Document Version

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.

Document Version

Publisher's PDF, also known as Version of record

Publication date:

2019

Link to publication in University of Groningen/UMCG research database

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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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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Chapter 2

Suruchi Nepal, Florian Bonn, Stefano Grasso, Tim Stobernack , Anne de Jong,

<ĂŝŚŽƵ͕ZŽŶĂůĚtĞĚĞŵĂ͕^ŝŐƌŝĚZŽƐĞŵĂ͕ŽƌƚĞĞĐŚĞƌ͕ŶĚƌĞĂƐKƩŽ͕:ŽŚŶt͘

Rossen, Jan Maarten van Dijl, and Erik Bathoorn

WƵďůŝƐŚĞĚŝŶsŝƌƵůĞŶĐĞ͘ϮϬϭϴ͖ϵ;ϭͿ͗ϭϯϳϳͲϭϯϴϵ

An ancient family of mobile genomic islands

introducing cephalosporinase and carbapenemase genes in

Enterobacteriaceae

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34 Abstract

The exchange of mobile genomic islands (MGIs) between microorganisms is ŽŌĞŶŵĞĚŝĂƚĞĚďLJƉŚĂŐĞƐ͕ǁŚŝĐŚŵĂLJƉƌŽǀŝĚĞďĞŶĞĮƚƐƚŽƚŚĞƉŚĂŐĞ͛ƐŚŽƐƚ͘dŚĞ

ƉƌĞƐĞŶƚƐƚƵĚLJƐƚĂƌƚĞĚǁŝƚŚƚŚĞŝĚĞŶƟĮĐĂƟŽŶŽĨEnterobacter cloacae, Klebsiella ppneumoniae and Escherichia coli ŝƐŽůĂƚĞƐ ǁŝƚŚ ĞdžĐĞƉƟŽŶĂů ĐĞƉŚĂůŽƐƉŽƌŝŶ ĂŶĚ i

 ĐĂƌďĂƉĞŶĞŵƌĞƐŝƐƚĂŶĐĞƉŚĞŶŽƚLJƉĞƐĨƌŽŵƉĂƟĞŶƚƐŝŶĂŶĞŽŶĂƚĂůǁĂƌĚ͘dŽŝĚĞŶƟĨLJ

 ƉŽƐƐŝďůĞ ŵŽůĞĐƵůĂƌ ĐŽŶŶĞĐƟŽŶƐ ďĞƚǁĞĞŶ ƚŚĞƐĞ ŝƐŽůĂƚĞƐ ĂŶĚ ƚŚĞŝƌ ɴͲůĂĐƚĂŵ

ƌĞƐŝƐƚĂŶĐĞ ƉŚĞŶŽƚLJƉĞƐ͕ ƚŚĞ ƌĞƐƉĞĐƟǀĞ ďĂĐƚĞƌŝĂů ŐĞŶŽŵĞ ƐĞƋƵĞŶĐĞƐ ǁĞƌĞ

 ĐŽŵƉĂƌĞĚ͘dŚŝƐƵŶǀĞŝůĞĚƚŚĞĞdžŝƐƚĞŶĐĞŽĨĂĨĂŵŝůLJŽĨĂŶĐŝĞŶƚD'/ƐƚŚĂƚǁĞƌĞ

ƉƌŽďĂďůLJĞdžĐŚĂŶŐĞĚďĞĨŽƌĞƚŚĞƐƉĞĐŝĞƐ E. cloacae, K. pneumoniae and E. coli i ĞŵĞƌŐĞĚĨƌŽŵƚŚĞŝƌĐŽŵŵŽŶĂŶĐĞƐƚƌLJ͘ƌĞƉƌĞƐĞŶƚĂƟǀĞD'/ĨƌŽŵ E. cloacae was ŶĂŵĞĚD/ZϭϳͲ'/͕ďĞĐĂƵƐĞŝƚŚĂƌďŽƌƐƚŚĞŶŽǀĞůɴͲůĂĐƚĂŵĂƐĞŐĞŶĞǀĂƌŝĂŶƚbla

͘

/ŵƉŽƌƚĂŶƚůLJ͕ŽƵƌŽďƐĞƌǀĂƟŽŶƐƐŚŽǁƚŚĂƚƚŚĞD/ZϭϳͲ'/ͲůŝŬĞD'/ƐŚĂƌďŽƌŐĞŶĞƐ

 ĂƐƐŽĐŝĂƚĞĚǁŝƚŚŚŝŐŚͲůĞǀĞůƌĞƐŝƐƚĂŶĐĞƚŽĐĞƉŚĂůŽƐƉŽƌŝŶƐ͘ŵŽŶŐƚŚĞŵ͕D/ZϭϳͲ'/

 ƐƚĂŶĚƐŽƵƚďĞĐĂƵƐĞD/ZϭϳĂůƐŽĚŝƐƉůĂLJƐĐĂƌďĂƉĞŶĞŵĂƐĞĂĐƟǀŝƚLJ͘ƐƐŚŽǁŶďLJ

ŵĂƐƐƐƉĞĐƚƌŽŵĞƚƌLJ͕ƚŚĞD/ZϭϳĐĂƌďĂƉĞŶĞŵĂƐĞŝƐĂŵŽŶŐƚŚĞŵŽƐƚĂďƵŶĚĂŶƚůLJ

ĞdžƉƌĞƐƐĞĚƉƌŽƚĞŝŶƐŽĨƚŚĞƌĞƐƉĞĐƟǀĞ E. cloacaeŝƐŽůĂƚĞ͘&ƵƌƚŚĞƌ͕ǁĞƐŚŽǁƚŚĂƚ D/ZϭϳͲ'/ͲůŝŬĞ ŝƐůĂŶĚƐ ĂƌĞ ĂƐƐŽĐŝĂƚĞĚ ǁŝƚŚ ŝŶƚĞŐƌĂƚĞĚ WϰͲůŝŬĞ ƉƌŽƉŚĂŐĞƐ͘ dŚŝƐ

 ŝŵƉůŝĐĂƚĞƐƉŚĂŐĞƐŝŶƚŚĞƐƉƌĞĂĚŽĨĐĞƉŚĂůŽƐƉŽƌŝŶĂŶĚĐĂƌďĂƉĞŶĞŵƌĞƐŝƐƚĂŶĐĞ

amongst Enterobacteriaceae ͘ dŚĞ ĚŝƐĐŽǀĞƌLJ ŽĨ ĂŶ ĂŶĐŝĞŶƚ ĨĂŵŝůLJ ŽĨ D'/Ɛ͕  ŵĞĚŝĂƟŶŐƚŚĞƐƉƌĞĂĚŽĨĐĞƉŚĂůŽƐƉŽƌŝŶĂƐĞĂŶĚĐĂƌďĂƉĞŶĞŵĂƐĞŐĞŶĞƐ͕ŝƐŽĨŚŝŐŚ

 ĐůŝŶŝĐĂůƌĞůĞǀĂŶĐĞ͕ďĞĐĂƵƐĞŚŝŐŚͲůĞǀĞůĐĞƉŚĂůŽƐƉŽƌŝŶĂŶĚĐĂƌďĂƉĞŶĞŵƌĞƐŝƐƚĂŶĐĞ

 ŚĂǀĞƐĞƌŝŽƵƐŝŵƉůŝĐĂƟŽŶƐĨŽƌƚŚĞƚƌĞĂƚŵĞŶƚŽĨƉĂƟĞŶƚƐǁŝƚŚĞŶƚĞƌŽďĂĐƚĞƌŝĂĐĞĂů ŝŶĨĞĐƟŽŶƐ͘

<ĞLJǁŽƌĚƐ͗ Enterobacter cloacae͕ƉƌŽƚĞŽŵĞ͕ĐĂƌďĂƉĞŶĞŵĂƐĞ͕ĐĞƉŚĂůŽƐƉŽƌŝŶĂƐĞ͕

mobile genomic island

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35

ŚĂƉƚ ĞƌϤ

/ŶƚƌŽĚƵĐƟŽŶ

dŚĞƐŚĂƉĞŽĨďĂĐƚĞƌŝĂůŐĞŶŽŵĞƐ͕ĂƐǁĞŬŶŽǁƚŚĞŵƚŽĚĂLJ͕ŝƐƚŚĞŽƵƚĐŽŵĞŽĨŵĂŶLJ ƐƵĐĐĞƐƐŝǀĞĞǀŽůƵƟŽŶĂƌLJĞǀĞŶƚƐƚŚĂƚŽĐĐƵƌƌĞĚĞǀĞƌƐŝŶĐĞƚŚĞƌĞƐƉĞĐƟǀĞďĂĐƚĞƌŝĂů ƐƉĞĐŝĞƐďƌĂŶĐŚĞĚŽīĨƌŽŵƚŚĞŝƌĐŽŵŵŽŶĂŶĐĞƐƚŽƌƐ

͘dŚŝƐŝƐĐůĞĂƌůLJĞǀŝĚĞŶĐĞĚ ƚŚƌŽƵŐŚĐŽŵƉĂƌŝƐŽŶƐŽĨƚŚĞŐĞŶŽŵĞƐĞƋƵĞŶĐĞƐŽĨŝŶĚŝǀŝĚƵĂůďĂĐƚĞƌŝĂůƐƉĞĐŝĞƐ͕

ǁŚĞƌĞĂĚŝƐƟŶĐƟŽŶĐĂŶďĞŵĂĚĞďĞƚǁĞĞŶƚŚĞĐŽƌĞŐĞŶŽŵĞĂŶĚƚŚĞĂĐĐĞƐƐŽƌLJ

ŐĞŶŽŵĞ͘,ĞƌĞ͕ƚŚĞĂĐĐĞƐƐŽƌLJŐĞŶŽŵĞƌĞŇĞĐƚƐƚŚŽƐĞĞůĞŵĞŶƚƐƚŚĂƚǁĞƌĞĞŝƚŚĞƌ

ůŽƐƚƚŚƌŽƵŐŚŐĞŶŽŵŝĐĞƌŽƐŝŽŶ͕ŽƌŐĂŝŶĞĚƚŚƌŽƵŐŚŚŽƌŝnjŽŶƚĂůŐĞŶĞƚƌĂŶƐĨĞƌ

͘

KĨŶŽƚĞ͕ƚŚĞĂĐĐĞƐƐŽƌLJŐĞŶŽŵĞŝƐŽŶůLJƚŚĞĞĐŚŽŽĨƚŚŽƐĞĞǀĞŶƚƐƚŚĂƚŚĂƉƉĞŶĞĚ ƌĞůĂƟǀĞůLJ ƌĞĐĞŶƚůLJ ŽŶ ƚŚĞ ĞǀŽůƵƟŽŶĂƌLJ ƟŵĞůŝŶĞ͕ ŶĂŵĞůLJ ĂŌĞƌ ƚŚĞ ƉĂƌƟĐƵůĂƌ ƐƉĞĐŝĞƐĞŵĞƌŐĞĚ͘/ŶƚŚŝƐĐŽŶƚĞdžƚ͕ŝƚŝƐĨƌĞƋƵĞŶƚůLJŽǀĞƌůŽŽŬĞĚƚŚĂƚĂůƐŽƚŚĞĐŽƌĞ

ŐĞŶŽŵĞŚĂƐďĞĞŶƐŚĂƉĞĚĨƌŽŵĞůĞŵĞŶƚƐƚŚĂƚǁĞƌĞƌĞĐƌƵŝƚĞĚƚŚƌŽƵŐŚŐĞŶĞƟĐ

ĞdžĐŚĂŶŐĞƐ͕ďƵƚďĞĨŽƌĞƐƉĞĐŝĂƟŽŶŽĐĐƵƌƌĞĚ͘dŚƵƐ͕ĂůƐŽƚŚĞĐŽƌĞŐĞŶŽŵĞŝƐƚŚĞ ŽīƐƉƌŝŶŐŽĨĞǀŽůƵƟŽŶĂŶĚŝŶĐůƵĚĞƐĂŶĐŝĞŶƚŵŽďŝůĞŐĞŶŽŵŝĐĞůĞŵĞŶƚƐ;D'/ƐͿ͕

ǁŚŝĐŚ ƚƵƌŶĞĚ ŽƵƚ ďĞŶĞĮĐŝĂů ĨŽƌ ƚŚĞ ƐƉĞĐŝĞƐ ǁŝƚŚŝŶ ŝƚƐ ĞĐŽůŽŐŝĐĂů ŶŝĐŚĞ ƚŽ ƚŚĞ ĞdžƚĞŶƚƚŚĂƚƚŚĞLJĂƌĞŶŽůŽŶŐĞƌƌĞĂĚŝůLJůŽƐƚ

͘ƐĂĐŽŶƐĞƋƵĞŶĐĞŽĨƚŚĞŝƌĐŽͲ ĞǀŽůƵƟŽŶǁŝƚŚƚŚĞƐƉĞĐŝĞƐ͕ƐƵĐŚĂŶĐŝĞŶƚD'/ƐŵĂLJŚĂǀĞůŽƐƚƉĂƌƟĐƵůĂƌƚƌĂŝƚƐŽĨ

ƉƌĞƐĞŶƚͲĚĂLJD'/Ɛ͕ƐƵĐŚĂƐĂĚŝīĞƌĞŶĐĞŝŶƚŚĞ'ĐŽŶƚĞŶƚ͘zĞƚ͕ŽƚŚĞƌƚƌĂŝƚƐĐĂŶƐƟůů ďĞĚŝƐĐĞƌŶĞĚ͕ƐƵĐŚĂƐƚŚĞƚLJƉŝĐĂůƐŝƚĞƐŽĨŝŶƚĞŐƌĂƟŽŶĂŶĚĂŶŽǀĞƌƌĞƉƌĞƐĞŶƚĂƟŽŶ of certain classes of genes

͘ /Ŷ ƚŚĞ ƉƌĞƐĞŶƚ ƐƚƵĚLJ ǁĞ ĨŽĐƵƐ ĂƩĞŶƟŽŶ ŽŶ ĂŶ ĂŶĐŝĞŶƚĨĂŵŝůLJŽĨD'/Ɛ͕ĮƌƐƚĚŝƐĐŽǀĞƌĞĚǁŝƚŚŝŶƚŚĞ͚ĐŽƌĞŐĞŶŽŵĞ͛ŽĨEnterobacter cloacae͘

E. cloacae ŝƐ Ă ƌŽĚͲƐŚĂƉĞĚ͕ ŶŽŶͲƐƉŽƌĞ ĨŽƌŵŝŶŐ͕ ĨĂĐƵůƚĂƟǀĞ ĂŶĂĞƌŽďŝĐ͕ 'ƌĂŵͲ ŶĞŐĂƟǀĞ ďĂĐƚĞƌŝƵŵ ďĞůŽŶŐŝŶŐ ƚŽ ƚŚĞ ĨĂŵŝůLJ ŽĨ Enterobacteriaceae͘ dŚĞ

ŽĐĐƵƌƌĞŶĐĞ ŽĨ E. cloacae ŝƐ ǁŝĚĞƐƉƌĞĂĚ͕ ƌĂŶŐŝŶŐ ĨƌŽŵ ƐŽŝů ĂŶĚ ƐĞǁĂŐĞ ƚŽ ƚŚĞ

ŚƵŵĂŶ ŐĂƐƚƌŽŝŶƚĞƐƟŶĂů ƚƌĂĐƚ͕ ǁŚĞƌĞ ŝƚ ŝƐ Ă ĨƌĞƋƵĞŶƚ ĐŽŵƉŽŶĞŶƚ ŽĨ ƚŚĞ ŐƵƚ

ŵŝĐƌŽďŝŽƚĂ͘ /ŵƉŽƌƚĂŶƚůLJ͕ E. cloacae ĐĂŶ ĐĂƵƐĞ ŽƉƉŽƌƚƵŶŝƐƟĐ ŝŶĨĞĐƟŽŶƐ ĂŶĚ ŚĂƐ ƌĞĐĞŶƚůLJ ĞŵĞƌŐĞĚ ĂƐ Ă ŶŽƐŽĐŽŵŝĂů ƉĂƚŚŽŐĞŶ͕ ĞƐƉĞĐŝĂůůLJ ŝŶ ŝŶƚĞŶƐŝǀĞ ĐĂƌĞ ƵŶŝƚƐ

