŶĚ ĐŽŶǀĞƌƐŝŽŶ͕ ĂŵŝŶŽ ĂĐŝĚ ƚƌĂŶƐƉŽƌƚĂŶĚŵĞƚĂďŽůŝƐŵ͕ĐĂƌLJĚƌĂƚĞƚƌĂŶƐƉŽƌƚĂŶĚŵĞƚĂďŽůŝƐŵ͕ĂŶĚƉŽƐƚͲ ƚƌĂŶƐůĂƟŽŶĂů ŵŽĚŝĮĐĂƟŽŶ͕ ƉƌŽƚĞŝŶ ƚƵƌŶŽǀĞƌ ĂŶĚ ĐŚĂƉĞƌŽŶĞƐ ;^ƵƉƉůĞŵĞŶƚĂƌLJ dĂďůĞ^ϯͿ͘/ŵƉŽƌƚĂŶƚůLJ͕ĨŽƌŝŶĚŝǀŝĚƵĂůŝƐŽůĂƚĞƐĂĚĚŝƟŽŶĂůƉƌŽƚĞŝŶƐǁĞƌĞŝĚĞŶƟĮĞĚ ƚŚĂƚǁĞƌĞƐŚĂƌĞĚǁŝƚŚĂůŝŵŝƚĞĚŶƵŵďĞƌŽĨŽƚŚĞƌŝƐŽůĂƚĞƐ͕ŽƌƚŚĂƚǁĞƌĞƵŶŝƋƵĞ ĨŽƌƚŚĞƌĞƐƉĞĐƟǀĞŝƐŽůĂƚĞ;&ŝŐƵƌĞϮͿ͘

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

Processed on: 13-11-2019 PDF page: 100PDF page: 100PDF page: 100PDF page: 100

100 &ŝŐƵƌĞ Ϯ͘ sĞŶŶ ĚŝĂŐƌĂŵ ƐŚŽǁŝŶŐ ƚŚĞ ŶƵŵďĞƌƐ ŽĨ ƵŶŝƋƵĞůLJ ĂŶĚ ĐŽŵŵŽŶůLJ ŝĚĞŶƟĮĞĚ ƉƌŽƚĞŝŶƐŽĨƚŚĞƐĞǀĞŶƐƚƵĚLJŝƐŽůĂƚĞƐďĞůŽŶŐŝŶŐƚŽƚŚĞE. cloacaeĐŽŵƉůĞdž͕ĂŶĚŽĨƚŚĞ E. cloacaedϭϯϬϰϳƚLJƉĞƐƚƌĂŝŶ͘&ŽƌƚŚŝƐĐŽŵƉĂƌŝƐŽŶ͕ƚŚĞϳϵϲƉƌŽƚĞŝŶƐŝĚĞŶƟĮĞĚŝŶ ŝƐŽůĂƚĞƐĂƐƐŽĐŝĂƚĞĚǁŝƚŚďŽŶĞŝŶĨĞĐƟŽŶƐǁĞƌĞŝŶĐůƵĚĞĚĂƐŽŶĞŐƌŽƵƉŵĂƌŬĞĚĂƐ͚ƚŽƚĂů ďŽŶĞ͛͘ dŽ ĂƉƉƌŽdžŝŵĂƚĞ ƉŽƚĞŶƟĂůůLJ ƐƉĞĐŝĮĐ ƉƌŽƚĞŝŶ ĨĞĂƚƵƌĞƐ ŽĨ ŐƌŽƵƉƐ ŽĨ ŝŶǀĞƐƟŐĂƚĞĚ ŝƐŽůĂƚĞƐ͕ ŝ͘Ğ͘ ƚŚĞ ĮǀĞ ďŽŶĞ ŝŶĨĞĐƟŽŶͲƌĞůĂƚĞĚ ŝƐŽůĂƚĞƐ versus the carbapenem resistant E. roggenkampii ŝƐŽůĂƚĞ;ϯϯϵϯϴϵ>Ϳ͕ƚŚĞŐƵƚŝƐŽůĂƚĞϭϰϭϬϮϰ<͕ŽƌƚŚĞdi ϭϯϬϰϳ ƚLJƉĞ ƐƚƌĂŝŶ͕ ƚŚĞ ŝĚĞŶƟĮĞĚ ƉƌŽƚĞŝŶƐ ŽĨ ĞĂĐŚ ŐƌŽƵƉ ĂŶĚ ƚŚĞŝƌ ĂŶŶŽƚĂƚĞĚ ĨƵŶĐƟŽŶƐǁĞƌĞĐŽŵƉĂƌĞĚ;^ƵƉƉůĞŵĞŶƚĂůdĂďůĞ^ϯͿ͘dŚŝƐƵŶǀĞŝůĞĚƚŚĞƉƌŽƚĞŝŶƐƚŚĂƚ ǁĞƌĞƵŶŝƋƵĞĨŽƌƚŚĞƌĞƐƉĞĐƟǀĞŐƌŽƵƉƐ͕ĂƚůĞĂƐƚƵŶĚĞƌƚŚĞƉƌĞƐĞŶƚƐƚĂŶĚĂƌĚŝnjĞĚ ĞdžƉĞƌŝŵĞŶƚĂůŐƌŽǁƚŚĐŽŶĚŝƟŽŶƐ͕ǁŚŝĐŚĂƌĞĚŝīĞƌĞŶƚĨƌŽŵƚŚĞĐŽŶĚŝƟŽŶƐĂƚƚŚĞ ƌĞƐƉĞĐƟǀĞƐŝƚĞƐŽĨŝƐŽůĂƟŽŶ͘/ƚƐŚŽƵůĚďĞŶŽƚĞĚƚŚĂƚŵĂŶLJŽĨƚŚĞƐĞƵŶŝƋƵĞƉƌŽƚĞŝŶƐ ĂƌĞƌĞůĂƚĞĚƚŽďĂĐƚĞƌŝĂůŵĞƚĂďŽůŝƐŵ͘,ŽǁĞǀĞƌ͕ĐĞƌƚĂŝŶĨĞĂƚƵƌĞƐǁĞƌĞĐŚĂƌĂĐƚĞƌŝƐƟĐ ĨŽƌ ƉĂƌƟĐƵůĂƌ ŝƐŽůĂƚĞƐ͘ dŚƵƐ͕ ĨŽƌ ƚŚĞ ĐĂƌďĂƉĞŶĞŵ ƌĞƐŝƐƚĂŶƚ E. roggenkampii ŝƐŽůĂƚĞ͕ƚŚĞŵƉͲƚLJƉĞɴͲůĂĐƚĂŵĂƐĞƌĞƐƉŽŶƐŝďůĞĨŽƌƚŚĞŽďƐĞƌǀĞĚĐĂƌďĂƉĞŶĞŵ ƌĞƐŝƐƚĂŶĐĞƐƚŽŽĚŽƵƚĂŵŽŶŐƚŚĞůŝƐƚŽĨŝĚĞŶƟĮĞĚƉƌŽƚĞŝŶƐ͕ĂŶĚƚŚĞƐĂŵĞǁĂƐƚƌƵĞ ĨŽƌƐĞǀĞƌĂůƉƌŽƚĞŝŶƐŝŶǀŽůǀĞĚŝŶĐŽƉƉĞƌŚŽŵĞŽƐƚĂƐŝƐ͘dŚĞƐĞƉƌŽƚĞŝŶƐǁĞƌĞŶĞŝƚŚĞƌ detectable in the E. cloacae ƚLJƉĞƐƚƌĂŝŶ͕ƚŚĞĐĂƌďĂƉĞŶĞŵƐƵƐĐĞƉƟďůĞŐƵƚŝƐŽůĂƚĞ͕ ŶŽƌƚŚĞŝƐŽůĂƚĞƐƌĞůĂƚĞĚƚŽďŽŶĞŝŶĨĞĐƟŽŶ;^ƵƉƉůĞŵĞŶƚĂůdĂďůĞ^ϯͿ͘KŶƚŚĞŽƚŚĞƌ

