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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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 5

Stefanie Desmet, Suruchi Nepal, Jan Maarten van Dijl, Marc Van Ranst, Monika A Chlebowicz, John W Rossen, Jeroen KJ Van Houdt, Piet Maes, Katrien Lagrou, Erik Bathoorn

WƵďůŝƐŚĞĚŝŶŶƟŵŝĐƌŽďŐĞŶƚƐŚĞŵŽƚŚĞƌĂƉLJ͘ϮϬϭϴDĂƌ͖ϲϮ;ϯͿ͘Ɖŝŝ͗ĞϬϭϰϰϴͲϭϳ͘ ĚŽŝ͗ϭϬ͘ϭϭϮϴͬ͘ϬϭϰϰϴͲϭϳ

ŶƟďŝŽƟĐƌĞƐŝƐƚĂŶĐĞƉůĂƐŵŝĚƐĐŽŝŶƚĞŐƌĂƚĞĚŝŶƚŽĂ

ŵĞŐĂƉůĂƐŵŝĚŚĂƌďŽƵƌŝŶŐ

ƚŚĞďůĂKyͲϰϮϳĐĂƌďĂƉĞŶĞŵĂƐĞŐĞŶĞ

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Abstract

KyͲϰϮϳŝƐĂŶĞǁĐůĂƐƐĐĂƌďĂƉĞŶĞŵĂƐĞĞŶĐŽƵŶƚĞƌĞĚŝŶĚŝīĞƌĞŶƚƐƉĞĐŝĞƐŽĨ

Enterobacteriaceae ŝŶĂĞůŐŝĂŶŚŽƐƉŝƚĂů͘dŽƐƚƵĚLJƚŚĞĚŝƐƉĞƌƐĂůŽĨƚŚŝƐŐĞŶĞ͕ǁĞ

ƉĞƌĨŽƌŵĞĚĂĐŽŵƉĂƌĂƟǀĞĂŶĂůLJƐŝƐŽĨƚǁŽƉůĂƐŵŝĚƐĐŽŶƚĂŝŶŝŶŐƚŚĞbla_OXA-427gene ŝƐŽůĂƚĞĚ ĨƌŽŵ Ă Klebsiella pneumoniae and an Enterobacter cloacae complex strain. The two IncA/C2 plasmids containing bla_OXA-427 ƐŚĂƌĞƚŚĞƐĂŵĞďĂĐŬďŽŶĞ͕ ŚŽǁĞǀĞƌ, in the K. pneumoniae ƐƚƌĂŝŶ͕ƚŚŝƐƉůĂƐŵŝĚŝƐĐŽŝŶƚĞŐƌĂƚĞĚŝŶƚŽĂŶ/ŶĐ&/ plasmid, ĨŽƌŵŝŶŐĂϯϮϭͲŬďŵĞŐĂƉůĂƐŵŝĚǁŝƚŚŵƵůƟƉůĞŵƵůƟͲƌĞƐŝƐƚĂŶĐĞƌĞŐŝŽŶƐ͘

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 dŚĞĚŝƐƐĞŵŝŶĂƟŽŶŽĨĐĂƌďĂƉĞŶĞŵƌĞƐŝƐƚĂŶĐĞŝŶEnterobacteriaceae is a major ƉƵďůŝĐŚĞĂůƚŚĐŽŶĐĞƌŶ͕ĂƐĐĂƌďĂƉĞŶĞŵĂŶƟďŝŽƟĐƐĂƌĞĂůĂƐƚƌĞƐŽƌƚƚŚĞƌĂƉLJĨŽƌ ƚŚĞƐĞĐŽŵŵŽŶƉĂƚŚŽŐĞŶƐ͘dŚĞŶĞǁKyͲϰϮϳĐĂƌďĂƉĞŶĞŵĂƐĞƉƌĞƐĞŶƚƐĂƉƌŽĮůĞ ŽĨǁŝĚĞƌĞƐŝƐƚĂŶĐĞƚŽɴͲůĂĐƚĂŵƐ, ŝŶĐůƵĚŝŶŐďƌŽĂĚƐƉĞĐƚƌƵŵƉĞŶŝĐŝůůŝŶƐ͕ĞdžƚĞŶĚĞĚͲ spectrum cephalosporins, and carbapenems1_{. The bla}