͘ /Ŷ ƚŚĞ ĐůŝŶŝĐĂů ƐĞƫŶŐ͕ E. cloacae ŚĂƐ ďĞĞŶ ŝĚĞŶƟĮĞĚ ĂƐ ƚŚĞ ĐĂƵƐĂƟǀĞ ĂŐĞŶƚŽĨƐŬŝŶĂŶĚƐŽŌƟƐƐƵĞŝŶĨĞĐƟŽŶƐ͕ƌĞƐƉŝƌĂƚŽƌLJĂŶĚƵƌŝŶĂƌLJƚƌĂĐƚŝŶĨĞĐƟŽŶƐ͕

ŝŶƚƌĂͲĂďĚŽŵŝŶĂů ŝŶĨĞĐƟŽŶƐ͕ ďĂĐƚĞƌĞŵŝĂ͕ ĞŶĚŽĐĂƌĚŝƟƐ͕ ƐĞƉƟĐ ĂƌƚŚƌŝƟƐ͕ ĂŶĚ

ŽƐƚĞŽŵLJĞůŝƟƐ ŝŶ ŝŵŵƵŶŽĐŽŵƉƌŽŵŝƐĞĚ ƉĂƟĞŶƚƐ

͘ dŚĞ ĂŶƟŵŝĐƌŽďŝĂů ƚŚĞƌĂƉLJ

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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ϯϲ

ŽĨ ƉĂƟĞŶƚƐ ǁŝƚŚ E. cloacae ŝŶĨĞĐƟŽŶƐ ŽŌĞŶ ĨĂĐĞƐ ĐŽŵƉůŝĐĂƟŽŶƐ ĚƵĞ ƚŽ ƚŚĞ ŝŶƚƌŝŶƐŝĐ ĚƌƵŐ ƌĞƐŝƐƚĂŶĐĞ ŽĨ ƚŚŝƐ ďĂĐƚĞƌŝƵŵ͕ ĂŶĚ ŝƚƐ ƉƌŽƉĞŶƐŝƚLJ ƚŽ ĂĐƋƵŝƌĞ

ŵƵůƟƉůĞƌĞƐŝƐƚĂŶĐĞŐĞŶĞƐ͘dŚƵƐ͕ƌĞƐŝƐƚĂŶĐĞŚĂƐďĞĞŶƌĞƉŽƌƚĞĚĂŐĂŝŶƐƚĂŵƉŝĐŝůůŝŶ͕

ĂŵŽdžŝĐŝůůŝŶ͕ĮƌƐƚͲŐĞŶĞƌĂƟŽŶĐĞƉŚĂůŽƐƉŽƌŝŶƐĂŶĚĐĞĨŽdžŝƟŶŽǁŝŶŐƚŽĐŽŶƐƟƚƵƟǀĞ

ĞdžƉƌĞƐƐŝŽŶŽĨƚŚĞŵƉɴͲůĂĐƚĂŵĂƐĞ͘E. cloacaeĂůƐŽĞdžŚŝďŝƚƐĂŚŝŐŚĨƌĞƋƵĞŶĐLJ

ŽĨĞŶnjLJŵĂƟĐƌĞƐŝƐƚĂŶĐĞƚŽďƌŽĂĚͲƐƉĞĐƚƌƵŵĐĞƉŚĂůŽƐƉŽƌŝŶƐ

͕ǁŚŝĐŚŝƐƚLJƉŝĐĂůůLJ

ĐĂƵƐĞĚ ďLJ ŽǀĞƌƉƌŽĚƵĐƟŽŶ ŽĨ ŵƉ ɴͲůĂĐƚĂŵĂƐĞƐ͘ ^ƵĐŚ ĞdžƚĞŶĚĞĚ ƐƉĞĐƚƌƵŵ

ĐĞƉŚĂůŽƐƉŽƌŝŶĂƐĞƐ;Ğ͘Ő͘d͕DzĂŶĚD/ZͿĐŽŶĨĞƌƌĞƐŝƐƚĂŶĐĞƚŽƚŚŝƌĚͲŐĞŶĞƌĂƟŽŶ

ĐĞƉŚĂůŽƐƉŽƌŝŶƐ͕ĂŶĚƚŚĞLJĂƌĞŶŽƚŝŶŚŝďŝƚĞĚďLJĐŽŵŵŽŶɴͲůĂĐƚĂŵĂƐĞŝŶŚŝďŝƚŽƌƐ

12

͘hŶƟůŶŽǁ͕ĨŽƵƌƚŚͲĂŶĚĮŌŚͲŐĞŶĞƌĂƟŽŶĐĞƉŚĂůŽƐƉŽƌŝŶƐŵĂŝŶƚĂŝŶƌĞĂƐŽŶĂďůĞ

ĂĐƟǀŝƚLJ ĂŐĂŝŶƐƚ ƐƵĐŚ ƐƚƌĂŝŶƐ͘ tŚŝůĞ d ĂŶĚ D/Z ŝŶ Enterobacter ƐƉĞĐŝĞƐ ĂƌĞ r ĞŶĐŽĚĞĚďLJŝŶƚƌŝŶƐŝĐĐŚƌŽŵŽƐŽŵĂůŐĞŶĞƐ͕DzŐĞŶĞƐŵĂLJďĞƉůĂƐŵŝĚͲďŽƌŶĞ

͘

/ŶŐĞŶĞƌĂů͕ĞdžƚĞŶĚĞĚƐƉĞĐƚƌƵŵĐĞƉŚĂůŽƐƉŽƌŝŶĂƐĞƐŽĨEnterobacteriaceae ŚĂǀĞ

ďĞĞŶĂƐƐŽĐŝĂƚĞĚǁŝƚŚŵŽďŝůĞŐĞŶŽŵŝĐĞůĞŵĞŶƚƐ͘

/ŶĐŽŶƚƌĂƐƚƚŽĐĞƉŚĂůŽƐƉŽƌŝŶĂƐĞƐ͕ĂĐƋƵŝƌĞĚĐĂƌďĂƉĞŶĂŵĂƐĞŐĞŶĞƐĂƌĞƐƟůůĨĂŝƌůLJ

ƵŶĐŽŵŵŽŶŝŶE. cloacae on a global scale

͘,ŽǁĞǀĞƌ͕ŵĂŶLJĞdžƚĞŶĚĞĚƐƉĞĐƚƌƵŵ ĐĞƉŚĂůŽƐƉŽƌŝŶĂƐĞƐ ŚĂǀĞ Ă ǁĞĂŬ ĂĸŶŝƚLJ ĨŽƌ ĐĂƌďĂƉĞŶĞŵƐ ĂŶĚ ĐĂŶ ŚLJĚƌŽůLJnjĞ ĐĂƌďĂƉĞŶĞŵƐ ǁŝƚŚ ůŽǁ ĞĸĐŝĞŶĐLJ͘ Ɛ Ă ĐŽŶƐĞƋƵĞŶĐĞ͕ ĐůŝŶŝĐĂů ƌĞƐŝƐƚĂŶĐĞ ŽĨ E.

cloacae ƚŽƚŚĞƐĞůĂƐƚͲůŝŶĞɴͲůĂĐƚĂŵĂŶƟďŝŽƟĐƐŝƐŵŽƐƚůLJďƌŽƵŐŚƚĂďŽƵƚďLJŽƵƚĞƌ

ŵĞŵďƌĂŶĞƉĞƌŵĞĂďŝůŝƚLJĚĞĨĞĐƚƐĐŽŵďŝŶĞĚǁŝƚŚĂĚĞƌĞƉƌĞƐƐŝŽŶŽĨĐŽŶƐƟƚƵƟǀĞ

ŵƉĐĞƉŚĂůŽƐƉŽƌŝŶĂƐĞƐ

͘

dŚĞƉƌĞƐĞŶƚƐƚƵĚLJƐƚĂƌƚĞĚǁŝƚŚƚŚĞŝĚĞŶƟĮĐĂƟŽŶŽĨE. cloacae, K. pneumoniae and E. coli ŝƐŽůĂƚĞƐǁŝƚŚĞdžĐĞƉƟŽŶĂůɴͲůĂĐƚĂŵƌĞƐŝƐƚĂŶĐĞƉŚĞŶŽƚLJƉĞƐ͕ŝŶĐůƵĚŝŶŐ i one E. cloacae ŝƐŽůĂƚĞ ǁŝƚŚ ĚĞĐƌĞĂƐĞĚ ƐƵƐĐĞƉƟďŝůŝƚLJ ĨŽƌ ĐĂƌďĂƉĞŶĞŵƐ͕ ŝŶ ƉĂƟĞŶƚƐ ŽŶ Ă ŶĞŽŶĂƚĂů ǁĂƌĚ ŽĨ ƚŚĞ hŶŝǀĞƌƐŝƚLJ DĞĚŝĐĂů ĞŶƚĞƌ 'ƌŽŶŝŶŐĞŶ

;hD'Ϳ͘ dŽ ĂƐƐĞƐƐ ƉŽƐƐŝďůĞ ŵŽůĞĐƵůĂƌ ĐŽŶŶĞĐƟŽŶƐ ďĞƚǁĞĞŶ ƚŚĞƐĞ ŝƐŽůĂƚĞƐ ĂŶĚ ƚŚĞŝƌ ĂŶƟďŝŽƟĐ ƌĞƐŝƐƚĂŶĐĞ ƉŚĞŶŽƚLJƉĞƐ͕ ƚŚĞ ƌĞƐƉĞĐƟǀĞ ďĂĐƚĞƌŝĂů ŐĞŶŽŵĞƐ

ǁĞƌĞĐŽŵƉĂƌĞĚ͘dŚŝƐůĞĚƚŽƚŚĞŝĚĞŶƟĮĐĂƟŽŶŽĨĨŽƵƌŚŝŐŚůLJĐŽŶƐĞƌǀĞĚD'/ƐŽĨ

ΕϭϰϬŬďŝŶƚŚĞŝŶǀĞƐƟŐĂƚĞĚŝƐŽůĂƚĞƐ͘dŚŝƐŽďƐĞƌǀĂƟŽŶŝƐŽĨŐĞŶĞƌĂůŝŵƉŽƌƚĂŶĐĞ͕

ƐŝŶĐĞŝƚĨŽĐƵƐĞƐĂƩĞŶƟŽŶŽŶĂĨĂŵŝůLJŽĨŐĞŶŽŵŝĐŝƐůĂŶĚƐĐĂƌƌLJŝŶŐĂĚŝǀĞƌƐŝƚLJŽĨ

ĐĞƉŚĂůŽƐƉŽƌŝŶĂƐĞͲĞŶĐŽĚŝŶŐ ŐĞŶĞƐ ƚŚĂƚ ǁĂƐ ĂůƌĞĂĚLJ ǁŝĚĞůLJ ƚƌĂŶƐĚƵĐĞĚ ďĞĨŽƌĞ

ƚŚĞƐƉĞĐŝĞƐE. cloacae, K. pneumoniae and E. coli emerged from their common i

ĂŶĐĞƐƚƌLJ͘

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: 37 PDF page: 37 PDF page: 37

37

ŚĂƉƚ ĞƌϤ

Results

ĞƐĐƌŝƉƟŽŶŽĨƚŚĞƐƚƵĚLJŝƐŽůĂƚĞƐĂŶĚĐůŝŶŝĐĂůďĂĐŬŐƌŽƵŶĚ

/ŶƚŚĞƉƌĞƐĞŶƚƐƚƵĚLJ͕ǁĞŝŶǀĞƐƟŐĂƚĞĚĨŽƵƌĞƉŝĚĞŵŝŽůŽŐŝĐĂůůLJůŝŶŬĞĚ'ƌĂŵͲŶĞŐĂƟǀĞ

ďĂĐƚĞƌŝĂůŝƐŽůĂƚĞƐǁŝƚŚĚĞĐƌĞĂƐĞĚƐƵƐĐĞƉƟďŝůŝƚLJƚŽĐĂƌďĂƉĞŶĞŵƐĂƐƌĞŇĞĐƚĞĚďLJ

ŵŝŶŝŵĂůŝŶŚŝďŝƚŽƌLJĐŽŶĐĞŶƚƌĂƟŽŶ;D/ͿǀĂůƵĞƐĨŽƌŵĞƌŽƉĞŶĞŵĂŶĚͬŽƌŝŵŝƉĞŶĞŵ шϬ͘ϱŵŐͬ>͘dĂďůĞϭƐƵŵŵĂƌŝnjĞƐƚŚĞƌĞƐƵůƚƐŽĨƚŚĞĂŶƟďŝŽƟĐƐƵƐĐĞƉƟďŝůŝƚLJƚĞƐƟŶŐ ŝŶƚŚĞƐĞĨŽƵƌŝƐŽůĂƚĞƐ͘

dŚĞĮƌƐƚƐƚƵĚLJŝƐŽůĂƚĞ;ŝƐŽůĂƚĞϭͿǁĂƐĂĐĂƌďĂƉĞŶĞŵͲƌĞƐŝƐƚĂŶƚ E. cloacae that was ŽďƚĂŝŶĞĚĨƌŽŵĂƌĞĐƚĂůƐǁĂďŽĨĂŶĞŽŶĂƚĞ;ƉĂƟĞŶƚϭͿ͕ǁŚŽŚĂĚďĞĞŶƌĞƉĂƚƌŝĂƚĞĚ

ĨƌŽŵƵƌĂĕĂŽƚŽƚŚĞhD'͘WƌŝŽƌƚŽĂĚŵŝƐƐŝŽŶŝŶƚŚĞhD'͕ƚŚĞƉĂƟĞŶƚŚĂĚ

ďĞĞŶƚƌĞĂƚĞĚǁŝƚŚŵĞƌŽƉĞŶĞŵ͘/ƐŽůĂƚĞϭƐŚŽǁĞĚĂŶĂƚLJƉŝĐĂůŐƌŽǁƚŚƉŚĞŶŽƚLJƉĞ

ƌĞƐƵůƟŶŐ ŝŶ ƐŵĂůů ĨĂƩLJ ĐŽůŽŶŝĞƐ ŽŶ ůŽŽĚ ŐĂƌ ;Ϳ ĂŶĚ DƵĞůůĞƌ ,ŝŶƚŽŶ ŐĂƌ

;D,ͿƉůĂƚĞƐ͘ƵƚŽŵĂƚĞĚƌĞƐŝƐƚĂŶĐĞĂŶĂůLJƐŝƐǁŝƚŚƚŚĞs/d<ϮƐLJƐƚĞŵƌĞǀĞĂůĞĚ ŝŶĐƌĞĂƐĞĚD/ǀĂůƵĞƐƚŽƚŚĞĐĂƌďĂƉĞŶĞŵƐŵĞƌŽƉĞŶĞŵ;ϴŵŐͬ>ͿĂŶĚŝŵŝƉĞŶĞŵ

;ϴŵŐͬ>Ϳ͕ǁŚŝĐŚǁĞƌĞƐƵďƐĞƋƵĞŶƚůLJĐŽŶĮƌŵĞĚďLJƚĞƐƚƐ͘/ŵŝƉĞŶĞŵƚĞƐƚƐŽŶ

D,ǁŝƚŚŽƌǁŝƚŚŽƵƚϮϱϬŵŐͬ>ĐůŽdžĂĐŝůůŝŶƌĞǀĞĂůĞĚƚŚĂƚŝŵŝƉĞŶĞŵƌĞƐŝƐƚĂŶĐĞ

ǁĂƐƐŝŐŶŝĮĐĂŶƚůLJƌĞĚƵĐĞĚŝŶƚŚĞƉƌĞƐĞŶĐĞŽĨĐůŽdžĂĐŝůůŝŶ͕ǁŚŝĐŚŝƐĂŶŝŶŚŝďŝƚŽƌŽĨ

ɴͲůĂĐƚĂŵĂƐĞƐŽĨƚŚĞŵƉͲƚLJƉĞ

͘^ƉĞĐŝĮĐĂůůLJ͕ƚŚĞD/ǁĂƐƌĞĚƵĐĞĚƚŽϬ͘ϭϮϱ ŵŐͬ>;&ŝŐƵƌĞϭͿ͘dŚŝƐƐŚŽǁĞĚƚŚĂƚƚŚĞƌĞĚƵĐĞĚĐĂƌďĂƉĞŶĞŵƐĞŶƐŝƟǀŝƚLJŽĨŝƐŽůĂƚĞ