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

Processed on: 13-11-2019 PDF page: 101PDF page: 101PDF page: 101PDF page: 101

101

ŚĂƉƚ

ĞƌϦ

ŚĂŶĚ͕ ƚŚĞ ůĂƩĞƌ ŐƌŽƵƉ ŽĨ ŝƐŽůĂƚĞƐ ƵŶŝƋƵĞůLJ ĨĞĂƚƵƌĞĚ ǀĂƌŝŽƵƐ ŇĂŐĞůůĂƌ ƉƌŽƚĞŝŶƐ ŝŶǀŽůǀĞĚŝŶďĂĐƚĞƌŝĂůŵŽƟůŝƚLJ;&ůŝ͕&ůŝ͕&ůŝ'͕&ůŐ<Ϳ͕ĂĮŵďƌŝĂůƉƌŽƚĞŝŶŝŶǀŽůǀĞĚŝŶ ĂĚŚĞƌĞŶĐĞƚŽƐƵƌĨĂĐĞƐĂŶĚƟƐƐƵĞƐ;^ĨŵͿ͕ƉƌŽƚĞŝŶƐŝŶǀŽůǀĞĚŝŶƚLJƉĞs/ƐĞĐƌĞƟŽŶ ƐLJƐƚĞŵƐ;ǀƉ͕/ŵƉ͕/ŵƉ͕/ŵƉͿĂŶĚŬŶŽǁŶĞīĞĐƚŽƌƉƌŽƚĞŝŶƐƚŚĂƚŚĂǀĞďĞĞŶ ŝŵƉůŝĐĂƚĞĚŝŶďĂĐƚĞƌŝĂůǀŝƌƵůĞŶĐĞ;/ŵƉ͕DǀŝDͿ͘/ŶƉĂƌƟĐƵůĂƌ͕ŐŝǀĞŶƚŚĞĐůŝŶŝĐĂů ƉƌĞƐĞŶƚĂƟŽŶŽĨƚŚĞŝƐŽůĂƚĞƐĂƐƐŽĐŝĂƚĞĚǁŝƚŚďŽŶĞŝŶĨĞĐƟŽŶƐ͕ƚŚĞŝĚĞŶƟĮĐĂƟŽŶ ŽĨ ƚLJƉĞ s/ ƐĞĐƌĞƟŽŶͲƌĞůĂƚĞĚ ƉƌŽƚĞŝŶƐ ĂŶĚ ĞīĞĐƚŽƌƐ͕ ǁŚŝĐŚ ĂƌĞ ŬŶŽǁŶ ƚŽ ďĞ ĂƐƐŽĐŝĂƚĞĚǁŝƚŚǀŝƌƵůĞŶĐĞŝƐǀĞƌLJŝŶƚƌŝŐƵŝŶŐ͘zĞƚ͕ǁŚŝůĞƚLJƉŝĐĂůĨŽƌƚŚĞŝŶǀĞƐƟŐĂƚĞĚ ŝƐŽůĂƚĞƐ͕ŝƚŚĂƐƚŽďĞŶŽƚĞĚŚĞƌĞƚŚĂƚƚŚĞƐĞǀŝƌƵůĞŶĐĞͲƌĞůĂƚĞĚƉƌŽƚĞŝŶƐĂƌĞŶŽƚ ƌĞƉƌĞƐĞŶƚĞĚŝŶĂůůŝŶĚŝǀŝĚƵĂůŝƐŽůĂƚĞƐĂƐƐŽĐŝĂƚĞĚǁŝƚŚďŽŶĞŝŶĨĞĐƟŽŶƐ͕ďƵƚƌĂƚŚĞƌ ĂƌĞĞdžƉƌĞƐƐĞĚďLJĂƚůĞĂƐƚŽŶĞŽĨƚŚĞĮǀĞƌĞƐƉĞĐƟǀĞŝƐŽůĂƚĞƐ;^ƵƉƉůĞŵĞŶƚĂůdĂďůĞ ^ϯͿϴ͕ϲ_{͘dŽƌĞĐŽŶĐŝůĞƚŚĞƐĞŽďƐĞƌǀĂƟŽŶƐǁŝƚŚƚŚĞŐĞŶŽŵĞƐĞƋƵĞŶĐĞƐ͕ǁĞŝŶƐƉĞĐƚĞĚ} ƚŚĞŽĐĐƵƌƌĞŶĐĞŽĨŇĂŐĞůůĂƌĂŶĚƚLJƉĞs/ƐĞĐƌĞƟŽŶƌĞůĂƚĞĚŐĞŶĞƐŝŶƚŚĞŐĞŶŽŵĞƐ ŽĨƚŚĞŝŶǀĞƐƟŐĂƚĞĚŝƐŽůĂƚĞƐ͘dŚŝƐƐŚŽǁĞĚƚŚĂƚƚŚĞLJĐŽŶƚĂŝŶĞĚƚǁŽƚŽƚŚƌĞĞƚLJƉĞ ///ƐĞĐƌĞƟŽŶƐLJƐƚĞŵƐĨŽƌŇĂŐĞůůĂƌďŝŽŐĞŶĞƐŝƐ͕ĂŶĚƚǁŽƚŽƚŚƌĞĞƚLJƉĞs/ƐĞĐƌĞƟŽŶ ƐLJƐƚĞŵƐ ;dĂďůĞ ϭͿ͘ Ɛ ĞdžĞŵƉůŝĮĞĚ ĨŽƌ ŝƐŽůĂƚĞ ϵϵϭϬϭ͕ ƚŚĞ ƌĞƐƉĞĐƟǀĞ ŐĞŶĞƐ ĂƌĞ ůŽĐĂƚĞĚĂƚĚŝīĞƌĞŶƚĐŚƌŽŵŽƐŽŵĂůƉŽƐŝƟŽŶƐ;&ŝŐƵƌĞϯͿ͘&ƵƌƚŚĞƌŵŽƌĞ͕ƚŚĞŐĞŶĞƐ ĨŽƌƚŚĞŝĚĞŶƟĮĞĚƚLJƉĞ///ĂŶĚƚLJƉĞs/ƐLJƐƚĞŵƐĂƌĞĂůůŝŶƚĞŐƌĂƚĞĚďĞƚǁĞĞŶƚZEͲ ĞŶĐŽĚŝŶŐ ŐĞŶĞƐ ĂƐ ƐƉĞĐŝĮĞĚ ŝŶ dĂďůĞ ϭ͕ ǁŚŝĐŚ ŝƐ ƐƵŐŐĞƐƟǀĞ ŽĨ ĂĐƋƵŝƐŝƟŽŶ ďLJ ŚŽƌŝnjŽŶƚĂůŐĞŶĞƚƌĂŶƐĨĞƌ͘/ƚƐŚŽƵůĚďĞŶŽƚĞĚ͕ŚŽǁĞǀĞƌ͕ƚŚĂƚƚŚĞƚZEŐĞŶĞƐĂƌĞ ŶŽƚĚŝƌĞĐƚůLJŇĂŶŬŝŶŐƚŚĞƐĞĐƌĞƟŽŶƐLJƐƚĞŵƐ͘

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

Processed on: 13-11-2019 PDF page: 102PDF page: 102PDF page: 102PDF page: 102

102

dĂďůĞϭ͘/ĚĞŶƟĮĞĚƚLJƉĞ///ĂŶĚƚLJƉĞs/ƐĞĐƌĞƟŽŶƐLJƐƚĞŵƐŝŶƚŚĞŝŶǀĞƐƟŐĂƚĞĚŝƐŽůĂƚĞƐ belonging to the E. cloacaeĐŽŵƉůĞdž͘

ďŽŶĞŝŶĨĞĐƟŽŶŝƐŽůĂƚĞƐ Type strain and gut isolates

/ĚĞŶƟĮĞĚůŽĐŝĨŽƌ type III or type VI ƐĞĐƌĞƟŽŶƐLJƐƚĞŵƐ ĂŶĚŇĂŶŬŝŶŐƚZE genes 99101 88701 55602 61202 44101 ATCC 13047 141024K 339389L ;>ĞƵͲͿdϯ^^;>ĞƵͲ'Ϳ н н Ͳ Ͳ Ͳ Ͳ Ͳ Ͳ ;^ĞƌͲ''Ϳdϯ^^;^ĞƌͲ''Ϳ н н Ͳ Ͳ Ͳ Ͳ Ͳ Ͳ ;'ůLJͲ'Ϳdϯ^^;ƐŶͲ''dͿ н н н Ͳ н Ͳ Ͳ н ;dŚƌͲd'dͿdϯ^^;'ůLJͲ'Ϳ Ͳ Ͳ Ͳ Ͳ Ͳ н Ͳ Ͳ ;^ĞƌͲ''Ϳdϯ^^;sĂůͲ'Ϳ Ͳ Ͳ н н н н н Ͳ ;ƐŶͲ''dͿdϯ^^;ƐŶͲ''dͿ Ͳ Ͳ Ͳ Ͳ Ͳ н н Ͳ ;dLJƌͲ'dͿdϯ^^;>ĞƵͲͿ Ͳ Ͳ н Ͳ Ͳ Ͳ Ͳ Ͳ ;'ůƵͲdͿdϯ^^;ƌŐͲ'Ϳ Ͳ Ͳ Ͳ н Ͳ Ͳ Ͳ Ͳ ;WŚĞͲ'Ϳdϯ^^;ƐŶͲ''dͿ Ͳ Ͳ Ͳ н Ͳ Ͳ Ͳ Ͳ ;ƌŐͲddͿdϲ^^;dLJƌͲ'dͿ Ͳ н Ͳ Ͳ Ͳ Ͳ Ͳ Ͳ ;dLJƌͲ'dͿdϲ^^;>ĞƵͲdͿ н н н н н н Ͳ н ;WŚĞͲ'Ϳdϲ^^;DĞƚͲdͿ н н Ͳ Ͳ Ͳ Ͳ Ͳ Ͳ ;sĂůͲ'Ϳdϲ^^;dLJƌͲ'dͿ Ͳ Ͳ н н Ͳ н н Ͳ ;^ĞƌͲ''Ϳdϲ^^;sĂůͲ'Ϳ Ͳ Ͳ Ͳ Ͳ н Ͳ н Ͳ ;'ůƵͲddͿdϲ^^;dŚƌͲ''dͿ Ͳ Ͳ Ͳ Ͳ Ͳ Ͳ Ͳ н н͕ŝĚĞŶƟĮĞĚ͖Ͳ͕ĂďƐĞŶƚ͖dϯ^^͕ƚLJƉĞ///ƐĞĐƌĞƟŽŶƐLJƐƚĞŵ͖dϲ^^͕ƚLJƉĞs/ƐĞĐƌĞƟŽŶƐ