OXA-427gene was detected

ŝŶ ĚŝīĞƌĞŶƚ Enterobacteriaceae ƐƉĞĐŝĞƐ ;ŶсϲͿ ĂŶĚ ŝŶ ĚŝīĞƌĞŶƚ ƉĂƟĞŶƚƐ ;ŶсϵͿ ŚŽƐƉŝƚĂůŝƐĞĚ Ăƚ Ă ƵŶŝǀĞƌƐŝƚLJ ŚŽƐƉŝƚĂů ŝŶ ĞůŐŝƵŵ͕ ŝŶ ĚŝīĞƌĞŶƚ ŚŽƐƉŝƚĂů ǁĂƌĚƐ͕ between March 2012 and June 2014 1_{͘dŚŝƐƐƵŐŐĞƐƚƐĂŚŝŐŚůĞǀĞůŽĨŝŶƚĞƌƐƉĞĐŝĞƐ} ƚƌĂŶƐŵŝƐƐŝŽŶŽĨƚŚŝƐƌĞƐŝƐƚĂŶĐĞŐĞŶĞ, probably associated with horizontal gene ƚƌĂŶƐĨĞƌŽĨĂƐƉĞĐŝĮĐƉůĂƐŵŝĚ͘ZĞĐĞŶƚƐƚƵĚŝĞƐ͕ďĂƐĞĚŽŶůŽŶŐͲƌĞĂĚƐĞƋƵĞŶĐŝŶŐ ĚĂƚĂ͕ƐƵŐŐĞƐƚƚŚĂƚŵŽďŝůŝƚLJŽĨƌĞƐŝƐƚĂŶĐĞŐĞŶĞƐŽĐĐƵƌƐĂƚŵƵůƟƉůĞŶĞƐƚĞĚŐĞŶĞƟĐ ůĞǀĞůƐǁŝƚŚƚƌĂŶƐŵŝƐƐŝŽŶŽĨƚŚĞƐƚƌĂŝŶƐďĞƚǁĞĞŶŝŶĚŝǀŝĚƵĂůƐ͕ƚƌĂŶƐĨĞƌŽĨƉůĂƐŵŝĚƐ ďĞƚǁĞĞŶƐƚƌĂŝŶƐĂŶĚƚƌĂŶƐƉŽƐŝƟŽŶŽĨƚŚĞƌĞƐŝƐƚĂŶĐĞŐĞŶĞďĞƚǁĞĞŶƉůĂƐŵŝĚƐ2,ϯ_. tĞ ƌĞƉŽƌƚ ƚŚĞ ĐŽŵƉĂƌĂƟǀĞ ĂŶĂůLJƐŝƐ ŽĨ ƚǁŽ ƉůĂƐŵŝĚƐ ĐŽŶƚĂŝŶŝŶŐ ƚŚĞ bla_OXA-427 ŐĞŶĞ͕ĨŽƌĨƵƚƵƌĞƐƚƵĚLJŽĨƚŚĞƉŽƚĞŶƟĂůŵĞĐŚĂŶŝƐŵŽĨŝŶƚĞƌƐƉĞĐŝĞƐƚƌĂŶƐŵŝƐƐŝŽŶ ŽĨƚŚĞbla_OXA-427 gene.

Two OXA-427 producing isolates, ŽŶĞ ŽĨ Klebsiella pneumoniae ;<>WEϱϳͿ͕ ĂŶĚ ƐĞĐŽŶĚ ŽĨ Enterobacter cloacae complex (ENCL58), ǁĞƌĞ ĐƵůƚƵƌĞĚ ĨƌŽŵ ĐůŝŶŝĐĂů ƐĂŵƉůĞƐ ŽĨ ƚǁŽ ĚŝīĞƌĞŶƚ ƉĂƟĞŶƚƐ ŝŶ :ĂŶƵĂƌLJ ϮϬϭϯ ĂŶĚ KĐƚŽďĞƌ ϮϬϭϯ͕ ƌĞƐƉĞĐƟǀĞůLJ͘ 'ĞŶŽŵŝĐ E ǁĂƐ ĞdžƚƌĂĐƚĞĚ ĨƌŽŵ ƚŚĞƐĞ ŝƐŽůĂƚĞƐ ĂŶĚ WĂĐŝŽ ƐĞƋƵĞŶĐŝŶŐǁĂƐƉĞƌĨŽƌŵĞĚĂƚƚŚĞ'ĞŶŽŵŝĐƐŽƌĞůĂďŽƌĂƚŽƌLJ;>ĞƵǀĞŶ͕ĞůŐŝƵŵͿ according to the protocol used by Conlan et al. 4_{.'ĞŶŽŵŝĐEǁĂƐƐŚĞĂƌĞĚƚŽ} ϭϬʹϭϱŬďĨƌĂŐŵĞŶƚƐĂŶĚĐŽŶǀĞƌƚĞĚŝŶƚŽ^DZdďĞůůƚĞŵƉůĂƚĞůŝďƌĂƌŝĞƐ͘ĂĐŚƐƚƌĂŝŶ ǁĂƐƐĞƋƵĞŶĐĞĚŽŶĂƐŝŶŐůĞ^DZdĐĞůůŽŶƚŚĞWĂĐŝŽZ^//ƵƐŝŶŐWϲƉŽůLJŵĞƌĂƐĞ ďŝŶĚŝŶŐ ĂŶĚ ϰ ƐĞƋƵĞŶĐŝŶŐ ŬŝƚƐ͘ dŚĞ ƐĞƋƵĞŶĐŝŶŐ ĐŽǀĞƌĂŐĞ ĨŽƌ ďŽƚŚ ƉůĂƐŵŝĚƐ ƌĂŶŐĞĚĨƌŽŵϭϲϬͲĨŽůĚƚŽϰϬϬͲĨŽůĚĐŽǀĞƌĂŐĞ.'ĞŶŽŵĞĂƐƐĞŵďůŝĞƐǁĞƌĞƉĞƌĨŽƌŵĞĚ ƵƐŝŶŐ,'WǀĞƌƐŝŽŶϯĂƐƉĂƌƚŽĨ^DZdŶĂůLJƐŝƐǀĞƌƐŝŽŶϮ͘ϯ͘Ϭ,ĂŶŶŽƚĂƟŽŶŽĨƚŚĞ ŐĞŶĞƐǁĂƐƉĞƌĨŽŵƌĞĚďLJƵƐŝŶŐZ^d;ŚƩƉ͗ͬͬƌĂƐƚ͘ŶŵƉĚƌ͘ŽƌŐͿ͕ĂŶĚƚŚĞĨƵŶĐƟŽŶĂů ĂƐƐŝŐŶŵĞŶƚƐĨŽƌƚŚĞƉƌĞĚŝĐƚĞĚKZ&ƐĐŽŶĮƌŵĞĚƵƐŝŶŐ>^dWŝŶƚŚĞWƌŽƚĞŝŶĂƚĂ ĂŶŬ;WͿĨƌŽŵE/;ŚƩƉ͗ͬͬǁǁǁ͘ŶĐďŝ͘Ŷůŵ͘ŶŝŚ͘ŐŽǀͿ͘/^ĮŶĚĞƌĂŶĚZĞƐ&ŝŶĚĞƌ ǁĞƌĞƵƐĞĚĨŽƌŝĚĞŶƟĮĐĂƟŽŶŽĨŵŽďŝůĞĞůĞŵĞŶƚƐĂŶĚĂŶƟďŝŽƟĐƌĞƐŝƐƚĂŶĐĞŐĞŶĞƐ͕ ƌĞƐƉĞĐƟǀĞůLJ5,6_{. WƵďD>^dǁĂƐƵƐĞĚĨŽƌŵƵůƟůŽĐƵƐƐĞƋƵĞŶĐĞƚLJƉŝŶŐ;D>^dͿ}7_{. The}