ϭŝƐůĂƌŐĞůLJĚƵĞƚŽƚŚĞƉƌŽĚƵĐƟŽŶŽĨĂĐĂƌďĂƉĞŶĞŵͲĚĞŐƌĂĚŝŶŐĞŶnjLJŵĞ͘KĨŶŽƚĞ͕

ĐŽŶŇƵĞŶƚƉůĂƟŶŐŽĨŝƐŽůĂƚĞϭŽŶD,ƌĞƐƵůƚƐŝŶĂƉĂƚĐŚLJŐƌŽǁƚŚƉŚĞŶŽƚLJƉĞƚŚĂƚ

ŝƐƐƵƉƉƌĞƐƐĞĚŝŶƚŚĞƉƌĞƐĞŶĐĞŽĨĐůŽdžĂĐŝůůŝŶ;&ŝŐƵƌĞϭͿ͘DƵůƟͲůŽĐƵƐƐĞƋƵĞŶĐĞƚLJƉĞ

;D>^dͿĂŶĂůLJƐŝƐĂƐƐŝŐŶƐŝƐŽůĂƚĞϭƚŽƚŚĞƐĞƋƵĞŶĐĞƚLJƉĞ;^dͲͿϮϯϮ;dnaA88͕ fusA25͕

gyrB49͕ leuS72͕pyrG49͕ rplB12Ϳ͕ǁŚĞƌĞ dnaA88ƌĞƉƌĞƐĞŶƚƐĂŶŽǀĞůdnaA ĂůůĞůĞ͘

WŚĞŶŽƚLJƉŝĐ^>ƚĞƐƚƐǁŝƚŚŝƐŽůĂƚĞϭǁĞƌĞŶĞŐĂƟǀĞ͘tŚŽůĞͲŐĞŶŽŵĞƐĞƋƵĞŶĐŝŶŐ

ƌĞǀĞĂůĞĚ Ă ŶŽǀĞů ɴͲůĂĐƚĂŵĂƐĞ ŐĞŶĞ ǀĂƌŝĂŶƚ͕ ǁŚŝĐŚ ŝƐ ŚŽŵŽůŽŐŽƵƐ ƚŽ ƚŚĞ D/Z

ůŝŶĞĂŐĞ͘/ŶĨĂĐƚ͕ƚŚŝƐŐĞŶĞŝƐϵϵ͘ϯйŝĚĞŶƟĐĂůƚŽƚŚĞ bla

gene from Klebsiella pneumoniae ;E ĂĐĐĞƐƐŝŽŶ Dϯϳϴϯϵ͘ϮͿ ĂŶĚ ŝƚ ǁĂƐ ƚŚƵƐ ĚĞƐŝŐŶĂƚĞĚ bla

;E/ĂĐĐĞƐƐŝŽŶϮϵϳϱϮ͘ϭͿ͘^ƉĞĐŝĮĐĂůůLJ͕D/ZϭϳŝƐĚŝƐƟŶĐƚĨƌŽŵD/ZϭĚƵĞƚŽ

ĮǀĞĂŵŝŶŽĂĐŝĚƐƵďƐƟƚƵƟŽŶƐ͘

hƉŽŶǁŝƚŚĚƌĂǁĂůŽĨŵĞƌŽƉĞŶĞŵƚƌĞĂƚŵĞŶƚĨŽƌϮǁĞĞŬƐ͕ĂĐĂƌďĂƉĞŶĞŵͲƐĞŶƐŝƟǀĞ

E. cloacae ŝƐŽůĂƚĞ;ŝ͘Ğ͘ŝƐŽůĂƚĞϮͿǁĂƐŽďƚĂŝŶĞĚĨƌŽŵĂŶŝŶƚƌĂǀĂƐĐƵůĂƌĐĂƚŚĞƚĞƌƟƉ

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: 38 PDF page: 38 PDF page: 38

38

ĨƌŽŵƉĂƟĞŶƚϭ͘dŚŝƐŝƐŽůĂƚĞϮǁĂƐƐƵƐĐĞƉƟďůĞƚŽƚŚŝƌĚͲŐĞŶĞƌĂƟŽŶĐĞƉŚĂůŽƐƉŽƌŝŶƐ

ĂŶĚĐĂƌďĂƉĞŶĞŵƐ͕ĂŶĚƚŚĞƐĞŶƐŝƟǀŝƚLJƚŽŝŵŝƉĞŶĞŵǁĂƐŶŽƚŝŶŇƵĞŶĐĞĚďLJƚŚĞ ƉƌĞƐĞŶĐĞŽĨĐůŽdžĂĐŝůůŝŶ;ŶŽƚƐŚŽǁŶͿ͘'ĞŶŽŵĞƐĞƋƵĞŶĐŝŶŐƌĞǀĞĂůĞĚƚŚĞƉƌĞƐĞŶĐĞ

of an ampC ŐĞŶĞĞŶĐŽĚŝŶŐƚŚĞdͲϱĐĞƉŚĂůŽƐƉŽƌŝŶĂƐĞ͘&ƵƌƚŚĞƌ͕D>^dĂŶĂůLJƐŝƐ C ƐŚŽǁĞĚƚŚĂƚŝƐŽůĂƚĞϮďĞůŽŶŐƐƚŽ^dͲϵϳ͘

ŌĞƌ ϯ ŵŽŶƚŚƐ ŽĨ ŚŽƐƉŝƚĂůŝnjĂƟŽŶ͕ ƉĂƟĞŶƚ ϭ ĂĐƋƵŝƌĞĚ Ă K. pneumoniae ;ŝ͘Ğ͘

ŝƐŽůĂƚĞ ϯͿ ǁŝƚŚ Ă ƌĞŵĂƌŬĂďůĞ ĂŶƟďŝŽƟĐ ƌĞƐŝƐƚĂŶĐĞ ƉĂƩĞƌŶ͘ ^ƉĞĐŝĮĐĂůůLJ͕ ŝƐŽůĂƚĞ

ϯǁĂƐƐŚŽǁŶƚŽďĞƌĞƐŝƐƚĂŶƚƚŽĐĞĨƵƌŽdžŝŵĞĂŶĚĐĞĨŽƚĂdžŝŵĞ͕ďƵƚƐƵƐĐĞƉƟďůĞƚŽ ĐĞŌĂnjŝĚŝŵĞ͘WŚĞŶŽƚLJƉŝĐ^>ͲƚĞƐƟŶŐǁĂƐŶĞŐĂƟǀĞ͘&ŽƌƚŚĞŵŽůĞĐƵůĂƌĚĞƚĞĐƟŽŶ

ŽĨƌĞƐŝƐƚĂŶĐĞŐĞŶĞƐ͕ĮƌƐƚĂŵŝĐƌŽĂƌƌĂLJĂŶĂůLJƐŝƐǁĂƐƉĞƌĨŽƌŵĞĚĂŶĚ͕ƐƵďƐĞƋƵĞŶƚůLJ͕

ŝƐŽůĂƚĞ ϯ ǁĂƐ ƐƵďũĞĐƚĞĚ ƚŽ ǁŚŽůĞ ŐĞŶŽŵĞ ƐĞƋƵĞŶĐŝŶŐ ĨŽůůŽǁĞĚ ďLJ ƐĐƌĞĞŶŝŶŐ

ŽĨƚŚĞƐĞƋƵĞŶĐĞĂŐĂŝŶƐƚƚŚĞZĞƐĮŶĚĞƌĚĂƚĂďĂƐĞ͘dŚŝƐƐŚŽǁĞĚƚŚĞƉƌĞƐĞŶĐĞŽĨ

Ă ɴͲůĂĐƚĂŵĂƐĞ ŐĞŶĞ ǀĂƌŝĂŶƚ ǁŝƚŚ ϵϵй ŝĚĞŶƟƚLJ ƚŽ ďůĂ

of K. pneumoniae͘

This gene was therefore designated bla

;E/ ĂĐĐĞƐƐŝŽŶ >Eϱϭϱϱϯϯ͘ϭͿ͘

^ƉĞĐŝĮĐĂůůLJ͕^,sϭϴϳŝƐĚŝƐƟŶĐƚĨƌŽŵ^,sϭϰϬĚƵĞƚŽƚǁŽEͲƚĞƌŵŝŶĂůĂŵŝŶŽĂĐŝĚ

ƐƵďƐƟƚƵƟŽŶƐ͘D>^dƐŚŽǁĞĚƚŚĂƚŝƐŽůĂƚĞϯďĞůŽŶŐƐƚŽ^dͲϮϬ͘

WĂƟĞŶƚϮǁĂƐŚŽƐƉŝƚĂůŝnjĞĚŝŶƚŚĞƐĂŵĞǁĂƌĚŽĨƚŚĞhD'ĂƐƉĂƟĞŶƚϭ͘ƵƌŝŶŐ ƚŚŝƐŚŽƐƉŝƚĂůƐƚĂLJ͕ƉĂƟĞŶƚϮĂĐƋƵŝƌĞĚĂŶ E. coli;ŝƐŽůĂƚĞϰͿƌĞƐŝƐƚĂŶƚƚŽĐĞĨŽdžŝƟŶĂŶĚ i ƚŚŝƌĚͲŐĞŶĞƌĂƟŽŶĐĞƉŚĂůŽƐƉŽƌŝŶƐ͘&ƵƌƚŚĞƌ͕ŝƐŽůĂƚĞϰĚŝƐƉůĂLJĞĚĂŵĞƌŽƉĞŶĞŵD/

ǀĂůƵĞŽĨϰŵŐͬ>͘^>ͲƚĞƐƚƐǁĞƌĞƉŽƐŝƟǀĞĂŶĚ͕ƵƉŽŶǁŚŽůĞŐĞŶŽŵĞƐĞƋƵĞŶĐŝŶŐ͕

ZĞƐĮŶĚĞƌĚĞƚĞĐƚĞĚƚǁŽĂĐƋƵŝƌĞĚɴͲůĂĐƚĂŵĂƐĞŐĞŶĞǀĂƌŝĂŶƚƐĚĞƐŝŐŶĂƚĞĚbla

1b

and bla

͘ĚĚŝƟŽŶĂůůLJ͕ĂŶͲϲŵƉɴͲůĂĐƚĂŵĂƐĞͲĞŶĐŽĚŝŶŐŐĞŶĞǁĂƐ

detected

͘/ƐŽůĂƚĞϰǁĂƐƐŚŽǁŶƚŽďĞůŽŶŐƚŽ^dͲϭϯϭ͘

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: 39 PDF page: 39 PDF page: 39

39

ŚĂƉƚ ĞƌϤ

dĂďůĞϭ

ŶƟďŝŽƟĐ Isolate 1

WĂƟĞŶƚϭ

E. cloacae

^dͲϮϯϮ

Isolate 2 WĂƟĞŶƚϭ

E. cloacae

^dͲϵϳ

Isolate 3 WĂƟĞŶƚϭ K.

pneumoniae

^dͲϮϬ

Isolate 4 WĂƟĞŶƚϮ

E. coli

^dͲϭϯϭ D/

;ŵŐͬ>Ϳ

D/;

ŵŐͬ>Ϳ

D/

;ŵŐͬ>Ϳ

D/

;ŵŐͬ>Ϳ

ŵŽdžŝĐŝůůŝŶнĐůĂǀƵůĂŶŝĐ acid

>32 >32 >32 >32

ĞĨƵƌŽdžŝŵĞ хϲϰ 32 хϲϰ хϲϰ

ĞĨŽƚĂdžŝŵĞ хϲϰ фϭ͘Ϭ ϭϲ хϲϰ

ĞŌĂnjŝĚŝŵĞ хϲϰ фϭ͘Ϭ 1 хϲϰ

ĞĨŽdžŝƟŶ хϲϰ хϲϰ хϲϰ 32

ĞĨĞƉŝŵĞ 2 фϭ͘Ϭ 2 ϭϲ

DĞƌŽƉĞŶĞŵ 8 фϬ͘Ϯϱ

Ϭ͘ϬϯϮ 4

/ŵŝƉĞŶĞŵ 8 фϬ͘Ϯϱ

Ϭ͘ϱ Ϭ͘ϳϱ

^>Ύ ŶĞŐĂƟǀĞ ŶĞŐĂƟǀĞ ŶĞŐĂƟǀĞ ƉŽƐŝƟǀĞ

ŶƟďŝŽƟĐ ƐƵƐĐĞƉƟďŝůŝƚLJ ŽĨ ƚŚĞ ĨŽƵƌ ďĂĐƚĞƌŝĂů ŝƐŽůĂƚĞƐ ĚĞƐĐƌŝďĞĚ ŝŶ ƚŚŝƐ ƐƚƵĚLJ͘ ůů ĨŽƵƌ

ŝƐŽůĂƚĞƐĐĂƌƌŝĞĚĂD/ZϭϳͲ'/ͲƌĞůĂƚĞĚD'/͘/ŶƚŚƌĞĞŽĨƚŚĞĨŽƵƌŝƐŽůĂƚĞƐ͕ĂĐĞƉŚĂůŽƐƉŽƌŝŶĂƐĞ gene (ampCͿǁĂƐŝŶƚĞŐƌĂƚĞĚŝŶƚŚŝƐŝƐůĂŶĚ͘

#

ĂƌďĂƉĞŶĞŵͲƌĞƐŝƐƚĂŶƚĐŽůŽŶLJǀĂƌŝĂŶƚƐŐƌĞǁŝŶƚŽƚŚĞĐĂƌďĂƉĞŶĞŵŝŶŚŝďŝƟŽŶnjŽŶĞ͘

Ύ^>ƉƌŽĚƵĐƟŽŶǁĂƐƚĞƐƚĞĚďLJͲƚĞƐƚƐĂŶĚŝŶĚŝĐĂƚĞĚďLJĂхϴͲĨŽůĚƌĞĚƵĐƟŽŶŝŶD/ŝŶ

ƚŚĞƉƌĞƐĞŶĐĞŽĨĐůĂǀƵůĂŶŝĐĂĐŝĚĐŽŵƉĂƌĞĚƚŽĐĞĨĞƉŝŵĞ͕ĐĞŌĂnjŝĚŝŵĞŽƌĐĞĨŽƚĂdžŝŵĞĂůŽŶĞ͘

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: 40 PDF page: 40 PDF page: 40

ϰϬ

&ŝŐƵƌĞϭ͘/ŵŝƉĞŶĞŵƚĞƐƚĨŽƌ E. cloacaeƐƚƵĚLJŝƐŽůĂƚĞϭŽŶDƵůůĞƌ,ŝŶƚŽŶĂŐĂƌǁŝƚŚŽƌ ǁŝƚŚŽƵƚĐůŽdžĂĐŝůůŝŶ͘

dŚĞŐĞŶŽŵŝĐŶĞŝŐŚďŽƌŚŽŽĚŽĨďůĂ

ŝƐĐŽŶƐĞƌǀĞĚŝŶEnterobacteriaceae

^ŝŶĐĞ ƚŚĞ ĐĞƉŚĂůŽƐƉŽƌŝŶĂƐĞ ŐĞŶĞƐ ŝĚĞŶƟĮĞĚ ŝŶ ƚŚĞ ĨŽƵƌ ƐƚƵĚLJ ŝƐŽůĂƚĞƐ ǁĞƌĞ

ĚŝƐƟŶĐƚ͕ƚŚĞƌĞƐƉĞĐƟǀĞĂŶƟďŝŽƟĐƌĞƐŝƐƚĂŶĐĞƉŚĞŶŽƚLJƉĞƐĐŽƵůĚŶŽƚďĞĚŝƌĞĐƚůLJ ƌĞůĂƚĞĚ ƚŽ ƌĞĐĞŶƚ ŚŽƌŝnjŽŶƚĂů ŐĞŶĞ ƚƌĂŶƐĨĞƌ ĞǀĞŶƚƐ ďĞƚǁĞĞŶ ƚŚĞƐĞ ŝƐŽůĂƚĞƐ͘