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

Processed on: 13-11-2019 PDF page: 103PDF page: 103PDF page: 103PDF page: 103

ϭϬϯ

ŚĂƉƚ

ĞƌϦ

&ŝŐƵƌĞϯ͘^ĐŚĞŵĂƟĐƌĞƉƌĞƐĞŶƚĂƟŽŶŽĨƚŚĞĐŚƌŽŵŽƐŽŵĂůůŽĐĂůŝnjĂƟŽŶŽĨŐĞŶĞƐĞŶĐŽĚŝŶŐ ƚLJƉĞ /// ĂŶĚ ƚLJƉĞ sŝ ƐĞĐƌĞƟŽŶ ƐLJƐƚĞŵƐ ŝŶ ƐƚƵĚLJ ŝƐŽůĂƚĞ ϵϵϭϬϭ͘ dŚĞ ƐĞĐƌĞƟŽŶ ƐLJƐƚĞŵƐ ǁĞƌĞĚĞƚĞĐƚĞĚǁŝƚŚƚŚĞŽŶůŝŶĞƚŽŽůdϯϰϲ,ƵŶƚĞƌ12_{͘dŚĞŽƌĂŶŐĞďŽdžĞƐƌĞƉƌĞƐĞŶƚƚLJƉĞs/} ƐĞĐƌĞƟŽŶƐLJƐƚĞŵƐĂŶĚƚŚĞďůƵĞďŽdžĞƐƌĞƉƌĞƐĞŶƚŇĂŐĞůůĂƌƚLJƉĞ///ƐĞĐƌĞƟŽŶƐLJƐƚĞŵƐ͘/Ŷ ŐƌĞĞŶ͕ƚŚĞƚZEŐĞŶĞƐĂƌĞƐŚŽǁŶ͘ Conclusion ůƚŽŐĞƚŚĞƌ͕ ƚŚĞ ŚĞƌĞ ƉƌĞƐĞŶƚĞĚ ƐƚƵĚLJ ŚŝŐŚůŝŐŚƚƐ ƚŚĞ ĚŝǀĞƌƐŝƚLJ ŽĨ ďĂĐƚĞƌŝĂ belonging to the E. cloacae ĐŽŵƉůĞdž͕ďŽƚŚĂƚƚŚĞŐĞŶŽŵŝĐĂŶĚƉƌŽƚĞŽŵŝĐůĞǀĞůƐ͘ dŚŝƐĚŝǀĞƌƐŝƚLJŝƐƐĞĞŵŝŶŐůLJƌĞŇĞĐƚĞĚŝŶƚŚĞŝƌĐůŝŶŝĐĂůƉƌĞƐĞŶƚĂƟŽŶ͘/ƚƐŚŽƵůĚďĞ ŶŽƚĞĚƚŚĂƚ͕ůŝŬĞƚŚĞŝŶǀĞƐƟŐĂƚĞĚĐĂƌďĂƉĞŶĞŵƌĞƐŝƐƚĂŶƚE. roggenkampii ŝƐŽůĂƚĞ͕i

ĂůůŽƚŚĞƌŝŶǀĞƐƟŐĂƚĞĚŝƐŽůĂƚĞƐĐĂƌƌLJampC ƚLJƉĞĐĞƉŚĂůŽƐƉŽƌŝŶĂƐĞŐĞŶĞƐ͘,ŽǁĞǀĞƌ͕C

ĐŽŶƚƌĂƌLJƚŽƚŚĞĐĂƌďĂƉĞŶĞŵƌĞƐŝƐƚĂŶƚŝƐŽůĂƚĞ͕ƚŚĞŽƚŚĞƌŝŶǀĞƐƟŐĂƚĞĚŝƐŽůĂƚĞƐĚŽ ŶŽƚƐĞĞŵƚŽĞdžƉƌĞƐƐƚŚĞƐĞƌĞƐŝƐƚĂŶĐĞŐĞŶĞƐƵŶĚĞƌƚŚĞƐƚĂŶĚĂƌĚŝnjĞĚĞdžƉĞƌŝŵĞŶƚĂů

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

Processed on: 13-11-2019 PDF page: 104PDF page: 104PDF page: 104PDF page: 104

104 ĐŽŶĚŝƟŽŶƐ ŽĨ ƚŚĞ ƉƌĞƐĞŶƚ ƐƚƵĚLJ͘ ŽŶǀĞƌƐĞůLJ͕ ƚŚĞ ĐĂƌďĂƉĞŶĞŵ ƌĞƐŝƐƚĂŶƚ E. roggenkampii ŝƐŽůĂƚĞĐĂƌƌŝĞƐŐĞŶĞƐĨŽƌƚǁŽƚLJƉĞs/ƐĞĐƌĞƟŽŶƐLJƐƚĞŵƐ͕ďƵƚŝƚĚŝĚi ƐĞĞŵŝŶŐůLJŶŽƚĞdžƉƌĞƐƐƚŚĞƐĞŐĞŶĞƐƵŶĚĞƌƚŚĞƚĞƐƚĞĚĐŽŶĚŝƟŽŶƐ͕ǁŚĞƌĞĂƐŝƐŽůĂƚĞƐ ĂƐƐŽĐŝĂƚĞĚǁŝƚŚďŽŶĞŝŶĨĞĐƟŽŶƐĚŝĚĞdžƉƌĞƐƐƉƌŽƚĞŝŶƐŽĨƚLJƉĞs/ƐĞĐƌĞƟŽŶƐLJƐƚĞŵƐ͘ dŚĞƐĞŽďƐĞƌǀĂƟŽŶƐĂĚĚĨƵĞůƚŽƚŚĞĂƐƐƵŵƉƟŽŶƚŚĂƚƚŚĞĚŝīĞƌĞŶƚŝƐŽůĂƚĞƐŚĂǀĞ ďĞĞŶƐƵďũĞĐƚƚŽĚŝīĞƌĞŶƚƐĞůĞĐƟǀĞƉƌĞƐƐƵƌĞƐƚŚĂƚĞŝƚŚĞƌůĞĚƚŽƌĞƐŝƐƚĂŶĐĞĂŐĂŝŶƐƚ ĐĂƌďĂƉĞŶĞŵƐ͕ŽƌƚŚĞĞdžƉƌĞƐƐŝŽŶŽĨĂƐĞĐƌĞƟŽŶƐLJƐƚĞŵƚŚĂƚŚĂƐďĞĞŶĂƐƐŽĐŝĂƚĞĚ ǁŝƚŚ ďĂĐƚĞƌŝĂů ǀŝƌƵůĞŶĐĞ͘ ƚ ƉƌĞƐĞŶƚ ǁĞ ĚŽ ŶŽƚ ŬŶŽǁ ƚŽ ǁŚĂƚ ĞdžƚĞŶƚ ƚŚĞƐĞ ĚŝīĞƌĞŶƚƉŚĞŶŽƚLJƉŝĐĂůƉƌĞƐĞŶƚĂƟŽŶƐĐĂŶďĞƌĞůĂƚĞĚƚŽĚŝīĞƌĞŶƚĐůĂĚĞƐǁŝƚŚŝŶ the E. cloacae ĐůƵƐƚĞƌ͘,ŽǁĞǀĞƌ͕ƚŚŝƐŝƐĚĞĮŶŝƚĞůLJƐŽŵĞƚŚŝŶŐǁŽƌƚŚŝŶǀĞƐƟŐĂƟŶŐ ŝŶĂĨƵƚƵƌĞƐƚƵĚLJďĞĐĂƵƐĞ͕ŝĨƚŚĞĚŝīĞƌĞŶƚĐůĂĚĞƐĐĂŶŝŶĚĞĞĚďĞĂƐƐŽĐŝĂƚĞĚǁŝƚŚ ĞŝƚŚĞƌŚŝŐŚĚƌƵŐƌĞƐŝƐƚĂŶĐĞŽƌǀŝƌƵůĞŶĐĞ͕ƚŚĞƉŽƐŝƟŽŶŽĨ͚E. cloacae’ isolates in ƚŚĞƉŚLJůŽŐĞŶLJŽĨƚŚŝƐĐŽŵƉůĞdžŵŝŐŚƚďĞĂƉƉůŝĐĂďůĞĨŽƌĂƉƉƌŽdžŝŵĂƟŶŐƚŚĞƌŝƐŬĨŽƌ ƌĞƐŝƐƚĂŶĐĞĂŐĂŝŶƐƚĐĂƌďĂƉĞŶĞŵƐ͕ƉĂƚŚŽŐĞŶŝĐŝƚLJ͕ŽƌďŽƚŚ͘/ŶƚŚŝƐƌĞƐƉĞĐƚ͕ŝƚǁŽƵůĚ ďĞƌĞĂůůLJŝŶƚĞƌĞƐƟŶŐƚŽƐĐƌĞĞŶĂĐŽůůĞĐƟŽŶŽĨĚŝīĞƌĞŶƚE. roggenkampii isolatesi

ĨŽƌƉŽƐƐŝďůĞĐĂƌďĂƉĞŶĞŵƌĞƐŝƐƚĂŶĐĞ͘

Materials and Methods

Bacterial isolates dŚĞďĂĐƚĞƌŝĂůŝƐŽůĂƚĞƐƵƐĞĚŝŶƚŚŝƐƐƚƵĚLJĂŶĚƚŚĞŝƌĂŶƟďŝŽƟĐƌĞƐŝƐƚĂŶĐĞĂƌĞůŝƐƚĞĚ ŝŶ^ƵƉƉůĞŵĞŶƚĂƌLJdĂďůĞϭ͘dŚĞŝŶŝƟĂůƐƉĞĐŝĞƐĚĞƚĞƌŵŝŶĂƟŽŶǁĂƐƉĞƌĨŽƌŵĞĚďLJ D>/ͲdK&D^͕ƵƐŝŶŐĂƌƵŬĞƌDŝĐƌŽŇĞdž;ƌƵŬĞƌŽƌƉŽƌƚĂƟŽŶ͕ŝůůĞƌŝĐĂ͕h^Ϳ͕ ƐŚŽǁŝŶŐƚŚĂƚƚŚĞLJĂůůďĞůŽŶŐĞĚƚŽƚŚĞE. cloacaeĐŽŵƉůĞdž͘ EƐĞƋƵĞŶĐĞĂŶĂůLJƐĞƐ EĂŶŽƉŽƌĞƐĞƋƵĞŶĐŝŶŐƉ Ƌ Ő͘ĂĐƚĞƌŝĂǁĞƌĞŐƌŽǁŶŽǀĞƌŶŝŐŚƚĂƚϯϳΣŽŶůŽŽĚŐĂƌ ;ͿƉůĂƚĞƐ͘dŚĞŶƐŝŶŐůĞĐŽůŽŶŝĞƐǁĞƌĞƉŝĐŬĞĚĨŽƌŽǀĞƌŶŝŐŚƚĐƵůƚƵƌĞĂƚϯϳΣŽŶ ƉůĂƚĞƐĂŐĂŝŶ͘&ŽƌEĞdžƚƌĂĐƟŽŶƚŚĞEĞĂƐLJhůƚƌĂůĞĂŶDŝĐƌŽďŝĂů<ŝƚ;YŝĂŐĞŶͿ ǁĂƐ ƵƐĞĚ ǁŝƚŚ ŵŝŶŽƌ ŵŽĚŝĮĐĂƟŽŶƐ͘  ϭϬ ђůͲůŽŽƉĨƵů ŽĨ ďĂĐƚĞƌŝĂ ǁĂƐ ĚŝƌĞĐƚůLJ ƚƌĂŶƐĨĞƌƌĞĚŝŶƚŽĂƚƵďĞǁŝƚŚŵŝĐƌŽďĞĂĚƐĂŶĚŵŝĐƌŽďĞĂĚƐƐŽůƵƟŽŶ͘dŚĞŝŶĐƵďĂƟŽŶ ƉĞƌŝŽĚǁĂƐƉƌŽůŽŶŐĞĚƚŽϮϬŵŝŶ͕ŝŶƐƚĞĂĚŽĨϱŵŝŶ͘dŚĞƋƵĂůŝƚLJĂŶĚƋƵĂŶƟƚLJŽĨ ŝƐŽůĂƚĞĚ E ǁĂƐ ĚĞƚĞƌŵŝŶĞĚ ƵƐŝŶŐ Ă YƵďŝƚΠ Ϯ͘Ϭ ŇƵŽƌŽŵĞƚĞƌ ;dŚĞƌŵŽ&ŝƐŚĞƌ