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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ƉůĂƐŵŝĚŵĂƉǁĂƐĚƌĂǁŶƵƐŝŶŐ^ŶĂƉŐĞŶĞƐŽŌǁĂƌĞ;ŚƩƉ͗ͬͬǁǁǁ͘ƐŶĂƉŐĞŶĞ͘ĐŽŵͿ͘ dŚĞ ŐĞŶŽŵŝĐ ĐŽŶƚĞŶƚ ŽĨ ƚŚĞ E. cloacae ƐƚƌĂŝŶ͕ E>ϱϴ͕ ĐŽŶƐŝƐƚƐ ŽĨ Ă ϱ͘Ϭ Dď ŐĞŶŽŵĞǁŝƚŚƐĞƋƵĞŶĐĞƚLJƉĞ;^dͲϭϳϭͿĂŶĚƚŚƌĞĞƉůĂƐŵŝĚƐ;ϭϳϳŬď͕ϭϮϭŬďĂŶĚϴϰ ŬďͿ͘dŚĞϭϳϳ͕ϬϯϳďƉƉůĂƐŵŝĚƉE>ϱϴͺϬϭ͕ĐĂƌƌLJŝŶŐƚŚĞ bla_OXA-427ŐĞŶĞ͕ŝƐĂŶ/ŶĐͬ ϮƉůĂƐŵŝĚŽĨƉůĂƐŵŝĚŵƵůƟůŽĐƵƐƐĞƋƵĞŶĐĞƚLJƉĞϯ͕ϭϬϬйŝĚĞŶƟĐĂůƚŽ<yϴϲϵϳϰϭ͘ϭ1 (&ŝŐƵƌĞϭͿ͘dŚĞŽƚŚĞƌƚǁŽƉůĂƐŵŝĚƐŽĨ/ŶĐ&/ď-,ĂŶĚ/ŶĐ&//ͲƚLJƉĞĐĂƌƌLJŶŽŬŶŽǁŶ ƌĞƐŝƐƚĂŶĐĞŐĞŶĞƐ͘dŚĞ/ŶĐͬϮƉůĂƐŵŝĚƐŚŽǁƐŽǀĞƌĂůůŚŝŐŚůĞǀĞůƐŽĨŚŽŵŽůŽŐLJ ǁŝƚŚ ŽƚŚĞƌ /ŶĐͬϮ ƉůĂƐŵŝĚƐ ĨŽƵŶĚ ŝŶ Enterobacter aerogenes ;&KϮϬϯϯϱϰ͘ϭ͖ ϵϵйƐĞƋƵĞŶĐĞŝĚĞŶƟƚLJ͕ϴϳйĐŽǀĞƌĂŐĞͿ͘

K.