,ŽǁĞǀĞƌ͕ĐŽŵƉĂƌŝƐŽŶŽĨƚŚĞǁŚŽůĞŐĞŶŽŵĞƐĞƋƵĞŶĐŝŶŐĚĂƚĂƵŶĐŽǀĞƌĞĚΕϭϰϬͲ

ŬďƌĞŐŝŽŶƐŽĨŚŝŐŚƐŝŵŝůĂƌŝƚLJŝŶƚŚĞĨŽƵƌŝƐŽůĂƚĞƐ͕ǁŚŝĐŚŚĂƌďŽƌampC genes in the C

ĐĂƐĞŽĨŝƐŽůĂƚĞƐϭ͕ϮĂŶĚϰ;&ŝŐƵƌĞϮͿ͘dŚŝƐƉƌŽŵƉƚĞĚƵƐƚŽŝŶǀĞƐƟŐĂƚĞƚŚĞƐƉƌĞĂĚ

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: 41 PDF page: 41 PDF page: 41

41

ŚĂƉƚ ĞƌϤ

ŽĨƚŚŝƐĐŽŶƐĞƌǀĞĚƌĞŐŝŽŶĂŵŽŶŐEnterobacteriaceae͘

ƐƐŚŽǁŶŝŶ&ŝŐƵƌĞϯĂŶĚ^ƵƉƉůĞŵĞŶƚĂů&ŝŐƵƌĞ^ϭ͕ƚŚĞampCʹĐŽŶƚĂŝŶŝŶŐƌĞŐŝŽŶ͕ CC ĂƐƉƌĞƐĞŶƚŝŶ E. coli͕ŝƐŚŝŐŚůLJĐŽŶƐĞƌǀĞĚŝŶƚŚĞŐĞŶĞƌĂCitrobacter, Escherichia, Enterobacter, Salmonella, Shigella and Klebsiella͕ǁŚĞƌĞƚŚĞƐĞƋƵĞŶĐĞƐĐůƵƐƚĞƌ ĂĐĐŽƌĚŝŶŐƚŽƚŚĞƌĞƐƉĞĐƟǀĞƐƉĞĐŝĞƐ͘KĨŶŽƚĞ͕ampC genes are absent from the C ƌĞƐƉĞĐƟǀĞĐŽŶƐĞƌǀĞĚƌĞŐŝŽŶƐŝŶ Salmonella and Klebsiella ƐƉĞĐŝĞƐ͕ĂƐŝƐƚŚĞĐĂƐĞ ŝŶ ŽƵƌ K. pneumoniae ƐƚƵĚLJ ŝƐŽůĂƚĞ ϯ ;&ŝŐƵƌĞ ϮͿ͘ &ƵƌƚŚĞƌ͕ ǁĞ ŽďƐĞƌǀĞĚ ƚŚĂƚ

ƉĂƌƚƐŽĨƚŚŝƐĐŽŶƐĞƌǀĞĚƌĞŐŝŽŶĂƌĞƉƌĞƐĞŶƚŝŶŽƚŚĞƌEnterobacteriaceae͕ƐƵĐŚĂƐ

ƌŽŶŽďĂĐƚĞƌ͕ŝĐŬLJĂ͕WƌŽƚĞƵƐ͕WƌŽǀŝĚĞŶĐŝĂ͕^ĞƌƌĂƟĂand YersiniaƐƉĞĐŝĞƐ͕ǁŚĞƌĞ

ŝƚůĂĐŬƐ ampC ĂŶĚĐĂŶďĞƉŽƐŝƟŽŶĞĚĂƚĚŝīĞƌĞŶƚĐŚƌŽŵŽƐŽŵĂůůŽĐŝ;ŶŽƚƐŚŽǁŶͿ͘ C dŚĞĐŽŶƐĞƌǀĞĚƌĞŐŝŽŶŝƐĐŽŵƉůĞƚĞůLJĂďƐĞŶƚĨƌŽŵŶŽŶͲĞŶƚĞƌŽďĂĐƚĞƌŝĂĐĞĂů'ƌĂŵͲ ŶĞŐĂƟǀĞďĂĐƚĞƌŝĂ͕ƐƵĐŚĂƐAcinetobacter, Haemophilus, Pasteurella, Prevotella and Pseudomonas ƐƉĞĐŝĞƐ͘ŽŵďŝŶĞĚǁŝƚŚƉƌĞǀŝŽƵƐƐƚƵĚŝĞƐŽŶƚŚĞƉŚLJůŽŐĞŶLJ

of Enterobacteriaceae

͕ƚŚĞƐĞŽďƐĞƌǀĂƟŽŶƐĂƌĞŝŶĚŝĐĂƟǀĞŽĨĂŶĂŶĐŝĞŶƚ D'/ ƚŚĂƚ ƐƉƌĞĂĚ ĂŵŽŶŐ ƚŚĞ ĞŶƚĞƌŽďĂĐƚĞƌŝĂĐĞĂů ĂŶĐĞƐƚƌLJ͕ ďĞĨŽƌĞ ŐĞŶĞƌĂ ůŝŬĞ

Enterobacter, Escherichia, and Klebsiella ĞǀŽůǀĞĚ͘

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: 42 PDF page: 42 PDF page: 42

42

&ŝŐƵƌĞϮ͘^ŝŵŝůĂƌŝƚLJĂŵŽŶŐD/ZϭϳͲ'/ͲůŝŬĞD'/ƐĂŶĚƐĐŚĞŵĂƟĐƌĞƉƌĞƐĞŶƚĂƟŽŶŽĨƚŚĞWϰͲ ůŝŬĞƉƌŽƉŚĂŐĞĂƚƚŚĞϯ͛ĞŶĚŽĨD/ZϭϳͲ'/͘

;ͿD/ZϭϳͲ'/ͲůŝŬĞD'/ƐŝĚĞŶƟĮĞĚŝŶƚŚĞĨŽƵƌƐƚƵĚLJŝƐŽůĂƚĞƐĂŶĚE. coli<ϭϮD'ϭϲϱϱ͘dŚĞ i ĂůŝŐŶŵĞŶƚŽĨD/ZϭϳͲ'/ͲůŝŬĞD'/ƐǁĂƐƉĞƌĨŽƌŵĞĚǁŝƚŚĂƐLJĮŐ;ŚƩƉ͗ͬͬŵũƐƵůů͘ŐŝƚŚƵď͘ŝŽͬ

ĂƐLJĮŐͬͿ͘dŚĞƉŽƐŝƟŽŶƐŽĨƌĞůĞǀĂŶƚŐĞŶĞƐĂƌĞŝŶĚŝĐĂƚĞĚ͘

;Ϳ'ĞŶĞƟĐŵĂƉŽĨƚŚĞĞĂƌůLJŽƉĞƌŽŶŽĨƚŚĞWϰͲůŝŬĞƉƌŽƉŚĂŐĞĂƐƐŽĐŝĂƚĞĚǁŝƚŚƚŚĞϯ͛ĞŶĚ ŽĨD/ZϭϳͲ'/͕ĨŽůůŽǁŝŶŐƚŚĞƚZEͲ>ĞƵͲŐĞŶĞ͘dŚĞƌĞůĂƟǀĞƉŽƐŝƟŽŶƐŽĨƚŚĞŐĞŶĞƐĨŽƌ

ƚŚĞ ƉŚĂŐĞ ŝŶƚĞŐƌĂƐĞ͕ ĂŶ dWͲĂƐĞ ŽĨ ƵŶŬŶŽǁŶ ĨƵŶĐƟŽŶ ;dWͲĂƐĞͿ͕ ƚŚĞ ƉŚĂŐĞ ƉŽůĂƌŝƚLJ

ƐƵƉƉƌĞƐƐŝŽŶƉƌŽƚĞŝŶ;ƉƐƵͿ͕ƚŚĞƉŚĂŐĞĐĂƉƐŝĚĂŶĚƐĐĂīŽůĚƉƌŽƚĞŝŶ;ƐŝĚͿ͕ƚŚĞƐŚĨĂŵŝůLJ ƐĞĐŽŶĚĂƌLJ ŝŵŵƵŶŝƚLJ ƌĞƉƌĞƐƐŽƌ ;cIͿ͖ ƚŚĞ E ƌĞƉůŝĐĂƟŽŶ ƉƌŽƚĞŝŶ ;repA)͕ ĂŶĚ ƚŚĞ E

ƉƌŝŵĂƐĞĂƌĞŝŶĚŝĐĂƚĞĚ͘EƵŵďĞƌƐĐŽƌƌĞƐƉŽŶĚƚŽƚŚĞĨŽůůŽǁŝŶŐŐĞŶĞĂŶŶŽƚĂƟŽŶƐ͗ϭ͕ůĂƚĞ

ŐĞŶĞƌĞŐƵůĂƚŽƌ͖Ϯ͕ƉŚĂŐĞEďŝŶĚŝŶŐƉƌŽƚĞŝŶ͖ϯ͕ƉŚĂŐĞŝŵŵƵŶŝƚLJƌĞƉƌĞƐƐŽƌƉƌŽƚĞŝŶ͖ϰ͕

ŝŵŵƵŶŝƚLJĚĞƌĞƉƌĞƐƐŝŽŶƉƌŽƚĞŝŶ͖ϱ͕ƉŚĂŐĞƉƌŽƚĞŝŶŽĨƵŶŬŶŽǁŶĨƵŶĐƟŽŶ͖ϲ͕ŚLJƉŽƚŚĞƟĐĂů

ƉƌŽƚĞŝŶĨŽƌŚĞŵĞƚƌĂŶƐĨĞƌĚƵƌŝŶŐĐLJƚŽĐŚƌŽŵĞĐďŝŽŐĞŶĞƐŝƐ͘

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: 43 PDF page: 43 PDF page: 43

43

ŚĂƉƚ ĞƌϤ

dŚĞďůĂ

ɴͲůĂĐƚĂŵĂƐĞŐĞŶĞŽĨE. cloacae ŝƐŽůĂƚĞϭŝƐůŽĐĂƚĞĚŽŶĂŶĂŶĐŝĞŶƚ

ŵŽďŝůĞŐĞŶŽŵŝĐŝƐůĂŶĚ

ŶĂůLJƐŝƐ ŽĨ ƚŚĞ ĐŽŶƐĞƌǀĞĚ ŐĞŶŽŵŝĐ ŶĞŝŐŚďŽƌŚŽŽĚ ŽĨ ƚŚĞ bla

gene in E.

cloacae ŝƐŽůĂƚĞ ϭ ƌĞǀĞĂůĞĚ ƚŚĞ ƉƌĞƐĞŶĐĞ ŽĨ ƐĞǀĞƌĂů ŐĞŶĞƐ ƚŚĂƚ ĂƌĞ ĐŽŵŵŽŶůLJ

ĨŽƵŶĚ ŽŶ D'/Ɛ͕ ĂƐ ĚĞƐĐƌŝďĞĚ ŝŶ ĚĞƚĂŝů ŝŶ ƚŚĞ ĨŽůůŽǁŝŶŐ ƉĂƌĂŐƌĂƉŚ͘ dŚĞ ŝĚĞĂ

that bla

ĐŽƵůĚ ďĞ ƉĂƌƚ ŽĨ Ă D'/ ǁĂƐ ĨƵƌƚŚĞƌ ĐŽƌƌŽďŽƌĂƚĞĚ ďLJ ŝŶƐƉĞĐƟŽŶ

ŽĨ ƚŚĞ ŐĞŶŽŵĞ ƐĞƋƵĞŶĐĞ ŽĨ E. cloacae ŝƐŽůĂƚĞ ϭ͕ ǁŚŝĐŚ ƌĞǀĞĂůĞĚ ƚŚĂƚ ŝŶĚĞĞĚ Ă ϭϰϬͲŬď D'/ ŝŶĐůƵĚŝŶŐ bla

ŚĂĚ ŝŶƚĞŐƌĂƚĞĚ ŝŶ ƚŚĞ WŚĞͲ' ƚZE ŐĞŶĞ͘

KĨ ŶŽƚĞ͕ ƚŚŝƐ ƚZE ŐĞŶĞ ĐŽŶƚĂŝŶƐ Ă ƉŚĂŐĞ WϰͲĂƐƐŽĐŝĂƚĞĚ ĂƩĂĐŚŵĞŶƚ ƐŝƚĞ (''d''dd''Ή

ŝŶƚŚĞϯ͛Ͳϱ͛ĚŝƌĞĐƟŽŶ;^ƵƉƉůĞŵĞŶƚĂů&ŝŐƵƌĞ^ϮͿ͘

^ŝŶĐĞƚŚĞŝĚĞŶƟĮĞĚD'/ĐĂƌƌŝĞƐƚŚĞbla

ŐĞŶĞ͕ǁĞŶĂŵĞĚŝƚD/ZϭϳͲ'/͘ƚƚŚĞ ϯ͛ĞŶĚŽĨƚŚĞD/ZϭϳͲ'/͕ĚŽǁŶƐƚƌĞĂŵŽĨĂ>ĞƵͲƚZEŐĞŶĞ͕ĂŶŝŶƚĞŐƌĂƚĞĚ

Wϰ ƉƌŽƉŚĂŐĞ ǁĂƐ ĚĞƚĞĐƚĞĚ ;&ŝŐƵƌĞ ϮͿ͘  >^dͲdž ĂŶĂůLJƐŝƐ ŽĨ ƚŚĞ ƌĞƉůŝĐĂƟǀĞ ŚĞůŝĐĂƐĞ ŐĞŶĞ ŽĨ ƚŚŝƐ Wϰ ƉƌŽƉŚĂŐĞ ŝŶĚŝĐĂƚĞĚ ƚŚĂƚ ŝƚ ŝƐ ŵŽƐƚ ĐůŽƐĞůLJ ƌĞůĂƚĞĚ ƚŽ

ĂWϰďĂĐƚĞƌŝŽƉŚĂŐĞŽĨSalmonella enterica;'/͗ϯϴϬϰϲϰϮϰϳ͕ϴϳйŝĚĞŶƟƚLJ͕ϭϬϬй ĐŽǀĞƌĂŐĞͿ͘

dŚĞϱ͛ƐŝĚĞŽĨD/ZϭϳͲ'/ŝƐƐĐŚĞŵĂƟĐĂůůLJƌĞƉƌĞƐĞŶƚĞĚŝŶ^ƵƉƉůĞŵĞŶƚĂů&ŝŐƵƌĞ^Ϯ͘

The bla

ŐĞŶĞůŽĐĂƚĞĚŽŶƚŚĞƌĞǀĞƌƐĞƐƚƌĂŶĚŝƐŇĂŶŬĞĚďLJĂŶ ampRƌĞŐƵůĂƚŽƌ ŐĞŶĞŽŶƚŚĞĨŽƌǁĂƌĚƐƚƌĂŶĚ͘dŚĞbla

ͲampRŐĞŶĞƐĂƌĞƉŽƐŝƟŽŶĞĚǁŝƚŚŝŶĂ ϯϮͲŬďƌĞŐŝŽŶŽĨD/ZϭϳͲ'/ƚŚĂƚŝƐŇĂŶŬĞĚďLJƚŚĞWŚĞͲ'ƚZEŐĞŶĞǁŝƚŚƚŚĞ