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

Processed on: 13-11-2019 PDF page: 105PDF page: 105PDF page: 105PDF page: 105

105

ŚĂƉƚ

ĞƌϦ

^ĐŝĞŶƟĮĐͿ͕ĂŶŐŝůĞŶƚdĂƉĞƐƚĂƟŽŶϮϮϬϬ;ŐŝůĞŶƚͿĂŶĚĂEĂŶŽƌŽƉ;dŚĞƌŵŽ&ŝƐŚĞƌ ^ĐŝĞŶƟĮĐͿ͘ >ŝďƌĂƌŝĞƐ ǁĞƌĞ ƉƌĞƉĂƌĞĚ ǁŝƚŚŽƵƚ ƐŚĞĂƌŝŶŐ ƚŽ ŵĂdžŝŵŝƐĞ ƐĞƋƵĞŶĐŝŶŐ ƌĞĂĚůĞŶŐƚŚ͘dŚĞůŝďƌĂƌLJǁĂƐƉƌĞƉĂƌĞĚƵƐŝŶŐƚŚĞϮůŝŐĂƟŽŶƐĞƋƵĞŶĐŝŶŐŬŝƚ;^Y<Ͳ >^<ϮϬϴͿ͘dŚĞƉƌŽƚŽĐŽůĨŽƌϮůŝŐĂƟŽŶƐĞƋƵĞŶĐŝŶŐŬŝƚǁĂƐĨŽůůŽǁĞĚĂƐĚĞƐĐƌŝďĞĚďLJ ƚŚĞŵĂŶƵĨĂĐƚƵƌĞƌ͘dŚĞĮŶĂůůŝďƌĂƌLJǁĂƐůŽĂĚĞĚŽŶƚŽĂ&>KͲD/EϭϬϲZϵ͘ϰŇŽǁĐĞůů͘ dŚĞƌƵŶǁĂƐƉĞƌĨŽƌŵĞĚŽŶĂDŝŶ/KEĚĞǀŝĐĞƵƐŝŶŐƚŚĞEͺϰϴ,ƌͺ^ĞƋƵĞŶĐŝŶŐͺ ZƵŶͺ&>KͲD/EϭϬϳͺ^Y<>^<ϮϬϴ ƉƌŽƚŽĐŽů ǁŝƚŚ ϵϳϲ ĂǀĂŝůĂďůĞ ƉŽƌĞƐ ;ϰϲϰ͕ ϯϭϰ͕ ϭϲϭ ĂŶĚ ϯϳ ƉŽƌĞƐ ƉĞƌ ŐƌŽƵƉͿ͘ dŚĞ ƌƵŶ ƉƌŽĐĞĞĚĞĚ ĨŽƌ ƚŚĞ ĨƵůů ϰϴ ŚŽƵƌƐ͘ ĂƐĞ ĐĂůůŝŶŐǁĂƐƉĞƌĨŽƌŵĞĚĂŌĞƌƚŚĞƌƵŶ͕ƵƐŝŶŐůďĂĐŽƌĞǀϭ͘Ϯ͘Ϯ;EĂŶŽƉŽƌĞͿǁŝƚŚƚŚĞ ƌϵϰͺϮϱϬďƉƐͺϮĚ͘ĐĨŐǁŽƌŬŇŽǁ͘>ĂƐƚůLJ͕ƚŚĞƋƵĂůŝƚLJŽĨƚŚĞĚĂƚĂǁĂƐĂŶĂůLJƐĞĚǁŝƚŚ WŽƌĞƚŽŽůƐǀϬ͘ϲ͘Ϭϭϯ_{ĂŶĚƚŚĞĨĂƐƚϱĮůĞƐǁĞƌĞƚƌĂŶƐĨŽƌŵĞĚŝŶƚŽĂĨĂƐƚƋĮůĞ͘} /ůůƵŵŝŶĂƐĞƋƵĞŶĐŝŶŐƋ Ő͘dŽƚĂůEĞdžƚƌĂĐƟŽŶĨŽƌǁŚŽůĞͲŐĞŶŽŵĞƐĞƋƵĞŶĐŝŶŐǁĂƐ ƉĞƌĨŽƌŵĞĚĚŝƌĞĐƚůLJĨƌŽŵĐŽůŽŶŝĞƐŽĨƚŚĞƌĞƐƉĞĐƟǀĞŝƐŽůĂƚĞƐƵƐŝŶŐƚŚĞhůƚƌĂĐůĞĂŶ DŝĐƌŽďŝĂůE/ƐŽůĂƟŽŶ<ŝƚ;DK/K>ĂďŽƌĂƚŽƌŝĞƐ͕ĂƌůƐďĂĚ͕͕h^ͿĂĐĐŽƌĚŝŶŐ ƚŽƚŚĞŵĂŶƵĨĂĐƚƵƌĞƌ͛ƐƉƌŽƚŽĐŽů͘EĐŽŶĐĞŶƚƌĂƟŽŶƐǁĞƌĞĚĞƚĞƌŵŝŶĞĚƵƐŝŶŐĂ YƵďŝƚΠϮ͘ϬŇƵŽƌŽŵĞƚĞƌĂŶĚƚŚĞĚƐE,^ĂŶĚͬŽƌZĂƐƐĂLJŬŝƚ;>ŝĨĞƚĞĐŚŶŽůŽŐŝĞƐ͕ ĂƌůƐďĂĚ͕ ͕ h^Ϳ͘ ^ƵďƐĞƋƵĞŶƚůLJ͕ E ůŝďƌĂƌŝĞƐ ǁĞƌĞ ƉƌĞƉĂƌĞĚ ƵƐŝŶŐ ƚŚĞ EĞdžƚĞƌĂydǀϯŬŝƚ;/ůůƵŵŝŶĂ͕^ĂŶŝĞŐŽ͕͕h^ͿĂĐĐŽƌĚŝŶŐƚŽƚŚĞŵĂŶƵĨĂĐƚƵƌĞƌ͛Ɛ ŝŶƐƚƌƵĐƟŽŶƐ͘^ĞƋƵĞŶĐĞĂŶĂůLJƐŝƐǁĂƐƉĞƌĨŽƌŵĞĚǁŝƚŚĂŶ/ůůƵŵŝŶĂDŝƐĞƋ^LJƐƚĞŵ ŐĞŶĞƌĂƟŶŐ ƉĂŝƌĞĚͲĞŶĚ ƌĞĂĚƐ ŽĨ ϯϬϬ ďƉ ĂƐ ĚĞƐĐƌŝďĞĚ ƉƌĞǀŝŽƵƐůLJ14_{͘ De novo} ĂƐƐĞŵďůLJŽĨƉĂŝƌĞĚͲĞŶĚƌĞĂĚƐǁĂƐƉĞƌĨŽƌŵĞĚƵƐŝŶŐ>'ĞŶŽŵŝĐƐtŽƌŬďĞŶĐŚ ǀϴ͘ϱ͘ϯ;Y/'E͕,ŝůĚĞŶ͕'ĞƌŵĂŶLJͿĂŌĞƌƋƵĂůŝƚLJƚƌŝŵŵŝŶŐ;YƐшϮϬͿĞƐƚĂďůŝƐŚŝŶŐ ĂǁŽƌĚƐŝnjĞŽĨϯϬ͘dŚĞƌĂǁt'^ĚĂƚĂƐĞƚƐŐĞŶĞƌĂƚĞĚŝŶƚŚĞĐƵƌƌĞŶƚƐƚƵĚLJĂƌĞ ĂǀĂŝůĂďůĞŝŶƚŚĞƵƌŽƉĞĂŶEƵĐůĞŽƟĚĞƌĐŚŝǀĞ;EͿƌĞƉŽƐŝƚŽƌLJƵŶĚĞƌŝŽƉƌŽũĞĐƚ WZ:ϮϮϭϭϵ;ŚƩƉ͗ͬͬǁǁǁ͘Ğďŝ͘ĂĐ͘ƵŬͬĞŶĂͬͿŽƌĂƌĞĂǀĂŝůĂďůĞŽŶƌĞƋƵĞƐƚ͘ ,LJďƌŝĚ ĂƐƐĞŵďůLJLJ LJ͘ dŚĞ ŚLJďƌŝĚ ĂƐƐĞŵďůLJ ŽĨ ƚŚĞ /ůůƵŵŝŶĂ ƐŚŽƌƚ ƌĞĂĚƐ ĂŶĚ ƚŚĞ DŝŶ/KEůŽŶŐƌĞĂĚƐŽĨƚŚĞĚŝīĞƌĞŶƚŝƐŽůĂƚĞƐǁĂƐƉĞƌĨŽƌŵĞĚƵƐŝŶŐ^WĚĞƐǀĞƌƐŝŽŶ ϯ͘ϭϬ͘ϭ;ŚƩƉ͗ͬͬďŝŽŝŶĨ͘ƐƉďĂƵ͘ƌƵͬĞŶͿ͕ŽƌhŶŝĐLJĐůĞƌǀĞƌƐŝŽŶϬ͘ϰ͘ϭ15_{͘ĂŶĚĂŐĞǀϬ͘ϴ͘ϭ} ǁĂƐƵƐĞĚƚŽǀŝƐƵĂůŝnjĞƚŚĞhŶŝĐLJĐůĞƌĂƐƐĞŵďůLJŐƌĂƉŚƐ16_{͘dŚĞƌĞƐƵůƟŶŐĂƐƐĞŵďůŝĞƐ} ĂƌĞƐƵďŵŝƩĞĚƚŽE/ƵŶĚĞƌĂĐĐĞƐƐŝŽŶŶƵŵďĞƌWϬϮϲϱϯϲŽƌĂƌĞĂǀĂŝůĂďůĞŽŶ ƌĞƋƵĞƐƚ͘^ĞƋƵĞŶĐĞĂŶĂůLJƐĞƐǁĞƌĞƉĞƌĨŽƌŵĞĚƵƐŝŶŐƚŚĞƌĂƉŝĚĂŶŶŽƚĂƟŽŶƵƐŝŶŐ ƐƵďƐLJƐƚĞŵƚĞĐŚŶŽůŽŐLJ;Z^dͿƐĞƌǀĞƌϰ͘Ϭ17_͘