KK pneumoniae ;'ĞŶĞ ďĂŶŬ ĂĐĐĞƐƐŝŽŶ ŶƵŵďĞƌ :YϬϭϬϵϴϰ͘ϭ͖ ϵϵй ƐĞƋƵĞŶĐĞ

ŝĚĞŶƟƚLJ͕ϴϲйĐŽǀĞƌĂŐĞͿĂŶĚSalmonella enterica;<DϲϳϬϯϯϲ͘ϭ͖ϵϵйƐĞƋƵĞŶĐĞ ŝĚĞŶƟƚLJ͕ϳϱйĐŽǀĞƌĂŐĞͿ͘dŚĞƉůĂƐŵŝĚƉE>ϱϴͺϬϭĐŽŶƚĂŝŶƐϮϮϯĐŽĚŝŶŐƐĞƋƵĞŶĐĞƐ ŝŶĐůƵĚŝŶŐƚŚƌĞĞĨƵŶĐƟŽŶĂůĂŶƟďŝŽƟĐƌĞƐŝƐƚĂŶĐĞŐĞŶĞƐ;bla_OXA-427͕bla_TEM-1B and strB) and two truncated resistance genes (strA and sul2). The bla_TEM-1 and the strB-strA ŐĞŶĞĐŽŵďŝŶĂƟŽŶŝƐůŽĐĂƚĞĚĂĚũĂĐĞŶƚƚŽƚŚĞbla_OXA-427resistance transposon in ƚŚĞĚŝƌĞĐƟŽŶĂǁĂLJĨƌŽŵ repA͕ĂŶĚďŽƚŚĂƌĞĂƐƐŽĐŝĂƚĞĚǁŝƚŚƚŚĞƉƌĞƐĞŶĐĞŽĨ/^ϭϱ ĞůĞŵĞŶƚƐ͕ŵĂŬŝŶŐŝƚĂƌĞƐŝƐƚĂŶĐĞŝƐůĂŶĚ;Z/Ϳ͘/ŶƚŚŝƐƉůĂƐŵŝĚ͕ƚǁŽƚƌĂŶƐĨĞƌƌĞŐŝŽŶƐ ǁĞƌĞŝĚĞŶƟĮĞĚ͕ůŽĐĂƚĞĚĨƌŽŵƉŽƐŝƟŽŶϰϵϯϭϯƚŽϱϳϯϮϰĂŶĚŝŶĐůƵĚŝŶŐϯtra genes and kfrA;ĨŽƌƉůĂƐŵŝĚŵĂŝŶƚĞŶĂŶĐĞͿĂŶĚŽŶĞĨƌŽŵƉŽƐŝƟŽŶƐϭϰϱϲϲϱƚŽϭϳϯϴϯϭ that includes 12 tra genes and trhF.

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ŚĂƉƚ

Ğƌϧ

&ŝŐƵƌĞ ϭ. ŝƌĐƵůĂƌ ƌĞƉƌĞƐĞŶƚĂƟŽŶ ŽĨ ƉE>ϱϴͺϬϭ͕ ŚĂƌďŽƵƌŝŶŐ bla_OXA-427.KŶůLJ ŐĞŶĞƐ ĨŽƌ ƉĂƌƟƟŽŶŝŶŐͬƌĞƉůŝĐĂƟŽŶ;LJĞůůŽǁͿ͕ĐŽŶũƵŐĂƟǀĞƚƌĂŶƐĨĞƌ;ŐƌĞĞŶͿ͕ĂŶƟďŝŽƟĐƌĞƐŝƐƚĂŶĐĞ;ƌĞĚͿ͕ and mobility (gray) are depicted. Prime symbols indicate truncated genes.

dŚĞĂƐƐĞŵďůLJŽĨƚŚĞK.KK pneumoniae͕<>WEϱϳ͕ƌĞĂĚƐƌĞƐƵůƚĞĚŝŶĂŐĞŶŽŵĞŽĨϱ͘Ϯϯ

DďǁŝƚŚ^d-ϭĂŶĚƚŚƌĞĞƉůĂƐŵŝĚƐ;ϯϮϭŬď͕ϭϳϱŬďĂŶĚϳϳŬďͿ͘dŚĞ bla_OXA-427gene ǁĂƐŝĚĞŶƟĮĞĚŽŶƚŚĞϯϮϭ͕ϰϱϱďƉƉůĂƐŵŝĚƉ<>WEϱϳͺϬϭƉůĂƐŵŝĚƚŚĂƚĐĂƌƌŝĞĚϰϭϳ ƉƌĞĚŝĐƚĞĚĐŽĚŝŶŐƐĞƋƵĞŶĐĞƐ;&ŝŐƵƌĞϮͿ͘dŚĞŽƚŚĞƌϮƉůĂƐŵŝĚƐĂƌĞŽĨƚLJƉĞ/ŶĐ&/ď with resistance genes bla_{dyͲDͲϭϱ͕}bla_dDͲϭ͕ĂƉŚ;ϯ͛ͿͲ/Ă, aadA2, sul1, ĂŶĚ ŽĨ ƚLJƉĞ /ŶĐ&//ǁŝƚŚ qnrS1.

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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&ŝŐƵƌĞϮ.ŝƌĐƵůĂƌƌĞƉƌĞƐĞŶƚĂƟŽŶŽĨƉ<>WEϱϳͺϬϭ͕ŚĂƌďŽƵƌŝŶŐbla_OXA-427. The plasmid parts ŝĚĞŶƟĐĂůƚŽƚŚŽƐĞŝŶƉE>ϱϴͺϬϭ;хϵϵйƐŝŵŝůĂƌŝƚLJͿĂƌĞŝŶĚŝĐĂƚĞĚŝŶďůƵĞ͘KŶůLJŐĞŶĞƐĨŽƌ ƉĂƌƟƟŽŶŝŶŐͬƌĞƉůŝĐĂƟŽŶ;LJĞůůŽǁͿ͕ĐŽŶũƵŐĂƟǀĞƚƌĂŶƐĨĞƌ;ŐƌĞĞŶͿ͕ĂŶƟďŝŽƟĐƌĞƐŝƐƚĂŶĐĞ;ƌĞĚͿ͕ and mobility (gray) are depicted. Prime symbols indicate truncated genes.

dŚĞƉůĂƐŵŝĚƉ<>WEϱϳͺϬϭĐŽŶƚĂŝŶĞĚƐĞǀĞŶƌĞƐŝƐƚĂŶĐĞŐĞŶĞƐĨŽƌĂŵŝŶŽŐůLJĐŽƐŝĚĞ resistance (aadA24͕ aacA4, strBͿ͕ ɴͲůĂĐƚĂŵ ƌĞƐŝƐƚĂŶĐĞ ;bla_OXA-10͕ bla_TEM-1B,

bla_OXA-427Ϳ͕ĐŚůŽƌĂŵƉŚĞŶŝĐŽůƌĞƐŝƐƚĂŶĐĞ;ŇŽZ;; ) and sulphonamide resistance (sul1).