WϰĂƩĂĐŚŵĞŶƚƐŝƚĞĂƚƚŚĞϱ͛ĞŶĚ͕ĂŶĚĂƚƌŝƉůĞƚŽĨ'ůLJͲ'ƚZEŐĞŶĞƐĂƚƚŚĞϯ͛

ĞŶĚ͘KĨŶŽƚĞ͕ƚŚŝƐƌĞŐŝŽŶĐŽŶƚĂŝŶƐƐĞǀĞƌĂůĂĚĚŝƟŽŶĂůƌĞƐŝƐƚĂŶĐĞŐĞŶĞƐ͕ƉŽƚĞŶƟĂůůLJ

ƉƌŽǀŝĚŝŶŐƌĞƐŝƐƚĂŶĐĞƚŽĐĂƟŽŶƐĂŶĚŚĞĂǀLJŵĞƚĂůƐ;cutA1͕ cutA2)

͕ŽƌƋƵĂƚĞƌŶĂƌLJ

ĂŵŵŽŶŝƵŵĐŽŵƉŽƵŶĚƐ;sugE)

͘&ƵƌƚŚĞƌ͕ŝƚŝŶĐůƵĚĞƐƚŚĞŐĞŶĞĨŽƌĂŵĞĐŚĂŶŽͲ ƐĞŶƐŝƟǀĞƉŽƚĂƐƐŝƵŵĞŋƵdžƉƵŵƉ;kefAͿƚŚĂƚĐŽƵůĚďĞŝŶǀŽůǀĞĚŝŶŽƐŵŽͲƉƌŽƚĞĐƟŽŶ

27

͕ ĂŶĚ ŐĞŶĞƐ ĨŽƌ ƚǁŽ ŽƵƚĞƌ ŵĞŵďƌĂŶĞ ůŝƉŽĐĂůŝŶƐ ;blc͕ yjelͿ ŝŵƉůŝĐĂƚĞĚ ŝŶ ƚŚĞ

ƚƌĂŶƐƉŽƌƚŽĨƐŵĂůůŚLJĚƌŽƉŚŽďŝĐŵŽůĞĐƵůĞƐ

͘dŚĞĮƌƐƚŐĞŶĞŽĨƚŚĞŝƐůĂŶĚĞŶĐŽĚĞƐ ĂƌĞŐƵůĂƚŽƌŽĨƚŚĞdĞƚZƚƌĂŶƐĐƌŝƉƟŽŶƌĞŐƵůĂƚŽƌĨĂŵŝůLJ͕ĂŶĚƚŚŝƐŐĞŶĞŝƐůŽĐĂƚĞĚ

downstream of the cutA1 and cutA2ŐĞŶĞƐ͘ZĞŐƵůĂƚŽƌƐŽĨƚŚĞdĞƚZĨĂŵŝůLJƌĞƉƌĞƐƐ

ŐĞŶĞƚƌĂŶƐĐƌŝƉƟŽŶ͕ĂŶĚƚƌĂŶƐĐƌŝƉƟŽŶŝƐĚĞƌĞƉƌĞƐƐĞĚŝŶƌĞƐƉŽŶƐĞƚŽƐƚƌĞƐƐ

͘

D/ZϭϳͲ'/ ĂůƐŽ ĐĂƌƌŝĞƐ ƐĞǀĞƌĂů ŐĞŶĞƐ ƚŚĂƚ ĂƌĞ ŬŶŽǁŶ ƚŽ ŚĂǀĞ ŚŽƵƐĞŬĞĞƉŝŶŐ

ĨƵŶĐƟŽŶƐ͘/ŶƉĂƌƟĐƵůĂƌ͕ŽŶƚŚĞĨŽƌǁĂƌĚƐƚƌĂŶĚ͕ƚŚĞgroESͲgroEL genes encode

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: 44 PDF page: 44 PDF page: 44

44

ĐŚĂƉĞƌŽŶĞƐ ŝŵƉůŝĐĂƚĞĚ ŝŶ ƉƌŽƚĞŝŶ ĨŽůĚŝŶŐ ĂŶĚ ĐĞůů ĐLJĐůĞ ƌĞŐƵůĂƟŽŶ

͘ KŶ ƚŚĞ

ƌĞǀĞƌƐĞƐƚƌĂŶĚŽĨD/ZϭϳͲ'/͕ƚŚĞƌĞĂƌĞƚŚĞfrdABCDŐĞŶĞƐƉŽƚĞŶƟĂůůLJŝŶǀŽůǀĞĚŝŶ

ĂŶĂĞƌŽďŝĐƌĞƐƉŝƌĂƟŽŶ͘^ƵĐŚ frd ŐĞŶĞƐǁĞƌĞƉƌĞǀŝŽƵƐůLJŝŵƉůŝĐĂƚĞĚŝŶƚƌĂŶƐĚƵĐƟŽŶ d ďLJ ƉŚĂŐĞƐ

͘ >ĂƐƚůLJ͕ D/ZϭϳͲ'/ ĐĂƌƌŝĞƐ ŐĞŶĞƐ ŝŶǀŽůǀĞĚ ŝŶ ŵĂŝŶƚĞŶĂŶĐĞ ŽĨ E

ĂŶĚŵŽďŝůĞŐĞŶĞƟĐĞůĞŵĞŶƚƐ͘dŚĞecnA and ecnB genes located downstream of bla

ĞŶĐŽĚĞƚŚĞŶƚĞƌŝĐŝĚŝŶĂŶĚƚŽdžŝŶͲĂŶƟƚŽdžŝŶƉĂŝƌƉƌĞǀŝŽƵƐůLJƌĞƉŽƌƚĞĚ

ƚŽ ƉƌĞǀĞŶƚ ůŽƐƐ ŽĨ ƉůĂƐŵŝĚƐ

͘ ůƐŽ ůŽĐĂƚĞĚ ĚŽǁŶƐƚƌĞĂŵ ŽĨ bla

͕ ƚŚĞ fxsA ŐĞŶĞĞŶĐŽĚĞƐĂƉŽůLJƚŽƉŝĐŵĞŵďƌĂŶĞƉƌŽƚĞŝŶƚŚĂƚƉƌĞǀĞŶƚƐďĂĐƚĞƌŝŽƉŚĂŐĞƐĨƌŽŵ

ĞdžĐůƵƐŝŽŶ

͘ hƉƐƚƌĞĂŵ ŽĨ bla

͕ ƚŚĞ E ƌĞƉĂŝƌ ŐĞŶĞ mutL and the high ĨƌĞƋƵĞŶĐLJŽĨůLJƐŽŐĞŶLJŽƉĞƌŽŶ ŚŇYy< ĂƌĞůŽĐĂƚĞĚ;^ƵƉƉůĞŵĞŶƚĂů&ŝŐƵƌĞ^ϮͿ͘  EŽƚĂďůLJ͕ƚŚĞĂďŽǀĞͲůŝƐƚĞĚŐĞŶĞƐƌĞƉƌĞƐĞŶƚƚŚĞĐŽŵŵŽŶĐŽŶƚĞdžƚŽĨĐŚƌŽŵŽƐŽŵĂů

ampC genes in C Citrobacter͕ r Escherichia͕ Enterobacter͕ ĂŶĚ r Shigella ƐƉĞĐŝĞƐ͕

ǁŚŝĐŚ ŝŵƉůŝĞƐ ƚŚĂƚ D/ZϭϳͲ'/ ŝƐ ĂŶ ĂŶĐŝĞŶƚ ŵŽďŝůĞ ŐĞŶŽŵŝĐ ĞůĞŵĞŶƚ ĂĐƋƵŝƌĞĚ ďĞĨŽƌĞƐƉĞĐŝĂƟŽŶ͘dŚĞůĂƩĞƌǀŝĞǁŝƐƐƵƉƉŽƌƚĞĚďLJƚŚĞĨĂĐƚƚŚĂƚƚŚĞ'ĐŽŶƚĞŶƚ

ŽĨƚŚĞD/ZϭϳͲ'/ĂŶĚŝƚƐůĞŌĂŶĚƌŝŐŚƚŇĂŶŬŝŶŐƐĞƋƵĞŶĐĞƐŝƐƋƵŝƚĞƐŝŵŝůĂƌĂŵŽŶŐ

Enterobacteriacaea͕ĂƐĞdžĞŵƉůŝĮĞĚŝŶ&ŝŐƵƌĞϰĨŽƌƚŚĞE. cloacae ƐƚƵĚLJŝƐŽůĂƚĞϭ and the E. coli<ϭϮƌĞĨĞƌĞŶĐĞƐƚƌĂŝŶD'ϭϲϱϱ͘dŚŝƐŝƐĐŽŶƐŝƐƚĞŶƚǁŝƚŚƚŚĞŶŽƟŽŶ i ƚŚĂƚŚŽƌŝnjŽŶƚĂůůLJƚƌĂŶƐĨĞƌƌĞĚEǁŝůůĂĚĂƉƚƚŽƚŚĞŚŽƐƚŐĞŶŽŵĞŽǀĞƌƟŵĞ͕Ă ƉƌŽĐĞƐƐŬŶŽǁŶĂƐŐĞŶŽŵĞĂŵĞůŝŽƌĂƟŽŶ

͘

>ĂŶĚŵĂƌŬĨĞĂƚƵƌĞƐŽĨD/ZϭϳͲ'/ͲůŝŬĞD'/ƐŝŶEnterobacteriaceae

^ĞǀĞƌĂů ͚ůĂŶĚŵĂƌŬ͛ ĨĞĂƚƵƌĞƐ ŽĨ ƚŚĞ D/ZϭϳͲ'/ ŚŽŵŽůŽŐŽƵƐ D'/Ɛ ĐĂŶ ďĞ

ĚŝƐƟŶŐƵŝƐŚĞĚ͘/ŶƚŚĞĮƌƐƚƉůĂĐĞ͕ƚŚĞƌĞƐƉĞĐƟǀĞƚZEŐĞŶĞƐĂƌĞŚŝŐŚůLJĐŽŶƐĞƌǀĞĚ

ŝŶĂůůƚŚĞƐĞŝƐůĂŶĚƐ͕ƐƚĂƌƟŶŐǁŝƚŚƚŚĞWŚĞͲ'ƚZEŐĞŶĞĂƚƚŚĞϱ͛ĞŶĚ͕ĨŽůůŽǁĞĚ

ďLJƚŚĞƚƌŝƉůĞƚ'ůLJͲ'ƚZEŐĞŶĞƐ͕ĂŶĚĞŶĚŝŶŐǁŝƚŚƚŚĞ>ĞƵͲƚZEŐĞŶĞĂƚ

ƚŚĞϯ͛ĞŶĚ͘dŚĞWŚĞͲ'ƚZEĂŶĚƚŚĞ>ĞƵͲƚZEŐĞŶĞƐĚĞĮŶĞƚŚĞďŽƌĚĞƌƐ

ŽĨƚŚĞD'/Ɛ͕ǁŚŝĐŚǀĂƌLJŝŶůĞŶŐƚŚĨƌŽŵϭϯϲƚŽϭϰϴŬďŝŶE. coli͕ E. cloacae and K.

pneumoniae;&ŝŐƵƌĞϮͿ͘&ƵƌƚŚĞƌ͕ƚŚĞƐĞD'/ƐƐŚĂƌĞƚŚĞĂĨŽƌĞͲŵĞŶƟŽŶĞĚ fxsA͕

groESͬEL͕ encB͕frdABCD and ŚŇYy<ŐĞŶĞƐ͕ĂƐǁĞůůĂƐĂƚƌĞŚĂůŽƐĞŽƉĞƌŽŶ͕Ă  ƉƵƚĂƟǀĞƐƵŐĂƌƚƌĂŶƐƉŽƌƚŐĞŶĞĐůƵƐƚĞƌ͕ĂƉƌŝŵŽƐŽŵĂůƌĞƉůŝĐĂƟŽŶƉƌŽƚĞŝŶEŐĞŶĞ

ĐůƵƐƚĞƌ͕ ĂŶĚ ĂŶ >ͲĂƐĐŽƌďĂƚĞ ƵƟůŝnjĂƟŽŶ ŐĞŶĞ ĐůƵƐƚĞƌ͘ >ĂƐƚůLJ͕ Ă WϰͲĂƐƐŽĐŝĂƚĞĚ

ŝŶƚĞŐƌĂƐĞŐĞŶĞŝƐůŽĐĂƚĞĚŶĞdžƚƚŽƚŚĞ>hͲƚZEŐĞŶĞ;&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: 45 PDF page: 45 PDF page: 45

45

ŚĂƉƚ ĞƌϤ

ŝǀĞƌƐŝƚLJŽĨampC ŐĞŶĞƐŝŶD/ZϭϳͲ'/ͲůŝŬĞD'/Ɛ C

The ampC ŐĞŶĞƐŽĨD/ZϭϳͲ'/ͲůŝŬĞD'/ƐĂƌĞŝŶƚĞŐƌĂƚĞĚĂƚƚŚĞƐĂŵĞůŽĐĂƟŽŶŝŶ C all Citrobacter͕r Escherichia͕ Enterobacter͕ĂŶĚ r ShigellaŐĞŶŽŵĞƐ͕ŶĞdžƚƚŽsugEͲ blcŐĞŶĞƐ͘EĞǀĞƌƚŚĞůĞƐƐ͕ƚŚĞƐĞƋƵĞŶĐĞƐŽĨƚŚĞƐĞ ampCŐĞŶĞƐĂƌĞƋƵŝƚĞĚŝǀĞƌƐĞ͘ C

ƐƐŚŽǁŶŝŶƚŚĞƉŚLJůŽŐĞŶĞƟĐƚƌĞĞŝŶ&ŝŐƵƌĞϱ͕ƚŚĞ ampC genes of C Citrobacter and Enterobacter ƐƉĞĐŝĞƐĂƌĞƌĞůĂƟǀĞůLJĐůŽƐĞůLJƌĞůĂƚĞĚƚŽĞĂĐŚŽƚŚĞƌ͕ĨŽƌŵŝŶŐĂ r ĚŝƐƟŶĐƚĐůƵƐƚĞƌĨƌŽŵƚŚĞampCŐĞŶĞƐĞŶĐŽƵŶƚĞƌĞĚŝŶ C Escherichia and Shigella ƐƉĞĐŝĞƐ͘tŝƚŚŝŶE. coli and i Shigella͕ƚŚĞĐůƵƐƚĞƌŝŶŐŽĨampC ŐĞŶĞƐĐŽƌƌĞƐƉŽŶĚƐ C ǁĞůů ǁŝƚŚ ƚŚĞ ĚŝīĞƌĞŶƚ ƉŚLJůŽŐĞŶĞƟĐ ŐƌŽƵƉƐ ĂŶĚ ƐƉĞĐŝĞƐ͕ ĂůƚŚŽƵŐŚ ƚŚĞ ampC genes of E. coli ŝƐŽůĂƚĞƐ ďĞůŽŶŐŝŶŐ ƚŽ ƉŚLJůŽŐĞŶĞƟĐ ŐƌŽƵƉ  ĂƌĞ ĚŝǀŝĚĞĚ ŽǀĞƌ i ĨŽƵƌĐůƵƐƚĞƌƐ;^ƵƉƉůĞŵĞŶƚĂů&ŝŐƵƌĞ^ϯͿ͘dŚĞƐĞĮŶĚŝŶŐƐĂƌĞŝŶĚŝĐĂƟǀĞŽĨƐĞƉĂƌĂƚĞ

ĂĐƋƵŝƐŝƟŽŶƐŽĨampC ďLJƚŚĞD/ZϭϳͲ'/ͲůŝŬĞD'/ƐŽǀĞƌƟŵĞ͘ C

džƉƌĞƐƐŝŽŶƉƌŽĮůĞŽĨD'/ͲĞŶĐŽĚĞĚƉƌŽƚĞŝŶƐĨƌŽŵŝƐŽůĂƚĞƐϭĂŶĚϮ

dŚĞ ĨƌĞƋƵĞŶƚ ŽĐĐƵƌƌĞŶĐĞ ŽĨ D/ZϭϳͲ'/ͲůŝŬĞ D'/Ɛ ŝŶ Enterobacteriaceae raised ƚŚĞ ƋƵĞƐƟŽŶ ƚŽ ǁŚĂƚ ĞdžƚĞŶƚ ƉƌŽƚĞŝŶƐ ĞŶĐŽĚĞĚ ďLJ ƐƵĐŚ D'/Ɛ ĂƌĞ ĞdžƉƌĞƐƐĞĚ͘

dŽ ĂƉƉƌŽdžŝŵĂƚĞ ƉƌŽƚĞŝŶ ĞdžƉƌĞƐƐŝŽŶ ĨƌŽŵ D/ZϭϳͲ'/ͲůŝŬĞ ŝƐůĂŶĚƐ͕ ĐĞůůƐ ŽĨ ƚŚĞ E.