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

Processed on: 13-11-2019 PDF page: 106PDF page: 106PDF page: 106PDF page: 106

106 WŚLJůŽŐĞŶĞƟĐĂŶĂůLJƐĞƐ  ŶĞŝŐŚďŽƌ ũŽŝŶŝŶŐ ƚƌĞĞ ǁĂƐ ĐŽŶƐƚƌƵĐƚĞĚ ďĂƐĞĚ ŽŶ ƚŚĞ ŐĞŶŽŵĞ ƐĞƋƵĞŶĐĞƐ ŽĨ ƚŚĞƐƚƵĚLJŝƐŽůĂƚĞƐ͕ƚŚĞE. cloacaeƚLJƉĞƐƚƌĂŝŶdϭϯϬϰϳĂŶĚϯϲƌĞƉƌĞƐĞŶƚĂƟǀĞ ƉƵďůŝĐůLJ ĂǀĂŝůĂďůĞ ŐĞŶŽŵĞ ƐĞƋƵĞŶĐĞƐ ŽĨ ďĂĐƚĞƌŝĂ ďĞůŽŶŐŝŶŐ ƚŽ ƚŚĞ E. cloacae ĐŽŵƉůĞdž ƵƐŝŶŐ ZŝĚŽŵ ^ĞƋ^ƉŚĞƌĞн ǀĞƌƐŝŽŶ ϱ͘ϭ͘Ϭ͘ ;DƺŶƐƚĞƌ͕ 'ĞƌŵĂŶLJͿ ǁŝƚŚ ĚĞĨĂƵůƚƐĞƫŶŐƐ18_{dŚĞƌĞůĂƚĞĚŶĞƐƐŽĨƚŚĞĚŝīĞƌĞŶƚŐĞŶŽŵĞƐǁĂƐĂŶĂůLJnjĞĚƵƐŝŶŐ} a local ad hocĐŐD>^dƐĐŚĞŵĞďĂƐĞĚŽŶϳϵϰƚĂƌŐĞƚƐ;ϳϰϵϮϱϵďĂƐĞƐͿĂŶĚϯϲϰϳc ƚĂƌŐĞƚƐǁĞƌĞƵƐĞĚĂƐĂĐĐĞƐƐŽƌLJƚĂƌŐĞƚƐ;ϯϰϵϯϰϲϰďĂƐĞƐͿ͕ĂŶĚƚŚĞŵŝƐƐŝŶŐǀĂůƵĞƐ ǁĞƌĞŝŐŶŽƌĞĚ;ƐĞĞ^ƵƉƉůĞŵĞŶƚĂůĂƚĂ^ϭͿ͘ Proteome analyses ll Enterobacter isolates r ǁĞƌĞĐƵůƚƵƌĞĚŝŶƚƌŝƉůŝĐĂƚĞŝŶďƌĂŝŶŚĞĂƌƚŝŶĨƵƐŝŽŶďƌŽƚŚ ;,/͖KdžŽŝĚ͕ĂƐŝŶŐƐƚŽŬĞ͕h<ͿĂƚϯϳo_{ǁŝƚŚǀŝŐŽƌŽƵƐƐŚĂŬŝŶŐĂƚϮϱϬƌƉŵ͘&Žƌ} ƐĂŵƉůĞƉƌĞƉĂƌĂƟŽŶ͕ĐĞůůƐǁĞƌĞĐŽůůĞĐƚĞĚŝŶƚŚĞŵŝĚͲĞdžƉŽŶĞŶƟĂůĂŶĚƐƚĂƟŽŶĂƌLJ ŐƌŽǁƚŚ ƉŚĂƐĞƐ ďLJ ĐĞŶƚƌŝĨƵŐĂƟŽŶ ĂŶĚ ĚŝƐƌƵƉƚĞĚ ďLJ ďĞĂĚͲďĞĂƟŶŐ ǁŝƚŚ ŐůĂƐƐ ďĞĂĚƐ;ΕϬ͘ϭŵŵĚŝĂŵĞƚĞƌͿŝŶĂWƌĞĐĞůůLJƐϮϰŚŽŵŽŐĞŶŝƐĂƚŽƌ;ĞƌƟŶdĞĐŚŶŽůŽŐŝĞƐ͕ &ƌĂŶĐĞͿĂƐĚĞƐĐƌŝďĞĚƉƌĞǀŝŽƵƐůLJϭϵ͕ϮϬ_{͘'ůĂƐƐďĞĂĚƐĂŶĚĐĞůůĚĞďƌŝƐǁĞƌĞƌĞŵŽǀĞĚ} ďLJ ĐĞŶƚƌŝĨƵŐĂƟŽŶ ;Ϯϭ͕ϬϬϬ п Ő͕ ϭϬ ŵŝŶ͕ ϰ ΣͿ͘ dŚĞ ĐĞůů ĞdžƚƌĂĐƚ ƉƌŽƚĞŝŶ ĨƌĂĐƟŽŶ ǁĂƐ ƉƌĞƉĂƌĞĚ ĂŶĚ ĂŶĂůLJnjĞĚ ďLJ >ͲD^ͬD^ ƵƐŝŶŐ ĂŶ KƌďŝƚƌĂƉ sĞůŽƐ WƌŽ ŵĂƐƐ ƐƉĞĐƚƌŽŵĞƚĞƌ ;dŚĞƌŵŽ&ŝƐŚĞƌ͕ tĂůƚŚĂŵ͕ D h^Ϳ ĂƐ ĚĞƐĐƌŝďĞĚ ƉƌĞǀŝŽƵƐůLJ21 ͘ ƌŝĞŇLJ͕ ƉƌŽƚĞŝŶƐ ǁĞƌĞ ĐŽŶĐĞŶƚƌĂƚĞĚ ǁŝƚŚ ^ƚƌĂƚĂĐůĞĂŶ ďĞĂĚƐ͕ ƐƵďƐĞƋƵĞŶƚůLJ ƌĞĚƵĐĞĚĂŶĚĂůŬLJůĂƚĞĚ͕ĚŝŐĞƐƚĞĚǁŝƚŚƚƌLJƉƐŝŶĂŶĚƚŚĞŶƉƵƌŝĮĞĚƚŚƌŽƵŐŚ^ƚĂŐĞdŝƉ ƉƵƌŝĮĐĂƟŽŶ͘ĞƐĂůƚĞĚƉĞƉƟĚĞƐǁĞƌĞůŽĂĚĞĚŽŶĂŶ ^zͲŶ>Ρ//ŶĂŶŽͲŇŽǁ> ƐLJƐƚĞŵ;dŚĞƌŵŽ&ŝƐŚĞƌͿǁŝƚŚϭϬђůďƵīĞƌ;Ϭ͘ϭй;ǀͬǀͿĂĐĞƟĐĂĐŝĚͿĂŶĚĂĐŽŶƐƚĂŶƚ ŇŽǁƌĂƚĞŽĨϬ͘ϱђ>ͬŵŝŶ͘ŌĞƌǁĂƌĚƐ͕ƚŚĞƉĞƉƟĚĞƐǁĞƌĞƐĞƉĂƌĂƚĞĚďLJƌĞǀĞƌƐĞĚ ƉŚĂƐĞĐŚƌŽŵĂƚŽŐƌĂƉŚLJǁŝƚŚĂϭϱϱŵŝŶŶŽŶͲůŝŶĞĂƌŐƌĂĚŝĞŶƚĨƌŽŵϭƚŽϱϬйďƵīĞƌ ;Ϭ͘ϭй;ǀͬǀͿĂĐĞƟĐĂĐŝĚŝŶĂĐĞƚŽŶŝƚƌŝůĞͿǁŝƚŚĂĐŽŶƐƚĂŶƚŇŽǁƌĂƚĞŽĨϬ͘ϯђůͬŵŝŶ ĂŶĚŝŶũĞĐƚĞĚŽŶůŝŶĞŝŶƚŽƚŚĞŵĂƐƐƐƉĞĐƚƌŽŵĞƚĞƌ͘dŚĞϮϬŵŽƐƚĂďƵŶĚĂŶƚƉƌĞĐƵƌƐŽƌ ŝŽŶƐǁĞƌĞƐĞůĞĐƚĞĚĨŽƌĐŽůůŝƐŝŽŶͲŝŶĚƵĐĞĚĚŝƐƐŽĐŝĂƟŽŶ;/ͿĨƌĂŐŵĞŶƚĂƟŽŶĂŌĞƌĂ ƐƵƌǀĞLJƐĐĂŶŝŶƚŚĞKƌďŝƚƌĂƉǁŝƚŚĂƌĞƐŽůƵƟŽŶŽĨϲϬ͕ϬϬϬĂŶĚĂĐƟǀĂƚĞĚůŽĐŬŵĂƐƐ ĐŽƌƌĞĐƟŽŶ͘D^ͬD^ƐĐĂŶƐǁĞƌĞƌĞĐŽƌĚĞĚŝŶƚŚĞĚƵĂůƉƌĞƐƐƵƌĞůŝŶĞĂƌŝŽŶƚƌĂƉĂŌĞƌ ĨƌĂŐŵĞŶƚĂƟŽŶǁĂƐƉĞƌĨŽƌŵĞĚĨŽƌϭϬŵƐĞĐǁŝƚŚĂŶŽƌŵĂůŝnjĞĚĐŽůůŝƐŝŽŶĞŶĞƌŐLJŽĨ ϯϱ͘