&ŝǀĞ ĂĚĚŝƟŽŶĂů ƌĞƐŝƐƚĂŶĐĞ ŐĞŶĞƐ ;strA͕ bla_OXA-2͕ sul2͕ bla_OXA-10 and aacA4) were ƚƌƵŶĐĂƚĞĚĂŶĚǁĞƌĞƉŽƚĞŶƟĂůůLJĚLJƐĨƵŶĐƟŽŶĂů͘/ŶƚĞƌĞƐƟŶŐůLJ,ƚǁŽƚLJƉĞƐŽĨrepA ŐĞŶĞƐĐŽĚŝŶŐĨŽƌƉůĂƐŵŝĚƌĞƉůŝĐĂƟŽŶƉƌŽƚĞŝŶƐǁĞƌĞĚĞƚĞĐƚĞĚŽŶƚŚĂƚƉůĂƐŵŝĚ͘ These repA ŐĞŶĞƐďĞůŽŶŐƚŽƚǁŽĚŝƐƟŶĐƚŝŶĐŽŵƉĂƟďŝůŝƚLJƉůĂƐŵŝĚŐƌŽƵƉƐŶĂŵĞůLJ͕ /ŶĐͬϮĂŶĚ/ŶĐ&/ď͘&ƵƌƚŚĞƌĂŶĂůLJƐŝƐĐŽŶĮƌŵĞĚƚŚĂƚƚŚĞƉ<>WEϱϳͺϬϭŝƐĐŽŵƉŽƐĞĚ

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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ϭϭϵ

ŚĂƉƚ

Ğƌϧ

ŽĨĂŶŽǀĞůƉůĂƐŵŝĚ ďĞůŽŶŐŝŶŐƚŽ/ŶĐ&/ď ŐƌŽƵƉ͕ĂŶĚ ĂŶ/ŶĐͬϮƉůĂƐŵŝĚ ŚŝŐŚůLJ ƐŝŵŝůĂƌƚŽƉE>ϱϴͺϬϭŝĚĞŶƟĮĞĚŝŶ͘ĐůŽĂĐĂĞE>ϱϴ;&ŝŐƵƌĞϯͿ͘ĚĚŝƟŽŶĂůůLJ͕ ƚŚĞ ƉůĂƐŵŝĚ ĐĂƌƌŝĞƐ Ϯ ƉĂƌƚƐ ŽĨ Ă ƌĞƉ ŐĞŶĞ ƌĞůĂƚĞĚ ƚŽ ĐŽůt ƐĞƉĂƌĂƚĞĚ ďLJ ƚŚĞ /ŶĐͬϮƌĞŐŝŽŶ͕ƚŽŐĞƚŚĞƌĐŽŵƉƌŝƐŝŶŐƚŚĞĐŽŵƉůĞƚĞƌĞƉůŝĐĂƟŽŶŐĞŶĞ͘ĂƐĞĚŽŶ ƚŚĞƉƌĞƐĞŶĐĞŽĨĚŝīĞƌĞŶƚƌĞƉŐĞŶĞƐĂŶĚƚŚĞůĂƌŐĞŶƵŵďĞƌŽĨŵŽďŝůĞŐĞŶĞƟĐ ĞůĞŵĞŶƚƐ͕ ƚŚĞ Ɖ<>WEϱϳͺϬϭ ƉůĂƐŵŝĚ ŝƐ ŝŶ ĨĂĐƚ Ă ŵĞŐĂƉůĂƐŵŝĚ ŽĨ ϯϮϭ Ŭď ĂŶĚ ĐŽƵůĚ ďĞ Ă ƌĞƐƵůƚ ŽĨ Ă ŵƵůƟƉůĞ ĨƵƐŝŽŶƐ ŽĨ ĚŝīĞƌĞŶƚ ƉůĂƐŵŝĚƐ ĂŶĚͬŽƌ ŵŽďŝůĞ ĞůĞŵĞŶƚƐ͘ dŚĞ /ŶĐ&/ď ƌĞŐŝŽŶ ƐŚŽǁĞĚ ŚŝŐŚĞƐƚ ŚŽŵŽůŽŐLJ ǁŝƚŚ <͘ ƉŶĞƵŵŽŶŝĂĞ plasmid CP021541.1 ;ϵϵйŝĚĞŶƟƚLJ͕ϲϴйĐŽǀĞƌĂŐĞͿ͕ĂŶĚƚŚĞŽůtƌĞŐŝŽŶǁŝƚŚ Achromobacter xylosoxidans subsp. ĚĞŶŝƚƌŝĮĐĂŶƐ ƉůĂƐŵŝĚ ,&ϲϳϵϮϳϵ͘ϭ ;ϭϬϬй ŝĚĞŶƟƚLJ͕ϯϳйĐŽǀĞƌĂŐĞͿ͘hŶĨŽƌƚƵŶĂƚĞůLJ͕ǁĞǁĞƌĞŶŽƚĂďůĞƚŽŝĚĞŶƟĨLJƚŚĞƐŽƵƌĐĞƐ ŽĨƚŚĞƐĞƉůĂƐŵŝĚĞůĞŵĞŶƚƐ͘ &ŝŐƵƌĞϯ͘WĂŝƌǁŝƐĞĐŽŵƉĂƌŝƐŽŶŽĨƉE>ϱϴͺϬϭ;ƵƉƉĞƌƐĞƋƵĞŶĐĞͿĂŶĚƉ<>WEϱϳͺϬϭ;ůŽǁĞƌ ƐĞƋƵĞŶĐĞͿ͘ dŚĞ intl1 ŐĞŶĞ ŽĨ ƉE>ϱϴͺϬϭ ŝƐ ƵƐĞĚ ĂƐ Ă ƐƚĂƌƟŶŐ ƉŽŝŶƚ͘ ƚ ƚŚĞ ƐƚĂƌƟŶŐ ƉŽƐŝƟŽŶ͕ ƚŚĞ ŚŽŵŽůŽŐŽƵƐ ƉůĂƐŵŝĚ ŝƐ ĚŝƐƌƵƉƚĞĚ ĂŶĚ ŝŶƚĞŐƌĂƚĞĚ ŝŶƚŽ ƚŚĞ Ɖ<>WEϱϳͺϬϭ ŵĞŐĂƉůĂƐŵŝĚ͘ŽŶŶĞĐƟŶŐďĂƌƐƌĞƉƌĞƐĞŶƚƌĞŐŝŽŶƐǁŝƚŚ>^dEŵĂƚĐŚĞƐŽĨхϵϵ͘ϵйŝŶƚŚĞ ƐĂŵĞ;ƌĞĚͿĂŶĚŝŶǀĞƌƚĞĚ;ďůƵĞͿŽƌŝĞŶƚĂƟŽŶƐ͕ǀŝƐƵĂůŝnjĞĚǁŝƚŚƚŚĞƌƚĞŵŝƐŽŵƉĂƌŝƐŽŶdŽŽů 8_.