cloacae ƐƚƵĚLJŝƐŽůĂƚĞƐϭĂŶĚϮǁĞƌĞŝŶǀĞƐƟŐĂƚĞĚƵƐŝŶŐůŝƋƵŝĚĐŚƌŽŵĂƚŽŐƌĂƉŚLJ

ĂŶĚƚĂŶĚĞŵŵĂƐƐƐƉĞĐƚƌŽŵĞƚƌLJ;>ͲD^ͬD^Ϳ͘ƚŽƚĂůŶƵŵďĞƌŽĨϭϯϬϬĚŝīĞƌĞŶƚ E.

cloacae ƉƌŽƚĞŝŶƐǁĂƐŝĚĞŶƟĮĞĚĨŽƌďŽƚŚƐƚƌĂŝŶƐ͕ŝŶĐůƵĚŝŶŐϴϱϳƉƌŽƚĞŝŶƐŽĨŝƐŽůĂƚĞ

ϭ ĂŶĚ ϭϭϭϲ ƉƌŽƚĞŝŶƐ ŽĨ ŝƐŽůĂƚĞ Ϯ ;^ƵƉƉůĞŵĞŶƚĂƌLJ dĂďůĞ ^ϭͿ͘ dŚĞ D^ ĂŶĂůLJƐŝƐ

ŝĚĞŶƟĮĞĚƚŚĞůĂƌŐĞƐƚŶƵŵďĞƌƐŽĨĚŝīĞƌĞŶƚƉƌŽƚĞŝŶƐŝŶƚŚĞƐƚĂƟŽŶĂƌLJƉŚĂƐĞŽĨ

ŐƌŽǁƚŚ;&ŝŐƵƌĞϲ͖^ƵƉƉůĞŵĞŶƚĂů&ŝŐƵƌĞ^ϰͿ͘&ƵƌƚŚĞƌ͕ůĂďĞůͲĨƌĞĞƋƵĂŶƟĮĐĂƟŽŶŽĨ

ƚŚĞƉƌŽƚĞŝŶƐŝĚĞŶƟĮĞĚďLJD^;ŝ͘Ğ͘ƐƉĞĐƚƌĂůĐŽƵŶƟŶŐͿƐŚŽǁĞĚƐŝŵŝůĂƌƉĂƩĞƌŶƐ

ŽĨ ƌĞƉƌĞƐĞŶƚĂƟŽŶ ŽĨ ƚŚĞ ƉƌŽƚĞŝŶƐ ĚĞƚĞĐƚĞĚ Ăƚ ƚŚĞ ŚŝŐŚĞƐƚ ůĞǀĞůƐ ĂŵŽŶŐ ƚŚĞ

ƚǁŽŝŶǀĞƐƟŐĂƚĞĚŝƐŽůĂƚĞƐ;&ŝŐƵƌĞϳͿ͘/ŶĐŽŶƚƌĂƐƚ͕ƚŚĞƌĞǁĂƐƐƵďƐƚĂŶƟĂůůLJŵŽƌĞ

ǀĂƌŝĂƟŽŶ ŝŶ ƚŚĞ ŝĚĞŶƟĮĞĚ ƉƌŽƚĞŝŶƐ ĨƌŽŵ ďŽƚŚ ŝƐŽůĂƚĞƐ ƚŚĂƚ ǁĞƌĞ ĚĞƚĞĐƚĂďůĞ

ĂƚƌĞůĂƟǀĞůLJůŽǁůĞǀĞůƐ͘/ŵƉŽƌƚĂŶƚůLJ͕ĨŽƌŝƐŽůĂƚĞϭ͕ĞůĞǀĞŶŽĨƚŚĞϰϱD/ZϭϳͲ'/Ͳ ĞŶĐŽĚĞĚ ƉƌŽƚĞŝŶƐ ǁĞƌĞ ŝĚĞŶƟĮĞĚ͕ ŝŶĐůƵĚŝŶŐ ƚŚĞ D/Zϭϳ ĐĂƌďĂƉĞŶĞŵĂƐĞ ƚŚĂƚ

ǁĂƐĞdžƉƌĞƐƐĞĚĂƚƚŚĞŚŝŐŚĞƐƚůĞǀĞůƐŝŶƚŚĞƐƚĂƟŽŶĂƌLJŐƌŽǁƚŚƉŚĂƐĞ;&ŝŐƵƌĞϳ͕

Ϳ͘/ŶĨĂĐƚ͕ũƵĚŐĞĚďLJƐƉĞĐƚƌĂůĐŽƵŶƟŶŐ͕D/ZϭϳŝƐŽŶĞŽĨƚŚĞϰϬŵŽƐƚĂďƵŶĚĂŶƚ

ƉƌŽƚĞŝŶƐ ƚŚĂƚ ǁĞƌĞ ŝĚĞŶƟĮĞĚ ŝŶ ŝƐŽůĂƚĞ ϭ͘ &ƵƌƚŚĞƌ͕ ƚŚĞ D/ZϭϳͲ'/ͲĞŶĐŽĚĞĚ

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: 46 PDF page: 46 PDF page: 46

ϰϲ

'ƌŽ>ƉƌŽƚĞŝŶǁĂƐƚŚĞƐĞĐŽŶĚŵŽƐƚĂďƵŶĚĂŶƚƉƌŽƚĞŝŶŝĚĞŶƟĮĞĚďLJD^͘EŝŶĞ ƉƌŽƚĞŝŶƐĞŶĐŽĚĞĚďLJƚŚĞD/ZϭϳͲ'/ͲůŝŬĞD'/ŽĨŝƐŽůĂƚĞϮǁĞƌĞŝĚĞŶƟĮĞĚ͘KĨŶŽƚĞ͕

ƚŚĞ dͲϱ ɴͲůĂĐƚĂŵĂƐĞ ŽĨ ŝƐŽůĂƚĞ Ϯ ƌĞŵĂŝŶĞĚ ƵŶĚĞƚĞĐƚĞĚ͕ ǁŚŝĐŚ ŝƐ ĐŽŶƐŝƐƚĞŶƚ ǁŝƚŚ ƚŚĞ ƐƵƐĐĞƉƟďŝůŝƚLJ ŽĨ ƚŚŝƐ ŝƐŽůĂƚĞ ƚŽ ƚŚŝƌĚͲŐĞŶĞƌĂƟŽŶ ĐĞƉŚĂůŽƐƉŽƌŝŶƐ ĂŶĚ ĐĂƌďĂƉĞŶĞŵƐ͘ _&ŝŐƵƌ

Ğ ϯ͗ WŚ LJůŽŐ ĞŶĞƟĐ ƚƌ ĞĞ ŽĨ Citrobact er ͕rE nterobact er ͕rEscherichia ͕Kleb siella ͕Salmonella ͕ĂŶĚ Shigella ƐƉĞĐŝ ĞƐ ĂŶĚ ĐŽŶƐĞƌ ǀĂ ƟŽŶ ŽĨ D/ZϭϳͲ'/ͲůŝŬ Ğ D'/Ɛ͘ dŚĞ ƉŚ LJůŽŐ ĞŶĞ ƟĐ ƚƌ ĞĞ ŽĨ ǁ ĂƐ Đƌ ĞĂ ƚĞĚ ǁŝƚŚ ƚŚĞ ZŝĚŽŵ ^ĞƋ^ƉŚĞƌ Ğн ƐŽŌǁ Ăƌ Ğ ǀϰ͘ϭ͘ϵ ƵƐŝŶŐ Ă ŶĞŝŐŚďŽƌ ũŽŝŶŝŶŐ ĂůŐ ŽƌŝƚŚŵ͘ /ƚ ŝƐ ďĂƐĞĚ ŽŶ Ă ^EW ĂŶĂůLJ ƐŝƐ ŽĨ ϯϵϰϯϬ ƚĂƌ ŐĞ ƚƐ ŽĨ ƚŚĞ D'/ Ĩƌ Žŵ ƚŚĞ ƌĞ ĨĞƌ ĞŶĐĞ genome of the E. coli <ϭϮƐ ƚƌ ĂŝŶD'ϭϲϱϱ͘dŚĞƐĐ

ĂůĞďĂƌƵŶĚĞƌƚŚĞƚƌ

ĞĞƌ ĞƉƌ ĞƐĞŶ ƚƐƚŚĞ ƉŚ LJůŽŐ ĞŶĞ ƟĐĚŝƐ ƚĂŶĐĞ;ŝŶйͿ͘  i dŚĞ ƌĞĚ ďĂƌ ƐŝŶĚŝĐ ĂƚĞ ƚŚĞ ĐŽŶƐĞƌ ǀĂ ƟŽŶ ŽĨ ƚŚĞ D/ZϭϳͲ'/ ƵƐŝŶŐ ƚŚĞ ƌĞƐƉĞĐƟǀ Ğ D'/ ŽĨ E. coli <ϭϮ Ɛƚƌ ĂŝŶ D'ϭϲϱϱ ĂƐ ƚŚĞ i ƌĞ ĨĞƌ ĞŶĐĞ͘ &Žƌ ƚŚĞ D'/ ĐŽŵƉĂƌŝƐŽŶƐ͕ ĂŶ ϴϬй E ƐŝŵŝůĂƌŝƚLJ ĐƵƚ ͲŽī ǁ ĂƐ ƵƐĞĚ ŝŶ E ƉůŽƩ Ğƌ ͘dŚĞ ƐĐ ĂůĞ ďĂƌ ƵŶĚĞƌ ƚŚĞ D'/ĂůŝŐŶŵĞŶ ƚŝŶĚŝĐ ĂƚĞƐ

ƚŚĞƐĞƋƵĞŶĐĞƉŽƐŝƟŽŶ

ŽĨƚŚĞƌ ĞĨĞƌ ĞŶĐĞD '/͘ĂĐƚ ĞƌŝĂůŝƐŽůĂ ƚĞƐĂŶĚƚŚĞƌ ĞƐƉĞĐƟǀ ĞŐ ĞŶŽŵĞ

ƐĞƋƵĞŶĐĞĂĐĐĞƐƐŝŽŶĐ

ŽĚĞƐĂƌ ĞƉƌ ĞƐĞŶ ƚĞĚŝŶ^ƵƉƉůĞŵĞŶ ƚĂůd ĂďůĞ^Ϯ ͘

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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47

ŚĂƉƚ ĞƌϤ

&ŝŐƵƌĞϰ͘'ĐŽŶƚĞŶƚŽĨƚŚĞD/ZϭϳͲ'/ĨƌŽŵE. cloacae and the related MGI from E. coli ĂŶĚƚŚĞŝƌŇĂŶŬŝŶŐƌĞŐŝŽŶƐ͘ZĞĚďĂƌƐŝŶĚŝĐĂƚĞƚŚĞƉŽƐŝƟŽŶŽĨƚŚĞƌĞƐƉĞĐƟǀĞD'/ĂŶĚďůƵĞ ďĂƌƐƚŚĞĚŝīĞƌĞŶƚŇĂŶŬŝŶŐƌĞŐŝŽŶƐ͘dŚĞŽǀĞƌĂůů'йƉĞƌƌĞŐŝŽŶŝƐŝŶĚŝĐĂƚĞĚĂŶĚƚŚĞƌĞĚͲ ďůƵĞĚŝĂŐƌĂŵƐŵĂƌŬǀĂƌŝĂƟŽŶƐŝŶƚŚĞƌĞƐƉĞĐƟǀĞ'ƉƌŽĮůĞƐ͘

&ŝŐƵƌĞϱ͘EĞŝŐŚďŽƌũŽŝŶŝŶŐƚƌĞĞŽĨƚŚĞampC genes in C Citrobacter͕ r Escherichia͕Enterobacter and ShigellaƐƉĞĐŝĞƐ͘dŚĞƉŚLJůŽŐĞŶĞƟĐƚƌĞĞŽĨampC genes in C Citrobacter͕ r Escherichia͕

Enterobacter and r Shigella ƐƉĞĐŝĞƐ ǁĂƐ ĐƌĞĂƚĞĚ ǁŝƚŚ ƚŚĞ ZŝĚŽŵ ^ĞƋ^ƉŚĞƌĞн ƐŽŌǁĂƌĞ

ǀϰ͘ϭ͘ϵ ƵƐŝŶŐ Ă ŶĞŝŐŚďŽƌ ũŽŝŶŝŶŐ ĂůŐŽƌŝƚŚŵ͘ dŚĞ ƐĐĂůĞ ďĂƌ ƌĞƉƌĞƐĞŶƚƐ ƚŚĞ й ĚŝīĞƌĞŶĐĞ

ĂŵŽŶŐϳϳŶƵĐůĞŽƟĚĞƐŽĨampC͕ƵƐŝŶŐƚŚĞ C ampC gene of the C E. coli<ϭϮƐƚƌĂŝŶD'ϭϲϱϱĂƐ i ĂƌĞĨĞƌĞŶĐĞ͘ĂĐƚĞƌŝĂůŝƐŽůĂƚĞƐĂŶĚƚŚĞƌĞƐƉĞĐƟǀĞŐĞŶŽŵĞƐĞƋƵĞŶĐĞĂĐĐĞƐƐŝŽŶĐŽĚĞƐĂƌĞ

ƉƌĞƐĞŶƚĞĚŝŶ^ƵƉƉůĞŵĞŶƚĂůdĂďůĞ^Ϯ͘

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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&ŝŐƵƌĞϲ͘EƵŵďĞƌƐŽĨĐŽŶƐŝƐƚĞŶƚůLJŽƌƵŶŝƋƵĞůLJŝĚĞŶƟĮĞĚƉƌŽƚĞŝŶƐŝŶƚŚĞE. cloacaeƐƚƵĚLJ ŝƐŽůĂƚĞƐ ϭ ĂŶĚ Ϯ ĚƵƌŝŶŐ ƚŚĞ ĞdžƉŽŶĞŶƟĂů ĂŶĚ ƐƚĂƟŽŶĂƌLJ ŐƌŽǁƚŚ ƉŚĂƐĞƐ͘ dŚĞ ĚŝĂŐƌĂŵƐ

ǁĞƌĞĐƌĞĂƚĞĚƵƐŝŶŐƚŚĞsĞŶŶĚŝĂŐƌĂŵǁĞďƚŽŽůŽĨƚŚĞs/ĂŶĚƚŚĞhŶŝǀĞƌƐŝƚLJŽĨ'ĞŶƚŝŶ

ĞůŐŝƵŵ;ŚƩƉ͗ͬͬďŝŽŝŶĨŽƌŵĂƟĐƐ͘ƉƐď͘ƵŐĞŶƚ͘ďĞͬǁĞďƚŽŽůƐͬsĞŶŶͬͿ

&ŝŐƵƌĞ ϳ͘ WƌŽƚĞŽŵŝĐ ƉƌŽĮůĞƐ ŽĨ ƚŚĞ ŝŶǀĞƐƟŐĂƚĞĚ E. cloacae ƐƚƵĚLJ ŝƐŽůĂƚĞƐ ϭ ĂŶĚ Ϯ ŝŶ