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

Processed on: 13-11-2019 PDF page: 107PDF page: 107PDF page: 107PDF page: 107

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ŚĂƉƚ

ĞƌϦ

ĂƚĂ ĂŶĂůLJƐŝƐ ǁĂƐ ƉĞƌĨŽƌŵĞĚ ĂĐĐŽƌĚŝŶŐ ƚŽ ŽŶŶ Ğƚ Ăů͘ Ϯϭ͕ϭϳ_{͘ /Ŷ ďƌŝĞĨ͕ ĚĂƚĂďĂƐĞ} ƐĞĂƌĐŚŝŶŐǁĂƐĚŽŶĞǁŝƚŚ^ŽƌĐĞƌĞƌͲ^Yh^dϰ;^ĂŐĞͲEZĞƐĞĂƌĐŚ͕DŝůƉŝƚĂƐ͕h^Ϳ͘ ŌĞƌĚĂƚĂĞdžƚƌĂĐƟŽŶĨƌŽŵƌĂǁĮůĞƐ͕ƚŚĞΎ͘ĚƚĂĮůĞƐǁĞƌĞƐĞĂƌĐŚĞĚǁŝƚŚ^ĞƋƵĞƐƚ ĂŐĂŝŶƐƚĂƚĂƌŐĞƚͲĚĞĐŽLJĚĂƚĂďĂƐĞǁŝƚŚĂƐĞƚŽĨĐŽŵŵŽŶůĂďŽƌĂƚŽƌLJĐŽŶƚĂŵŝŶĂŶƚƐ͘ ŶŽŶͲƌĞĚƵŶĚĂŶƚĚĂƚĂďĂƐĞĨŽƌƉĞƉƟĚĞͬƉƌŽƚĞŝŶƐĞĂƌĐŚĞƐǁĂƐĐƌĞĂƚĞĚĨƌŽŵƚŚĞ ŐĞŶŽŵĞ ƐĞƋƵĞŶĐĞƐ ŽĨ ƚŚĞ ƐĞǀĞŶ ƐƚƵĚLJ ŝƐŽůĂƚĞƐ ĂŶĚ ƚŚĞŐĞŶŽŵĞ ƐĞƋƵĞŶĐĞ ŽĨ the E. cloacae ƚLJƉĞ ƐƚƌĂŝŶ d ϭϯϬϰϳ ĂƐ ĚŽǁŶůŽĂĚĞĚ ĨƌŽŵ hŶŝƉƌŽƚ ;ŚƩƉ͗ͬͬ ǁǁǁ͘ƵŶŝƉƌŽƚ͘ŽƌŐ͖ Ϯϯrd _{ŽĨ KĐƚŽďĞƌ ϮϬϭϱͿ͘ dŚĞ ƵƐĞĚ ĚĂƚĂďĂƐĞ ŝŶĐůƵĚĞƐ ƉƌŽƚĞŝŶ} ƐĞƋƵĞŶĐĞƐƚŚĂƚĚŝīĞƌŝŶĂƚůĞĂƐƚϭĂŵŝŶŽĂĐŝĚ͕ĂŶĚŝƚĐŽŶƚĂŝŶƐϯϬϰϴϲƉƌŽƚĞŝŶƐ ŝŶƚŽƚĂů͘KŶůLJƐƚƌŝĐƚƚƌLJƉƟĐƉĞƉƟĚĞƐǁŝƚŚƵƉƚŽƚǁŽŵŝƐƐĞĚĐůĞĂǀĂŐĞƐǁĞƌĞƵƐĞĚ ĨŽƌƚŚĞĚĂƚĂďĂƐĞƐĞĂƌĐŚ͘&ŝdžĞĚŵŽĚŝĮĐĂƟŽŶƐǁĞƌĞŶŽƚĐŽŶƐŝĚĞƌĞĚ͘KdžŝĚĂƟŽŶŽĨ ŵĞƚŚŝŽŶŝŶĞĂŶĚĐĂƌďĂŵŝĚŽŵĞƚŚLJůĂƟŽŶŽĨĐLJƐƚĞŝŶĞǁĞƌĞĐŽŶƐŝĚĞƌĞĚĂƐǀĂƌŝĂďůĞ ŵŽĚŝĮĐĂƟŽŶƐ͘ DĂƐƐ ƚŽůĞƌĂŶĐĞ ĨŽƌ ƉƌĞĐƵƌƐŽƌ ŝŽŶƐ ǁĂƐ ƐĞƚ ƚŽ ϭϬ ƉƉŵ͕ ĂŶĚ ĨŽƌ ĨƌĂŐŵĞŶƚŝŽŶƐƚŽϬ͘ϱĂ͘sĂůŝĚĂƟŽŶŽĨƚŚĞD^ͬD^ͲďĂƐĞĚƉĞƉƟĚĞĂŶĚƉƌŽƚĞŝŶ ŝĚĞŶƟĮĐĂƟŽŶ ǁĂƐ ƉĞƌĨŽƌŵĞĚ ǁŝƚŚ ^ĐĂīŽůĚ ǀ͘ϰ͘ϰ͘ϭ͘ϭ ;WƌŽƚĞŽŵĞ ^ŽŌǁĂƌĞ͕ WŽƌƚůĂŶĚ͕ h^Ϳ͘ WĞƉƟĚĞ ŝĚĞŶƟĮĐĂƟŽŶƐ ǁĞƌĞ ŽŶůLJ ĂĐĐĞƉƚĞĚ ŝĨ ƚŚĞLJ ĞdžĐĞĞĚĞĚ ƚŚĞ ĨŽůůŽǁŝŶŐ ƐƉĞĐŝĮĐ ĚĂƚĂďĂƐĞ ƐĞĂƌĐŚ ĞŶŐŝŶĞ ƚŚƌĞƐŚŽůĚƐ͗ ƚŚĞ ^Yh^d ŝĚĞŶƟĮĐĂƟŽŶƐƌĞƋƵŝƌĞĚĂƚůĞĂƐƚĚĞůƚĂŶƐĐŽƌĞƐŽĨхϬ͘ϭĂŶĚyŽƌƌƐĐŽƌĞƐŽĨх Ϯ͘Ϯ͕ϯ͘ϯĂŶĚϯ͘ϳĨŽƌĚŽƵďůLJ͕ƚƌŝƉůLJĂŶĚĂůůŚŝŐŚĞƌĐŚĂƌŐĞĚƉĞƉƟĚĞƐ͕ƌĞƐƉĞĐƟǀĞůLJ͘ WƌŽƚĞŝŶ ŝĚĞŶƟĮĐĂƟŽŶƐ ;^ƵƉƉůĞŵĞŶƚĂů dĂďůĞ ^ϮͿ ǁĞƌĞ ĂĐĐĞƉƚĞĚ ŝĨ Ăƚ ůĞĂƐƚ Ϯ ŝĚĞŶƟĮĞĚƉĞƉƟĚĞƐǁĞƌĞĚĞƚĞĐƚĞĚǁŝƚŚĂďŽǀĞŵĞŶƟŽŶĞĚĮůƚĞƌĐƌŝƚĞƌŝĂŝŶϮŽƵƚ ŽĨϯďŝŽůŽŐŝĐĂůƌĞƉůŝĐĂƚĞƐ͘dŚŝƐƌĞƐƵůƚĞĚŝŶĂĨĂůƐĞͲƉŽƐŝƟǀĞĚŝƐĐŽǀĞƌLJƌĂƚĞ;&ZͿ ďĞůŽǁϬ͘ϮйŽŶƉƌŽƚĞŝŶůĞǀĞůĂƐǁĂƐǀĞƌŝĮĞĚďLJĂƐĞĂƌĐŚĂŐĂŝŶƐƚĂĐŽŶĐĂƚĞŶĂƚĞĚ ƚĂƌŐĞƚͲƉƐĞƵĚŽƌĞǀĞƌƐĞĚĚĞĐŽLJĚĂƚĂďĂƐĞ͘ůůD^ĚĂƚĂŚĂǀĞďĞĞŶĚĞƉŽƐŝƚĞĚƚŽƚŚĞ WƌŽƚĞŽŵĞyĐŚĂŶŐĞŽŶƐŽƌƟƵŵǀŝĂƚŚĞWZ/ƉĂƌƚŶĞƌƌĞƉŽƐŝƚŽƌLJǁŝƚŚƚŚĞĚĂƚĂƐĞƚ ŝĚĞŶƟĮĞƌWyϬϬϳϭϭϯ22_͘