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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dŚĞ ŐĞŶĞƟĐ ĞŶǀŝƌŽŶŵĞŶƚ ĚŽǁŶƐƚƌĞĂŵ ŽĨ ƚŚĞ bla_OXA-427 ŐĞŶĞ͕ ĐŚĂƌĂĐƚĞƌŝƐĞĚ ďLJ /^ϭϯϮϲĂŶĚĂdŶϯĨĂŵŝůLJƚƌĂŶƐƉŽƐŽŶ͕ŝƐŝĚĞŶƟĐĂůŝŶƚŚĞƚǁŽƉůĂƐŵŝĚƐ͘hƉƐƚƌĞĂŵ ŽĨ ƚŚĞ bla_OXA-427ŐĞŶĞ͕ ŚŽǁĞǀĞƌ͕ ŝŶ Ɖ<>WEϱϳͺϬϭ͕ ĐŽŵƉĂƌĞĚ ƚŽ ƉE>ϱϴͺϬϭ͕ ĂĚĚŝƟŽŶĂůƌĞƐŝƐƚĂŶĐĞŐĞŶĞƐĂƌĞƉƌĞƐĞŶƚ;&ŝŐƵƌĞϭĂŶĚ&ŝŐƵƌĞϮͿ. /Ŷ&ŝŐƵƌĞϯ͕ƚŚĞĂůŝŐŶŵĞŶƚŽĨƉE>ϱϴĂŶĚƉ<>WEϱϳŝƐƐŚŽǁŶ͘/ŶƚŚĞƉ<>WEϱϳͺϬϭ ŵĞŐĂƉůĂƐŵŝĚ͕ƚŚĞ/ŶĐͬϮƌĞŐŝŽŶŝƐŇĂŶŬĞĚďLJŝĚĞŶƟĐĂůĐůĂƐƐ/ŝŶƚĞŐƌŽŶŝŶƚĞŐƌĂƐĞ ŐĞŶĞƐ͕ IntI1͕ĂƚďŽƚŚƐŝĚĞƐ͘ƚƚŚĞϱ͛ƐŝĚĞ͕ƚŚĞ IntI1ŐĞŶĞŝƐĨƵƐĞĚǁŝƚŚĂƌĞƐŝƐƚĂŶĐĞ ŝŶƚĞŐƌŽŶ͘ƚƚŚĞϯ͛ƐŝĚĞ͕ƚŚĞIntI1 ŐĞŶĞŽĨ/ŶĐͬϮŝƐĨƵƐĞĚǁŝƚŚĂƉĂƌƟĂůcolW-WW like repA ŐĞŶĞ ďLJ Ă ƚƌĂŶƐƉŽƐĂƐĞ ŐĞŶĞ ŽĨ ƚŚĞ dŶϯͲĨĂŵŝůLJ ƚƌĂŶƐƉŽƐĂƐĞƐ ĂŶĚ ĂŶ /^ϲŝŶƐĞƌƟŽŶƐĞƋƵĞŶĐĞ͘/ŶďŽƚŚƌĞŐŝŽŶƐŽĨĨƵƐŝŽŶ͕ƚŚĞƌĞŝƐĂŶĂďƵŶĚĂŶĐĞŽĨdŶϯ ĨĂŵŝůLJƚƌĂŶƐƉŽƐĂƐĞƐ͕/^ϲ͕ĂŶĚ/^ϭϭϬŝŶƐĞƌƟŽŶƐĞƋƵĞŶĐĞƐ͘dŚĞƌĞŐŝŽŶŚŽŵŽůŽŐŽƵƐ ƚŽƉE>ϱϴŝƐĚŝǀŝĚĞĚŝŶƚǁŽƉĂƌƚƐďLJĂƚƌĂŶƐƉŽƐŽŶŇĂŶŬĞĚŽŶďŽƚŚƐŝĚĞƐďLJ/^ϵϭ͕ carrying the resistance gene ŇŽZ.