ƚŚĞĞdžƉŽŶĞŶƟĂůĂŶĚƐƚĂƟŽŶĂƌLJŐƌŽǁƚŚƉŚĂƐĞƐ͘;ͿĂƌĚŝĂŐƌĂŵĚĞƉŝĐƟŶŐƚŚĞƌĞůĂƟǀĞ

ĂŵŽƵŶƚƐŽĨϱϬϬͬϭϯϬϬŝĚĞŶƟĮĞĚE. cloacaeƉƌŽƚĞŝŶƐŝŶƚŚĞĞdžƉŽŶĞŶƟĂů;ͿĂŶĚƐƚĂƟŽŶĂƌLJ

;^ͿŐƌŽǁƚŚƉŚĂƐĞƐďĂƐĞĚŽŶŶŽƌŵĂůŝnjĞĚƐƉĞĐƚƌĂůĐŽƵŶƚƐ͘ZĞůĂƟǀĞĂŵŽƵŶƚƐŽĨŝĚĞŶƟĮĞĚ

ƉƌŽƚĞŝŶƐĞŶĐŽĚĞĚďLJƚŚĞD/ZϭϳͲ'/;Ϳ͕ĂŶĚƚŚĞŝƌƌĞƐƉĞĐƟǀĞĨƵŶĐƟŽŶƐ;Ϳ͘

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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Discussion

dŚĞƉƌĞƐĞŶƚƐƚƵĚLJŚŝŐŚůŝŐŚƚƐƚŚĞŝĚĞŶƟĮĐĂƟŽŶŽĨĂŶĂŶĐŝĞŶƚD'/ͲĨĂŵŝůLJŝŶƚĞŐƌĂƟŶŐ

ampCͲůŝŬĞĐĞƉŚĂůŽƐƉŽƌŝŶĂƐĞŐĞŶĞƐŝŶĂǁŝĚĞƌĂŶŐĞŽĨ CC Enterobacteriaceae͘dŚŝƐ

D'/ǁĂƐĮƌƐƚŝĚĞŶƟĮĞĚŝŶĂĐĂƌďĂƉĞŶĞŵͲƌĞƐŝƐƚĂŶƚĐůŝŶŝĐĂůŝƐŽůĂƚĞŽĨE. cloacae͕

ǁŚĞƌĞŝƚǁĂƐƐŚŽǁŶƚŽĐĂƌƌLJƚŚĞ bla

ŐĞŶĞĨŽƌĂŶŵƉͲůŝŬĞĐĞƉŚĂůŽƐƉŽƌŝŶĂƐĞ͘

Ĩ

ĐĐŽƌĚŝŶŐůLJ͕ ƚŚĞ ƌĞƐƉĞĐƟǀĞ D'/ ǁĂƐ ŶĂŵĞĚ D/ZϭϳͲ'/͘ &ƵƌƚŚĞƌ ŝŶƐƉĞĐƟŽŶ ŽĨ

D/ZϭϳͲ'/ƌĞǀĞĂůĞĚĂŚŝŐŚĂďƵŶĚĂŶĐĞŽĨĚŝīĞƌĞŶƚƌĞƐŝƐƚĂŶĐĞŐĞŶĞƐŽĨǁŚŝĐŚƚŚĞ

D/ZϭϳĞŶnjLJŵĞǁĂƐĨŽƵŶĚƚŽďĞŚŝŐŚůLJĞdžƉƌĞƐƐĞĚ͘/ŵƉŽƌƚĂŶƚůLJ͕D/ZϭϳĚŝƐƉůĂLJƐ

ĐĂƌďĂƉĞŶĞŵĂƐĞĂĐƟǀŝƚLJƚŚĂƚĐĂŶďĞŝŶŚŝďŝƚĞĚǁŝƚŚĐůŽdžĂĐŝůůŝŶ͕ƐƵŐŐĞƐƟŶŐƚŚĂƚƚŚĞ ĐĂƌďĂƉĞŶĞŵƌĞƐŝƐƚĂŶĐĞŽĨŝƐŽůĂƚĞϭŚĂƐƚŽďĞĂƩƌŝďƵƚĞĚƚŽƚŚŝƐĞŶnjLJŵĞ͘

/ŶƚĞƌĞƐƟŶŐůLJ͕ƚŚĞD/ZϭϳͲ'/ŽĨƐƚƵĚLJŝƐŽůĂƚĞϭǁĂƐĨŽƵŶĚƚŽďĞĂƐƐŽĐŝĂƚĞĚǁŝƚŚĂ

WϰƉƌŽƉŚĂŐĞ͘dŚŝƐƐƵŐŐĞƐƚƐƚŚĂƚƚŚĞƚƌĂŶƐŵŝƐƐŝŽŶŽĨD/ZϭϳͲ'/ͲůŝŬĞD'/ƐďĞƚǁĞĞŶ

Enterobacteriaceae ŵĂLJŚĂǀĞďĞĞŶƉŚĂŐĞͲŵĞĚŝĂƚĞĚ͘ŽŶƐŝƐƚĞŶƚǁŝƚŚƚŚŝƐŝĚĞĂ͕

 ĂůůD/ZϭϳͲ'/ͲůŝŬĞD'/ƐǁĞƌĞĨŽƵŶĚƚŽďĞŝŶƚĞŐƌĂƚĞĚŝŶƚŽWŚĞͲ'ƚZEŐĞŶĞƐ

ƚŚĂƚŝŶĐůƵĚĞĂƉŚĂŐĞWϰͲĂƐƐŽĐŝĂƚĞĚĂƩĂĐŚŵĞŶƚƐŝƚĞĂƚƚŚĞϱ͛ĞŶĚŽĨƚŚĞD'/͘

DŽƌĞŽǀĞƌ͕ƚŚĞLJĂƌĞĂůůŇĂŶŬĞĚďLJĂĐŽŶƐĞƌǀĞĚWϰŝŶƚĞŐƌĂƐĞŐĞŶĞ͕ǁŝƚŚƚŚĞ>ĞƵͲ



ƚZEŐĞŶĞĂƐĂƩĂĐŚŵĞŶƚƐŝƚĞĂƚƚŚĞϯ͛ĞŶĚ͘dŚĞƐĞĨĞĂƚƵƌĞƐĂƌĞƚLJƉŝĐĂůĨŽƌ

ĐŚƌŽŵŽƐŽŵĂůWϰͲůŝŬĞŐĞŶĞĐůƵƐƚĞƌƐ

͘

ZĞƐŝƐƚĂŶĐĞŐĞŶĞƐĂƌĞĨƌĞƋƵĞŶƚůLJĞdžĐŚĂŶŐĞĚďĞƚǁĞĞŶŵŝĐƌŽŽƌŐĂŶŝƐŵƐǀŝĂD'/Ɛ

ĂŶĚƚŚŝƐƐĞĞŵƐƚŽďĞƚŚĞĐĂƐĞĂůƐŽĨŽƌƚŚĞD/ZϭϳͲ'/ͲůŝŬĞŝƐůĂŶĚƐŽĨǁŚŝĐŚŵŽƐƚ͕ďƵƚ

ŶŽƚĂůů͕ǁĞƌĞĨŽƵŶĚƚŽĐĂƌƌLJĂŶampCͲůŝŬĞĐĞƉŚĂůŽƐƉŽƌŝŶĂƐĞŐĞŶĞ͘^ƵĐŚƌĞƐŝƐƚĂŶĐĞ CC ŐĞŶĞƐƚŚĂƚĂƌĞĐĂƌƌŝĞĚĂŶĚŝŶƚĞŐƌĂƚĞĚŝŶƚŽƚŚĞďĂĐƚĞƌŝĂůĐŚƌŽŵŽƐŽŵĞďLJƉŚĂŐĞƐ

ĂƌĞƐŽŵĞƟŵĞƐƌĞĨĞƌƌĞĚƚŽĂƐ͚ŵŽƌŽŶƐ͛͘/ŶŐĞŶĞƌĂů͕ŵŽƌŽŶƐĂƌĞĐŽŵƉŽƐĞĚŽĨŽŶĞ

 ŽƌŵŽƌĞŐĞŶĞƐƚŚĂƚŚĂǀĞŶŽƉĂƌƟĐƵůĂƌĨƵŶĐƟŽŶŝŶƚŚĞƉŚĂŐĞ͛ƐůLJƐŽŐĞŶŝĐĐLJĐůĞ͕

ďƵƚƚŚĂƚŵĂLJƉƌŽǀŝĚĞďĞŶĞĮƚƐƚŽƚŚĞƉŚĂŐĞ͛ƐŚŽƐƚ͘dŚŝƐĐŽĞǀŽůƵƟŽŶŽĨƉƌŽƉŚĂŐĞƐ

ĂŶĚďĂĐƚĞƌŝĂǁŽƵůĚďĞŝŵƉŽƌƚĂŶƚĨŽƌƐƉĞĐŝĞƐƚŽĂĚĂƉƚƚŽǀĂƌŝŽƵƐĞŶǀŝƌŽŶŵĞŶƚĂů niches

͘/ŶƚŚĞĐĂƐĞŽĨD/Zϭϳ͕ƚŚĞďĞŶĞĮƚĨŽƌƚŚĞ E. cloacae ŚŽƐƚǁŽƵůĚďĞ  ƚŚĞ ĂĐƋƵŝƐŝƟŽŶ ŽĨ ƌĞƐŝƐƚĂŶĐĞ ƚŽ ĐĞƉŚĂůŽƐƉŽƌŝŶƐ Žƌ ĞǀĞŶ ĐĂƌďĂƉĞŶĞŵƐ͘ /Ŷ ƚŚŝƐ

ƌĞƐƉĞĐƚ͕ŝƚŝƐŝŵƉŽƌƚĂŶƚƚŽŶŽƚĞƚŚĂƚƐƵĐŚĂŶƟďŝŽƟĐƐĂƌĞƉƌŽĚƵĐĞĚŝŶŶĂƚƵƌĞďLJ

ĨƵŶŐŝĂŶĚ StreptomycetesƚŚĂƚŵĂLJĐŽŵƉĞƚĞĨŽƌƚŚĞƐĂŵĞĞĐŽůŽŐŝĐĂůŶŝĐŚĞƐĂƐE.

cloacae͘DŽƌŽŶƐĂƌĞĨƌĞƋƵĞŶƚůLJŇĂŶŬĞĚďLJƚZEƐĂŶĚƚŚŝƐǁĂƐĂůƐŽĨŽƵŶĚƚŽďĞ

the case for bla

͘dŚĞŝĚĞĂƚŚĂƚ bla

ŝƐĂŵŽƌŽŶŝƐĨƵƌƚŚĞƌƐƵƉƉŽƌƚĞĚďLJ

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: 50 PDF page: 50 PDF page: 50

ϱϬ

 ƚŚĞŽďƐĞƌǀĂƟŽŶƚŚĂƚŝƚŝƐůŽĐĂůŝnjĞĚŝŶƚŚĞǀŝĐŝŶŝƚLJŽĨƚŚĞďĂĐƚĞƌŝĂůŚŝŐŚĨƌĞƋƵĞŶĐLJ

ůLJƐŽŐĞŶLJŽƉĞƌŽŶŚŇYy<

͘

>ĂƐƚůLJ͕ ǁĞ ŽďƐĞƌǀĞĚ Ă ĚŝǀĞƌƐŝƚLJ ŽĨ ŐĞŶĞƐ ĞŶĐŽĚŝŶŐ ĐĞƉŚĂůŽƐƉŽƌŝŶĂƐĞƐ ĂŵŽŶŐ ƚŚĞŝĚĞŶƟĮĞĚD/ZϭϳͲ'/ͲůŝŬĞŵŽďŝůĞŐĞŶŽŵŝĐŝƐůĂŶĚƐ͘dŚĞŝŶƚĞŐƌĂƟŽŶŽĨďƌŽĂĚ

ƐƉĞĐƚƌƵŵĐĞƉŚĂůŽƐƉŽƌŝŶĂƐĞŐĞŶĞƐ͕ĂƐĨŽƵŶĚŝŶƚŚĞE. cloacaeƐƚƵĚLJŝƐŽůĂƚĞƐŝƐŽĨ

ŵĂũŽƌĐůŝŶŝĐĂůŝŵƉŽƌƚĂŶĐĞ͘WĂƌƟĐƵůĂƌůLJ͕ƵƉŽŶŚŝŐŚͲůĞǀĞůĞdžƉƌĞƐƐŝŽŶ͕ďŽƚŚƚŚĞdͲ

ĂŶĚƚŚĞD/ZͲĨĂŵŝůŝĞƐŽĨɴͲůĂĐƚĂŵĂƐĞƐĂƌĞĐĂƉĂďůĞŽĨŚLJĚƌŽůLJnjŝŶŐƚŚŝƌĚͲŐĞŶĞƌĂƟŽŶ

ĐĞƉŚĂůŽƐƉŽƌŝŶƐ

͘DŽƌĞŽǀĞƌ͕ŽƵƌƐƚƵĚLJƐŚŽǁƐƚŚĂƚƚŚĞĐĂƌďĂƉĞŶĞŵƌĞƐŝƐƚĂŶĐĞ

of E. cloacae ƐƚƵĚLJŝƐŽůĂƚĞϭĐĂŶďĞĂƩƌŝďƵƚĞĚƚŽƚŚĞŶĞǁĂůůĞůĞbla

and the ŚŝŐŚͲůĞǀĞůƉƌŽĚƵĐƟŽŶŽĨƚŚĞĞŶĐŽĚĞĚD/ZϭϳĞŶnjLJŵĞ͘dŚĞĐĂƌďĂƉĞŶĞŵƌĞƐŝƐƚĂŶĐĞ ŽĨƐƚƵĚLJŝƐŽůĂƚĞϭŵĂLJďĞĨƵƌƚŚĞƌĞŶŚĂŶĐĞĚďLJĂĐŚĂŶŐĞŝŶŽƵƚĞƌŵĞŵďƌĂŶĞ

ƉƌŽƚĞŝŶ ĞdžƉƌĞƐƐŝŽŶ͘ /Ŷ ƉĂƌƟĐƵůĂƌ͕ ƚŚĞ ompF ŐĞŶĞ ŽĨ ŝƐŽůĂƚĞ ϭ͕ ŚŽŵŽůŽŐŽƵƐ ƚŽ F ompK35͕ĐŽŶƚĂŝŶƐƚǁŽƉŽŝŶƚŵƵƚĂƟŽŶƐĐŽŵƉĂƌĞĚƚŽƚŚĞƌĞĨĞƌĞŶĐĞ ompF gene F of E. cloacaeD',ϱϰ͕ŽŶĞŽĨǁŚŝĐŚƌĞƐƵůƚƐŝŶĂƐƚŽƉĐŽĚŽŶĂƚƉŽƐŝƟŽŶϮϬϬ͘

dŚĞƌĞĨŽƌĞ͕ ĂŶ ĂĚĚŝƟŽŶĂů ĨĂĐƚŽƌ ĐŽŶƚƌŝďƵƟŶŐ ƚŽ ƚŚĞ ĐĂƌďĂƉĞŶĞŵ ƌĞƐŝƐƚĂŶĐĞ ŽĨ

ŝƐŽůĂƚĞϭĐŽƵůĚďĞƚŚĞĂďƐĞŶĐĞŽĨĂŶŽƵƚĞƌŵĞŵďƌĂŶĞƉŽƌŝŶ͕ĞƐƉĞĐŝĂůůLJKŵƉ&͘

dŚĞůĂƩĞƌǁŽƵůĚďĞĐŽŶƐŝƐƚĞŶƚǁŝƚŚƉƌĞǀŝŽƵƐƐƚƵĚŝĞƐŽŶƚŚĞƌŽůĞŽĨƉŽƌŝŶƐŝŶƚŚĞ

resistance of Enterobacteriaceae ƚŽĂŶƟďŝŽƟĐƐ

͘

/ŶĐŽŶĐůƵƐŝŽŶ͕ǁĞŝĚĞŶƟĮĞĚĂŶŽǀĞůĨĂŵŝůLJŽĨD'/ƐŚĂƌďŽƌŝŶŐŐĞŶĞƐĂƐƐŽĐŝĂƚĞĚ

ǁŝƚŚƚŚĞƌĞƐŝƐƚĂŶĐĞƚŽĐĞƉŚĂůŽƐƉŽƌŝŶƐ͕ƉŚĂŐĞŝŵŵƵŶŝƚLJ͕ĂŶĚĂĐĐĞƐƐŽƌLJŵĞƚĂďŽůŝĐ

ĨƵŶĐƟŽŶƐ͘dŚĞƐĞD/ZϭϳͲ'/ͲůŝŬĞŝƐůĂŶĚƐĂƌĞƉƌĞƐĞŶƚŝŶǀĂƌŝŽƵƐEnterobacteriaceae͕

ŝŶĐůƵĚŝŶŐ K. pneumoniae͕ E. coli͕ ĂŶĚ ͘ ĐůŽĂĐĂĞ͘ /ŵƉŽƌƚĂŶƚůLJ͕ ƚŚĞ D/ZϭϳͲ'/Ͳ ůŝŬĞŝƐůĂŶĚƐĂƌĞĂƐƐŽĐŝĂƚĞĚǁŝƚŚŝŶƚĞŐƌĂƚĞĚWϰͲůŝŬĞƉƌŽƉŚĂŐĞƐ͕ǁŚŝĐŚŝŵƉůŝĐĂƚĞ