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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108 Ethics statement dŚĞďĂĐƚĞƌŝĂůŝƐŽůĂƚĞƐƵƐĞĚĨŽƌƚŚĞƉƌĞƐĞŶƚĂŶĂůLJƐĞƐǁĞƌĞĐŽůůĞĐƚĞĚŝŶƚŚĞĐŽƵƌƐĞ ŽĨƌŽƵƟŶĞĚŝĂŐŶŽƐƟĐƐĂŶĚŝŶĨĞĐƟŽŶƉƌĞǀĞŶƟŽŶĐŽŶƚƌŽů͘KƌĂůĐŽŶƐĞŶƚĨŽƌƚŚĞƵƐĞ ŽĨƐƵĐŚĐůŝŶŝĐĂůƐĂŵƉůĞƐĨŽƌƌĞƐĞĂƌĐŚƉƵƌƉŽƐĞƐŝƐƌŽƵƟŶĞůLJŽďƚĂŝŶĞĚƵƉŽŶƉĂƟĞŶƚ ĂĚŵŝƐƐŝŽŶƚŽƚŚĞhD'͕ŝŶĂĐĐŽƌĚĂŶĐĞǁŝƚŚƚŚĞŐƵŝĚĞůŝŶĞƐŽĨƚŚĞDĞĚŝĐĂůƚŚŝĐƐ ŽŵŵŝƩĞĞŽĨƚŚĞhŶŝǀĞƌƐŝƚLJDĞĚŝĐĂůĞŶƚĞƌ'ƌŽŶŝŶŐĞŶ͘ůůĞdžƉĞƌŝŵĞŶƚƐǁĞƌĞ ƉĞƌĨŽƌŵĞĚŝŶĂĐĐŽƌĚĂŶĐĞǁŝƚŚƚŚĞŐƵŝĚĞůŝŶĞƐŽĨƚŚĞĞĐůĂƌĂƟŽŶŽĨ,ĞůƐŝŶŬŝĂŶĚ ƚŚĞŝŶƐƟƚƵƟŽŶĂůƌĞŐƵůĂƟŽŶƐ͕ĂŶĚĂůůƐĂŵƉůĞƐǁĞƌĞĂŶŽŶLJŵŝnjĞĚ͘ Funding dŚŝƐǁŽƌŬǁĂƐƐƵƉƉŽƌƚĞĚďLJƚŚĞ'ƌĂĚƵĂƚĞ^ĐŚŽŽůĨŽƌDĞĚŝĐĂů^ĐŝĞŶĐĞƐŽĨƚŚĞ hŶŝǀĞƌƐŝƚLJ ŽĨ 'ƌŽŶŝŶŐĞŶ ;ƚŽ ^͘E͘ ĂŶĚ ^͘'͘Ϳ͕ ƚŚĞ WĞŽƉůĞ WƌŽŐƌĂŵŵĞ ;DĂƌŝĞ ^ŬųŽĚŽǁƐŬĂͲƵƌŝĞĐƟŽŶƐͿŽĨƚŚĞƵƌŽƉĞĂŶhŶŝŽŶ͛Ɛ,ŽƌŝnjŽŶϮϬϮϬWƌŽŐƌĂŵŵĞ ƵŶĚĞƌZŐƌĂŶƚĂŐƌĞĞŵĞŶƚ;ŶŽ͘ϲϰϮϴϯϲƚŽ^͘'͕͘͘ĂŶĚ:͘D͘ǀ͘͘Ϳ͕ƚŚĞĞƵƚƐĐŚĞ &ŽƌƐĐŚƵŶŐƐŐĞŵĞŝŶƐĐŚĂŌ ;^&ͬdZZ ϯϰ ĨƌĂŵĞǁŽƌŬ ƚŽ ͕͘͘ &͘͘ ĂŶĚ ͘K͘Ϳ͕ ƚŚĞ 'ĞƌŵĂŶ&ĞĚĞƌĂůDŝŶŝƐƚƌLJŽĨĚƵĐĂƟŽŶĂŶĚZĞƐĞĂƌĐŚ;^ĞƉƚŽŵŝĐƐƚŽ&͘͘Ϳ͕ĂŶĚƚŚĞ &ƵŶĚĂŵĞŶƚĂůZĞƐĞĂƌĐŚ&ƵŶĚƐĨŽƌƚŚĞĞŶƚƌĂůhŶŝǀĞƌƐŝƟĞƐ;ϮϬϭϲ&ϳϬϬϴƚŽ<Ϳ͘ dŚĞĨƵŶĚĞƌƐŚĂĚŶŽƌŽůĞŝŶƐƚƵĚLJĚĞƐŝŐŶ͕ĚĂƚĂĐŽůůĞĐƟŽŶĂŶĚĂŶĂůLJƐŝƐ͕ĚĞĐŝƐŝŽŶƚŽ ƉƵďůŝƐŚ͕ŽƌƉƌĞƉĂƌĂƟŽŶŽĨƚŚĞŵĂŶƵƐĐƌŝƉƚ͘ ŝƐĐůŽƐƵƌĞŽĨWŽƚĞŶƟĂůŽŶŇŝĐƚƐŽĨ/ŶƚĞƌĞƐƚ :͘t͘͘Z͘ŝƐďĞŝŶŐĞŵƉůŽLJĞĚďLJƚŚĞĐŽŵƉĂŶLJ/ďLJE͘dŚĞŽƚŚĞƌĂƵƚŚŽƌƐĚĞĐůĂƌĞ ƚŚĂƚƚŚĞLJŚĂǀĞŶŽĮŶĂŶĐŝĂůĂŶĚŶŽŶͲĮŶĂŶĐŝĂůĐŽŵƉĞƟŶŐŝŶƚĞƌĞƐƚƐŝŶƌĞůĂƟŽŶƚŽ ƚŚĞĚŽĐƵŵĞŶƚĞĚƌĞƐĞĂƌĐŚ͘

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

ϭ͘ :ŽƐĞƉŚ t͘ ŚŽǁ͕ zƵ͕ D͘ Enterobacter ĂĐƚĞƌĞŵŝĂ͗ ůŝŶŝĐĂů &ĞĂƚƵƌĞƐ ĂŶĚr

ŵĞƌŐĞŶĐĞŽĨŶƟďŝŽƟĐZĞƐŝƐƚĂŶĐĞĚƵƌŝŶŐdŚĞƌĂƉLJ͘ Ann Intern Med. 115͕ϱϴϱʹ ϱϵϬ;ϭϵϵϭͿ͘

Ϯ͘ ^Ăďŝƌ͕ E͘ et al. ĂĐƚĞƌŝĂů ďŝŽĮůŵͲďĂƐĞĚ ĐĂƚŚĞƚĞƌͲĂƐƐŽĐŝĂƚĞĚ ƵƌŝŶĂƌLJ ƚƌĂĐƚ

ŝŶĨĞĐƟŽŶƐ͗ĂƵƐĂƟǀĞƉĂƚŚŽŐĞŶƐĂŶĚĂŶƟďŝŽƟĐƌĞƐŝƐƚĂŶĐĞ͘Am. J. Infect. Control

45͕ϭϭϬϭʹϭϭϬϱ;ϮϬϭϳͿ͘ ϯ͘ <ĂŵŝŷƐŬĂ͕t͕͘WĂƚnjĞƌ͕:͘ΘnjŝĞƌnjĂŶŽǁƐŬĂ͕͘hƌŝŶĂƌLJƚƌĂĐƚŝŶĨĞĐƟŽŶƐĐĂƵƐĞĚďLJ ĞŶĚĞŵŝĐŵƵůƟͲƌĞƐŝƐƚĂŶƚ Enterobacter cloacaeŝŶĂĚŝĂůLJƐŝƐĂŶĚƚƌĂŶƐƉůĂŶƚĂƟŽŶ ƵŶŝƚ͘ J. Hosp. Infect. 51͕ϮϭϱʹϮϮϬ;ϮϬϬϮͿ͘ ϰ͘ tĞƐƚďůŽŵ͕d͘h͘ΘŽŐŐŝŶƐ͕D͘͘KƐƚĞŽŵLJĞůŝƟƐĐĂƵƐĞĚďLJ Enterobacter taylorae͕ ĨŽƌŵĞƌůLJĞŶƚĞƌŝĐŐƌŽƵƉϭϵ͘J. Clin. Microbiol. 25͕ϮϰϯϮʹϮϰϯϯ;ϭϵϴϳͿ͘

ϱ͘ ŝƐƐĞ͕,͘et al.dƌĞĂƚŵĞŶƚŽĨďŽŶĞĂŶĚũŽŝŶƚŝŶĨĞĐƟŽŶƐĐĂƵƐĞĚďLJ Enterobacter

cloacae ǁŝƚŚĂŇƵŽƌŽƋƵŝŶŽůŽŶĞʹĐŽƚƌŝŵŽdžĂnjŽůĞĐŽŵďŝŶĂƟŽŶ͘/Ŷƚ͘:͘ŶƟŵŝĐƌŽď͘ Agents 54͕ϮϰϱʹϮϰϴ;ϮϬϭϵͿ͘

ϲ͘ WĂĂƵǁ͕͘et al. /ĚĞŶƟĮĐĂƟŽŶŽĨƌĞƐŝƐƚĂŶĐĞĂŶĚǀŝƌƵůĞŶĐĞĨĂĐƚŽƌƐŝŶĂŶĞƉŝĚĞŵŝĐ

Enterobacter hormaechei ŽƵƚďƌĞĂŬƐƚƌĂŝŶ͘ Microbiology 155͕ϭϰϳϴʹϭϰϴϴ;ϮϬϬϵͿ͘

ϳ͘ ,ŽƌŵĂĞĐŚĞ͕ ͕ ͘ථ͖ ĚǁĂƌĚƐ͕ W͘ Z͘  WƌŽƉŽƐĞĚ 'ĞŶƵƐ Enterobacter͘ Int. Bull.