We conclude that the megaplasmid in the K.KK pneumoniaeŝƐŽůĂƚĞŝƐĂƌĞƐƵůƚŽĨ

ĐŽŝŶƚĞŐƌĂƟŽŶŽĨĂŶ/ŶĐͬϮͲƚLJƉĞƉůĂƐŵŝĚŚĂƌďŽƵƌŝŶŐbla_OXA-427ǁŝƚŚĂŶ/ŶĐ&ϭďͲƚLJƉĞ ƉůĂƐŵŝĚ͘dŚĞĂďŝůŝƚLJŽĨdŶϯĂŶĚŝŶƐĞƌƟŽŶƐĞƋƵĞŶĐĞƐƚŽŵĞĚŝĂƚĞŝŶĐŽŝŶƚĞŐƌĂƟŽŶ ŽĨƉůĂƐŵŝĚƐŝƐǁĞůůŬŶŽǁŶϵ_{.WůĂƐŵŝĚĐŽŝŶƚĞŐƌĂƚĞƐŚĂǀĞďĞĞŶƌĞƉŽƌƚĞĚƉƌĞǀŝŽƵƐůLJ} in carbapenemase producing Enterobacteriaceae strains4,10_{͖ ƚŚĞLJ ƐŚŽǁĞĚ} /ŶĐ&/ĂͲƚLJƉĞ ƉůĂƐŵŝĚ ďĂĐŬŐƌŽƵŶĚƐ͕ ĂŶĚ /^Ϯϲ ŝŶƐĞƌƟŽŶ ƐĞƋƵĞŶĐĞƐ ŇĂŶŬŝŶŐ ƚŚĞ ƌĞŐŝŽŶƐŽĨĨƵƐŝŽŶ͘dŽŽƵƌŬŶŽǁůĞĚŐĞ͕ŚŽǁĞǀĞƌ͕ĐŽŝŶƚĞŐƌĂƚĞƐĨŽƌŵĞĚĨƌŽŵůĂƌŐĞ ƌĞƐŝƐƚĂŶĐĞƉůĂƐŵŝĚƐǁŝƚŚƐŝnjĞƐŽĨхϯϬϬŬď͕ŚĂǀĞŶŽƚďĞĞŶƌĞƉŽƌƚĞĚƉƌĞǀŝŽƵƐůLJ͘ dŚĞƉůĂƐŵŝĚĐŽŝŶƚĞŐƌĂƟŽŶƐĞĞŵƐƚŽƌĞƉƌĞƐĞŶƚĂŶŝŵƉŽƌƚĂŶƚŵŽůĞĐƵůĂƌƉĂƚŚǁĂLJ ĨŽƌŝŶƚĞƌͲƐƉĞĐŝĞƐƚƌĂŶƐĨĞƌŽĨƉůĂƐŵŝĚƐĂŶĚƚŚĞŝƌƌĞƐŝƐƚĂŶĐĞŐĞŶĞƐ͘ŽŝŶƚĞŐƌĂƟŽŶ ǁŝƚŚĚŝƐƌƵƉƟŽŶŽĨƌĞƉůŝĐŽŶƐ͕ĂƐŽďƐĞƌǀĞĚŝŶƉ<>WEϱϳͺϬϭ͕ŵĂLJĂůůŽǁƉůĂƐŵŝĚ ŝŶĐŽŵƉĂƟďŝůŝƚLJƚŽďĞŽǀĞƌĐŽŵĞ11_{.EĞdžƚ͕ĐŽŝŶƚĞŐƌĂƟŽŶǁŝƚŚĂƉůĂƐŵŝĚƚŚĂƚŚĂƐ} ĐŽͲĞǀŽůǀĞĚŝŶ K.KK pneumoniae ŵĂLJƉƌŽǀŝĚĞƚŚĞƚƌĂŶƐĨĞƌƌĞĚƉůĂƐŵŝĚǁŝƚŚƐƉĞĐŝĮĐ ĐŽŶũƵŐĂƟŽŶ ĂŶĚ ƌĞƐƚƌŝĐƟŽŶ ŵŽĚŝĮĐĂƟŽŶ ŵĂĐŚŝŶĞƌLJ11_{. dŚĞ ĐŽŝŶƚĞŐƌĂƟŽŶ ŽĨ} ƉůĂƐŵŝĚƐ ŝŶƚŽ Ɖ<>WEϱϳͺϬϭ ŚĂƐ ƉƌŽďĂďůLJ ƚĂŬĞŶ ƉůĂĐĞ ŝŶ ƌĞĐĞŶƚ LJĞĂƌƐ͘ &ƵƚƵƌĞ ƐƚƵĚŝĞƐǁŝůůŶĞĞĚƚŽĐůĂƌŝĨLJǁŚĞƚŚĞƌŵĞŐĂƉůĂƐŵŝĚƐŽĨƚŚĞƐŝnjĞƉƌĞƐĞŶƚĞĚŝŶŽƵƌ ƐƚƵĚLJĂƌĞƉĞƌƐŝƐƚĞŶƚ͕ŽƌƚŚĂƚƚŚĞLJƌĞƉƌĞƐĞŶƚĂƚĞŵƉŽƌĂƌLJŝŶŝƟĂůƐƚĂŐĞŝŶĂŶƟďŝŽƟĐ ƌĞƐŝƐƚĂŶĐĞƉůĂƐŵŝĚĞǀŽůƵƟŽŶ͘