ƉŚĂŐĞƐ ŝŶ ƚŚĞ ƐƉƌĞĂĚ ŽĨ ŚŝŐŚͲůĞǀĞů ĐĞƉŚĂůŽƐƉŽƌŝŶ ĂŶĚ ĐĂƌďĂƉĞŶĞŵ ƌĞƐŝƐƚĂŶĐĞ

amongst Enterobacteriaceae͘ /Ŷ ǀŝĞǁ ŽĨ ƚŚĞ ƐĞƌŝŽƵƐ ĐŽŶƐĞƋƵĞŶĐĞƐ ŽĨ ŚŝŐŚͲ ůĞǀĞůĐĞƉŚĂůŽƐƉŽƌŝŶĂŶĚĐĂƌďĂƉĞŶĞŵƌĞƐŝƐƚĂŶĐĞĨŽƌƚƌĞĂƚŵĞŶƚŽĨƉĂƟĞŶƚƐǁŝƚŚ ĞŶƚĞƌŽďĂĐƚĞƌŝĂĐĞĂůŝŶĨĞĐƟŽŶƐ͕ƚŚĞĚŝƐĐŽǀĞƌLJŽĨĂŶĂŶĐŝĞŶƚŵŽďŝůĞŐĞŶŽŵŝĐŝƐůĂŶĚ

ƚŚĂƚŚĂƐĨĂĐŝůŝƚĂƚĞĚƚŚĞƐƉƌĞĂĚŽĨ͚ĐĞƉŚĂůŽƐƉŽƌŝŶĂƐĞĂŶĚĐĂƌďĂƉĞŶĞŵĂƐĞŵŽƌŽŶƐ͛

ŝƐŽĨŚŝŐŚĐůŝŶŝĐĂůƌĞůĞǀĂŶĐĞ͘

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: 51 PDF page: 51 PDF page: 51

51

ŚĂƉƚ ĞƌϤ

DĂƚĞƌŝĂůƐĂŶĚDĞƚŚŽĚƐ

ĂĐƚĞƌŝĂůŝƐŽůĂƚĞƐ

dŚĞ ďĂĐƚĞƌŝĂů ŝƐŽůĂƚĞƐ ƵƐĞĚ ŝŶ ƚŚŝƐ ƐƚƵĚLJ ĂŶĚ ƚŚĞŝƌ ĂŶƟďŝŽƟĐ ƌĞƐŝƐƚĂŶĐĞƐ ĂƌĞ

ƐƵŵŵĂƌŝnjĞĚŝŶdĂďůĞϭ͘^ƉĞĐŝĞƐĚĞƚĞƌŵŝŶĂƟŽŶǁĂƐƉĞƌĨŽƌŵĞĚďLJ ŵĂƚƌŝdžͲĂƐƐŝƐƚĞĚ ůĂƐĞƌ ĚĞƐŽƌƉƟŽŶͬŝŽŶŝnjĂƟŽŶͲƟŵĞͲŽĨͲŇŝŐŚƚ ;D>/ͲdK&Ϳ D^͕ ƵƐŝŶŐ Ă ƌƵŬĞƌ DŝĐƌŽŇĞdž;ƌƵŬĞƌŽƌƉŽƌĂƟŽŶ͕ŝůůĞƌŝĐĂ͕h^Ϳ͘

ŶƟďŝŽƟĐƐƵƐĐĞƉƟďŝůŝƚLJƚĞƐƟŶŐ

ŶƟďŝŽƟĐ ƐƵƐĐĞƉƟďŝůŝƚLJǁĂƐƌŽƵƟŶĞůLJĚĞƚĞƌŵŝŶĞĚǁŝƚŚƚŚĞs/d<ϮƐLJƐƚĞŵǁŝƚŚ

ĂŶ ^dŶϯϰϰ ĐĂƌĚ ;ďŝŽDĠƌŝĞƵdž͕ DĂƌĐLJ ů͛ƚŽŝů͕ &ƌĂŶĐĞͿ͘ dŚĞ s/d< Ϯ ŵŝŶŝŵƵŵ

ŝŶŚŝďŝƚŽƌLJ ĐŽŶĐĞŶƚƌĂƟŽŶ ƌĞƐƵůƚƐ ǁĞƌĞ ŝŶƚĞƌƉƌĞƚĞĚ ĂĐĐŽƌĚŝŶŐ ƚŽ ƚŚĞ ĂĚǀĂŶĐĞĚ

ĞdžƉĞƌƚƐLJƐƚĞŵĨŽůůŽǁŝŶŐh^dŐƵŝĚĞůŝŶĞƐ;ǁǁǁ͘ĞƵĐĂƐƚ͘ŽƌŐͿ͘dŚĞƐƵƐĐĞƉƟďŝůŝƚLJ

ƚŽŵĞƌŽƉĞŶĞŵŽƌŝŵŝƉĞŶĞŵǁĂƐƐƵďƐĞƋƵĞŶƚůLJǀĞƌŝĮĞĚŽŶD,ƵƐŝŶŐƚŚĞ

ŝŽĚŝƐŬ ƚĞƐƚ ĂĐĐŽƌĚŝŶŐ ƚŽ ŵĂŶƵĨĂĐƚƵƌĞƌ͛Ɛ ŐƵŝĚĞůŝŶĞƐ ; ŝŽĚŝƐŬ͕ DĂŶŶŚĞŝŵ͕

'ĞƌŵĂŶLJͿ͘ dŚĞ ƉƌĞƐĞŶĐĞ ŽĨ ŐĞŶĞƐ ĨŽƌ ĐĂƌďĂƉĞŶĞŵĂƐĞ͕ ^> ĂŶĚ ŵƉ ǁĂƐ

ǀĞƌŝĮĞĚ ǁŝƚŚ ƚŚĞ ŚĞĐŬͲDZ dϭϬϯ y> ŵŝĐƌŽĂƌƌĂLJ ĂƐƐĂLJ ;ŚĞĐŬͲWŽŝŶƚƐ͕

tĂŐĞŶŝŶŐĞŶ͕ƚŚĞEĞƚŚĞƌůĂŶĚƐͿ͘

EƐĞƋƵĞŶĐĞĂŶĂůLJƐĞƐ

EĂŶŽƉŽƌĞƐĞƋƵĞŶĐŝŶŐ Ɖ 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

^ĐŝĞŶƟĮĐͿ͕ĂŶŐŝůĞŶƚdĂƉĞƐƚĂƟŽŶϮϮϬϬ;ŐŝůĞŶƚͿĂŶĚĂEĂŶŽƌŽƉ;dŚĞƌŵŽ&ŝƐŚĞƌ

^ĐŝĞŶƟĮĐͿ͘ >ŝďƌĂƌŝĞƐ ǁĞƌĞ ƉƌĞƉĂƌĞĚ ǁŝƚŚŽƵƚ ƐŚĞĂƌŝŶŐ ƚŽ ŵĂdžŝŵŝƐĞ ƐĞƋƵĞŶĐŝŶŐ

ƌĞĂĚůĞŶŐƚŚ͘dŚĞůŝďƌĂƌLJǁĂƐƉƌĞƉĂƌĞĚƵƐŝŶŐƚŚĞϮůŝŐĂƟŽŶƐĞƋƵĞŶĐŝŶŐŬŝƚ;^Y<Ͳ

>^<ϮϬϴͿ͘dŚĞƉƌŽƚŽĐŽůĨŽƌϮůŝŐĂƟŽŶƐĞƋƵĞŶĐŝŶŐŬŝƚǁĂƐĨŽůůŽǁĞĚĂƐĚĞƐĐƌŝďĞĚ

ďLJ ƚŚĞ ŵĂŶƵĨĂĐƚƵƌĞƌ͘ dŚĞ ĮŶĂů ůŝďƌĂƌLJ ǁĂƐ ůŽĂĚĞĚ ŽŶƚŽ ĂŶ &>KͲD/EϭϬϲ Zϵ͘ϰ

ŇŽǁ ĐĞůů͘ dŚĞ ƌƵŶ ǁĂƐ ƉĞƌĨŽƌŵĞĚ ŽŶ Ă DŝŶ/KE ĚĞǀŝĐĞ ƵƐŝŶŐ ƚŚĞ Eͺϰϴ,ƌͺ

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: 52 PDF page: 52 PDF page: 52

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^ĞƋƵĞŶĐŝŶŐͺZƵŶͺ&>KͲD/EϭϬϳͺ^Y<>^<ϮϬϴ ƉƌŽƚŽĐŽů ǁŝƚŚ ϵϳϲ ĂǀĂŝůĂďůĞ ƉŽƌĞƐ

;ϰϲϰ͕ϯϭϰ͕ϭϲϭĂŶĚϯϳƉŽƌĞƐƉĞƌŐƌŽƵƉͿ͘dŚĞƌƵŶƉƌŽĐĞĞĚĞĚĨŽƌƚŚĞĨƵůůϰϴŚŽƵƌƐ͘

ĂƐĞĐĂůůŝŶŐǁĂƐƉĞƌĨŽƌŵĞĚĂŌĞƌƚŚĞƌƵŶ͕ƵƐŝŶŐůďĂĐŽƌĞǀϭ͘Ϯ͘Ϯ;EĂŶŽƉŽƌĞͿǁŝƚŚ ƚŚĞƌϵϰͺϮϱϬďƉƐͺϮĚ͘ĐĨŐǁŽƌŬŇŽǁ͘>ĂƐƚůLJ͕ƚŚĞƋƵĂůŝƚLJŽĨƚŚĞĚĂƚĂǁĂƐĂŶĂůLJƐĞĚ

ǁŝƚŚWŽƌĞƚŽŽůƐǀϬ͘ϲ͘Ϭ

ĂŶĚƚŚĞĨĂƐƚϱĮůĞƐǁĞƌĞƚƌĂŶƐĨŽƌŵĞĚŝŶƚŽĂĨĂƐƚƋĮůĞ͘

/ůůƵŵŝŶĂƐĞƋƵĞŶĐŝŶŐ Ƌ 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 ƉƌĞǀŝŽƵƐůLJ

͘  Ğ ŶŽǀŽ ĂƐƐĞŵďůLJŽĨƉĂŝƌĞĚͲĞŶĚƌĞĂĚƐǁĂƐƉĞƌĨŽƌŵĞĚƵƐŝŶŐ>'ĞŶŽŵŝĐƐtŽƌŬďĞŶĐŚ ǀϴ͘ϱ͘ϯ;Y/'E͕,ŝůĚĞŶ͕'ĞƌŵĂŶLJͿĂŌĞƌƋƵĂůŝƚLJƚƌŝŵŵŝŶŐ;YƐшϮϬͿĞƐƚĂďůŝƐŚŝŶŐ

ĂǁŽƌĚƐŝnjĞŽĨϯϬ͘dŚŝƐƌĞƐƵůƚĞĚŝŶϭϱϵĂŶĚϲϲĐŽŶƟŐƐ;шϱϬϬďƉͿǁŝƚŚĂŶĂǀĞƌĂŐĞ

ĐŽǀĞƌĂŐĞ ŽĨ ϳϮͲĨŽůĚ͘ dŚĞ ĂĐƋƵŝƌĞĚ ĂŶƟŵŝĐƌŽďŝĂů ƌĞƐŝƐƚĂŶĐĞ ŐĞŶĞƐ ĂŶĚ ŵƵůƟͲ ůŽĐƵƐ ƐĞƋƵĞŶĐĞ ƚLJƉĞƐ ;D>^dͿ ǁĞƌĞ ŝĚĞŶƟĮĞĚ ďLJ ƵƉůŽĂĚŝŶŐ ƚŚĞ ĂƐƐĞŵďůĞĚ

ŐĞŶŽŵĞƐĞƋƵĞŶĐĞƐŽŶƚŽƚŚĞZĞƐĮŶĚĞƌƐĞƌǀĞƌǀϮ͘ϭ͘

ĂŶĚƚŚĞD>^dϭ͘ϳƐĞƌǀĞƌ

44

͕ ƌĞƐƉĞĐƟǀĞůLJ͘ dŚĞ ƌĂǁ t'^ ĚĂƚĂƐĞƚƐ ŐĞŶĞƌĂƚĞĚ ŝŶ ƚŚĞ ĐƵƌƌĞŶƚ ƐƚƵĚLJ ĂƌĞ

ĂǀĂŝůĂďůĞŝŶƚŚĞƵƌŽƉĞĂŶEƵĐůĞŽƟĚĞƌĐŚŝǀĞ;EͿƌĞƉŽƐŝƚŽƌLJƵŶĚĞƌŝŽƉƌŽũĞĐƚ WZ:ϮϮϭϭϵ;ŚƩƉ͗ͬͬǁǁǁ͘Ğďŝ͘ĂĐ͘ƵŬͬĞŶĂͬͿ͘

,LJďƌŝĚĂƐƐĞŵďůLJ LJ LJ͘dŚĞŚLJďƌŝĚĂƐƐĞŵďůLJŽĨƚŚĞ/ůůƵŵŝŶĂƐŚŽƌƚƌĞĂĚƐĂŶĚƚŚĞDŝŶ/KE

ůŽŶŐ ƌĞĂĚƐ ŽĨ ŝƐŽůĂƚĞ ϯϯϵϯϴϵ> ǁĂƐ ƉĞƌĨŽƌŵĞĚ ƵƐŝŶŐ ^WĚĞƐ ǀĞƌƐŝŽŶ͗ ϯ͘ϭϬ͘ϭ

;ŚƩƉ͗ͬͬďŝŽŝŶĨ͘ƐƉďĂƵ͘ƌƵͬĞŶͿ͘dŚĞƌĞƐƵůƟŶŐĂƐƐĞŵďůLJǁĂƐƐƵďŵŝƩĞĚƚŽE/ƵŶĚĞƌ ĂĐĐĞƐƐŝŽŶŶƵŵďĞƌWϬϮϲϱϯϲ͘^ĞƋƵĞŶĐĞƐŝŵŝůĂƌŝƚLJĂŶĂůLJƐĞƐďĞƚǁĞĞŶD/ZϭϳĂŶĚ

D'/Ɛ ĨƌŽŵ ƐĐŚĞƌŝĐŚŝĂ ĐŽůŝ ǁĞƌĞ ƉĞƌĨŽƌŵĞĚ ƵƐŝŶŐ ƚŚĞ ƌĂƉŝĚ ĂŶŶŽƚĂƟŽŶ ƵƐŝŶŐ

ƐƵďƐLJƐƚĞŵƚĞĐŚŶŽůŽŐLJ;Z^dͿƐĞƌǀĞƌϰ͘Ϭ

͘ WŚLJůŽŐĞŶĞƟĐĂŶĂůLJƐĞƐ

WŚLJůŽŐĞŶĞƟĐ ƚƌĞĞƐ ďĂƐĞĚ ŽŶ ƐŝŶŐůĞ ŶƵĐůĞŽƟĚĞ ƉŽůLJŵŽƌƉŚŝƐŵƐ ;^EWƐͿ ǁĞƌĞ

ďƵŝůƚ ƵƐŝŶŐ ƚŚĞ ^EW ĐŽŵƉĂƌŝƐŽŶ ƚŽŽů ŝŶ ZŝĚŽŵ ^ĞƋ^ƉŚĞƌĞ ǀϰ͘ϭ͘ϵ ;DƺŶƐƚĞƌ͕