Bacteriol. Nomencl. Taxon. ;ϭϵϲϬͿ͘

ϴ͘ WĂĂƵǁ͕ ͘ et al. 'ĞŶŽŵŝĐ ŝǀĞƌƐŝƚLJ ǁŝƚŚŝŶ ƚŚĞ Enterobacter cloacae Complex͘

PLoS One 3͕ĞϯϬϭϴ;ϮϬϬϴͿ͘

ϵ͘ WĂǀůŽǀŝĐ͕D͘et al. ĚƵĂůĂƉƉƌŽĂĐŚĞŵƉůŽLJŝŶŐD>/ͲdK&D^ĂŶĚƌĞĂůͲƟŵĞWZ

ĨŽƌĨĂƐƚƐƉĞĐŝĞƐŝĚĞŶƟĮĐĂƟŽŶǁŝƚŚŝŶƚŚĞ Enterobacter cloacae ĐŽŵƉůĞdž͘ FEMS

DŝĐƌŽďŝŽů͘>ĞƩ͘ 328͕ϰϲʹϱϯ;ϮϬϭϮͿ͘

ϭϬ͘ ,ŽīŵĂŶŶ͕ ,͘ Θ ZŽŐŐĞŶŬĂŵƉ͕ ͘ WŽƉƵůĂƟŽŶ ŐĞŶĞƟĐƐ ŽĨ ƚŚĞ ŶŽŵĞŶƐƉĞĐŝĞƐ

Enterobacter cloacae. Appl. Environ. Microbiol. 69͕ϱϯϬϲʹϱϯϭϴ;ϮϬϬϯͿ͘

ϭϭ͘ EĞƉĂů͕ ^͘ et al. Ŷ ĂŶĐŝĞŶƚ ĨĂŵŝůLJ ŽĨ ŵŽďŝůĞ ŐĞŶŽŵŝĐ ŝƐůĂŶĚƐ ŝŶƚƌŽĚƵĐŝŶŐ

cephalosporinase and carbapenemase genes in Enterobacteriaceae͘ Virulence 9͕ ϭϯϳϳʹϭϯϴϵ;ϮϬϭϴͿ͘ ϭϮ͘ DĂƌơŶĞnjͲ'ĂƌĐşĂ͕ W͘ D͕͘ ZĂŵŽƐ͕ ͘ Θ ZŽĚƌşŐƵĞnjͲWĂůĞŶnjƵĞůĂ͕ W͘ dϯϰϲ,ƵŶƚĞƌ͗  EŽǀĞůtĞďͲĂƐĞĚdŽŽůĨŽƌƚŚĞWƌĞĚŝĐƟŽŶŽĨdLJƉĞ///͕dLJƉĞ/sĂŶĚdLJƉĞs/^ĞĐƌĞƟŽŶ ^LJƐƚĞŵƐŝŶĂĐƚĞƌŝĂů'ĞŶŽŵĞƐ͘PLoS One 10͕ĞϬϭϭϵϯϭϳ;ϮϬϭϱͿ͘ ϭϯ͘ >ŽŵĂŶ͕ E͘ :͘ Θ YƵŝŶůĂŶ͕ ͘ Z͘ WŽƌĞƚŽŽůƐ͗  ƚŽŽůŬŝƚ ĨŽƌ ĂŶĂůLJnjŝŶŐ ŶĂŶŽƉŽƌĞ ƐĞƋƵĞŶĐĞĚĂƚĂ͘ŝŽŝŶĨŽƌŵĂƟĐƐ 30͕ϯϯϵϵʹϯϰϬϭ;ϮϬϭϰͿ͘ ϭϰ͘ ŚŽƵ͕ <͘ et al. dŚĞ ŵŽƐĂŝĐ ŐĞŶŽŵĞ ƐƚƌƵĐƚƵƌĞ ĂŶĚ ƉŚLJůŽŐĞŶLJ ŽĨ ^ŚŝŐĂ ƚŽdžŝŶͲ

producing Escherichia coli KϭϬϰ͗,ϰ ŝƐ ĚƌŝǀĞŶ ďLJ ƐŚŽƌƚͲƚĞƌŵ ĂĚĂƉƚĂƟŽŶ͘ Clin.

Microbiol. Infect. 21͕ϰϲϴ͘ĞϳͲϰϲϴ͘Ğϭϴ;ϮϬϭϱͿ͘

ϭϱ͘ tŝĐŬ͕Z͘Z͕͘:ƵĚĚ͕>͘D͕͘'ŽƌƌŝĞ͕͘>͘Θ,Žůƚ͕<͘͘hŶŝĐLJĐůĞƌ͗ZĞƐŽůǀŝŶŐďĂĐƚĞƌŝĂů

ŐĞŶŽŵĞĂƐƐĞŵďůŝĞƐĨƌŽŵƐŚŽƌƚĂŶĚůŽŶŐƐĞƋƵĞŶĐŝŶŐƌĞĂĚƐ͘PLoS Comput. Biol.

13͕ϭʹϮϮ;ϮϬϭϳͿ͘

ϭϲ͘ tŝĐŬ͕Z͘Z͕͘^ĐŚƵůƚnj͕D͕͘͘ŽďĞů͕:͘Θ,Žůƚ͕<͘͘ĂŶĚĂŐĞ͗/ŶƚĞƌĂĐƟǀĞǀŝƐƵĂůŝnjĂƟŽŶ

ŽĨĚĞŶŽǀŽŐĞŶŽŵĞĂƐƐĞŵďůŝĞƐ͘ŝŽŝŶĨŽƌŵĂƟĐƐ 31͕ϯϯϱϬʹϯϯϱϮ;ϮϬϭϱͿ͘

ϭϳ͘ njŝnj͕Z͘<͘ et al.dŚĞZ^d^ĞƌǀĞƌ͗ZĂƉŝĚĂŶŶŽƚĂƟŽŶƐƵƐŝŶŐƐƵďƐLJƐƚĞŵƐƚĞĐŚŶŽůŽŐLJ͘

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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110 ϭϴ͘ >ĞŽƉŽůĚ͕^͘Z͕͘'ŽĞƌŝŶŐ͕Z͘s͕͘tŝƩĞŶ͕͕͘,ĂƌŵƐĞŶ͕͘ΘDĞůůŵĂŶŶ͕͘ĂĐƚĞƌŝĂů ǁŚŽůĞͲŐĞŶŽŵĞ ƐĞƋƵĞŶĐŝŶŐ ƌĞǀŝƐŝƚĞĚ͗ WŽƌƚĂďůĞ͕ ƐĐĂůĂďůĞ͕ ĂŶĚ ƐƚĂŶĚĂƌĚŝnjĞĚ ĂŶĂůLJƐŝƐĨŽƌƚLJƉŝŶŐĂŶĚĚĞƚĞĐƟŽŶŽĨǀŝƌƵůĞŶĐĞĂŶĚĂŶƟďŝŽƟĐƌĞƐŝƐƚĂŶĐĞŐĞŶĞƐ͘ J. Clin. Microbiol. 52͕ϮϯϲϱʹϮϯϳϬ;ϮϬϭϰͿ͘ ϭϵ͘ ŽŶŶ͕&͘et al.'ůŽďĂůĂŶĂůLJƐŝƐŽĨƚŚĞŝŵƉĂĐƚŽĨůŝŶĞnjŽůŝĚŽŶƚŽǀŝƌƵůĞŶĐĞĨĂĐƚŽƌ

ƉƌŽĚƵĐƟŽŶŝŶ^͘ĂƵƌĞƵƐh^ϯϬϬ͘Int. J. Med. Microbiol. 306͕ϭϯϭʹϭϰϬ;ϮϬϭϲͿ͘

ϮϬ͘ DĂĂƐƐ͕^͘et al. ĸĐŝĞŶƚ͕ŐůŽďĂůͲƐĐĂůĞƋƵĂŶƟĮĐĂƟŽŶŽĨĂďƐŽůƵƚĞƉƌŽƚĞŝŶĂŵŽƵŶƚƐ ďLJŝŶƚĞŐƌĂƟŽŶŽĨƚĂƌŐĞƚĞĚŵĂƐƐƐƉĞĐƚƌŽŵĞƚƌLJĂŶĚƚǁŽͲĚŝŵĞŶƐŝŽŶĂů ŐĞůͲďĂƐĞĚ ƉƌŽƚĞŽŵŝĐƐ͘ Anal. Chem. 83͕ϮϲϳϳʹϮϲϴϰ;ϮϬϭϭͿ͘ Ϯϭ͘ ŽŶŶ͕&͘et al. WŝĐŬŝŶŐǀĂŶŝƐŚĞĚƉƌŽƚĞŝŶƐĨƌŽŵƚŚĞǀŽŝĚ͗,ŽǁƚŽĐŽůůĞĐƚĂŶĚƐŚŝƉͬ ƐŚĂƌĞĞdžƚƌĞŵĞůLJĚŝůƵƚĞƉƌŽƚĞŝŶƐŝŶĂƌĞƉƌŽĚƵĐŝďůĞĂŶĚŚŝŐŚůLJĞĸĐŝĞŶƚŵĂŶŶĞƌ͘ Anal. Chem. 86͕ϳϰϮϭʹϳϰϮϳ;ϮϬϭϰͿ͘ ϮϮ͘ WĞƌĞnjͲZŝǀĞƌŽů͕z͘et al.dŚĞWZ/ĚĂƚĂďĂƐĞĂŶĚƌĞůĂƚĞĚƚŽŽůƐĂŶĚƌĞƐŽƵƌĐĞƐŝŶ

ϮϬϭϵ͗/ŵƉƌŽǀŝŶŐƐƵƉƉŽƌƚĨŽƌƋƵĂŶƟĮĐĂƟŽŶĚĂƚĂ͘Nucleic Acids Res. 47͕ϰϰϮʹ ϰϱϬ;ϮϬϭϵͿ͘

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