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

ĐĐĞƐƐŝŽŶŶƵŵďĞƌƐ dŚĞ ĂŶŶŽƚĂƚĞĚ ƉůĂƐŵŝĚ ƐĞƋƵĞŶĐĞƐ ŽĨ Ɖ<>WEϱϳͺϬϭ ĂŶĚ ƉE>ϱϴͺϬϭ ǁĞƌĞ ĚĞƉŽƐŝƚĞĚĂƚ:ͬD>ͬ'ĞŶĂŶŬƵŶĚĞƌƚŚĞĂĐĐĞƐƐŝŽŶŶƵŵďĞƌƐ>dϴϴϮϲϵϴĂŶĚ >dϴϴϮϲϵϵ͕ƌĞƐƉĞĐƟǀĞůLJ͘ ĐŬŶŽǁůĞĚŐĞŵĞŶƚ tĞŬŝŶĚůLJƚŚĂŶŬƌƚƵƌ^ĂďĂƚ;hŶŝǀĞƌƐŝƚLJŽĨ'ƌŽŶŝŶŐĞŶ͕hŶŝǀĞƌƐŝƚLJDĞĚŝĐĂů ĞŶƚĞƌ'ƌŽŶŝŶŐĞŶ͕EĞƚŚĞƌůĂŶĚƐͿĨŽƌŚŝƐƌĞǀŝĞǁŽĨƚŚĞƐĞƋƵĞŶĐĞĂŶĂůLJƐŝƐ͕ĂŶĚDr. z͘WĨĞŝĨĞƌĨƌŽŵƚŚĞZŽďĞƌƚ<ŽĐŚ/ŶƐƟƚƵƚĞĨŽƌŬŝŶĚůLJƉƌŽǀŝĚŝŶŐƚŚĞE. coliũϱϯĂnjŝĚZi 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

Processed on: 13-11-2019 PDF page: 122PDF page: 122PDF page: 122PDF page: 122

122 References 1. ŽŐĂĞƌƚƐ͕W͘ et al. KyͲϰϮϳ͕ĂŶĞǁƉůĂƐŵŝĚͲďŽƌŶĞĐĂƌďĂƉĞŶĞŵͲŚLJĚƌŽůLJƐŝŶŐ ĐůĂƐƐɴͲůĂĐƚĂŵĂƐĞŝŶ Enterobacteriaceae. :͘ŶƟŵŝĐƌŽď͘ŚĞŵŽƚŚĞƌ͘ 72͕ ϮϰϲϵʹϮϰϳϳ;ϮϬϭϳͿ͘ Ϯ͘ ŶŶĂ͘^ŚĞƉƉĂƌĚ͕ĂEŝĐŽůĞ^ƚŽĞƐƐĞƌ͕ĂŶŝĞů:͘tŝůƐŽŶ͕ĂZŽďĞƌƚ^͘EĞƐƚĞĚ ZƵƐƐŝĂŶ ŽůůͲ>ŝŬĞ 'ĞŶĞƟĐ DŽďŝůŝƚLJ ƌŝǀĞƐ ZĂƉŝĚŝƐƐĞŵŝŶĂƟŽŶ ŽĨ ƚŚĞ ĂƌďĂƉĞŶĞŵ ZĞƐŝƐƚĂŶĐĞ 'ĞŶĞďůĂ<W͘ Am. Soc. Microbioloy 60͕ ϯϳϲϳʹ ϯϳϳϴ;ϮϬϭϲͿ͘

ϯ͘ DĂƚŚĞƌƐ͕ ͘ :͘ et al. Klebsiella pneumoniae carbapenemase (KPC)-producing K. pneumoniae ĂƚĂƐŝŶŐůĞŝŶƐƟƚƵƟŽŶ͗/ŶƐŝŐŚƚƐŝŶƚŽĞŶĚĞŵŝĐŝƚLJ ĨƌŽŵ tŚŽůĞͲŐĞŶŽŵĞ ƐĞƋƵĞŶĐŝŶŐ͘ ŶƟŵŝĐƌŽď͘ ŐĞŶƚƐ ŚĞŵŽƚŚĞƌ͘ 59͕ ϭϲϱϲʹϭϲϲϯ;ϮϬϭϱͿ͘

ϰ͘ ŽŶůĂŶ͕^͘et al.^ŝŶŐůĞͲŵŽůĞĐƵůĞƐĞƋƵĞŶĐŝŶŐƚŽƚƌĂĐŬƉůĂƐŵŝĚĚŝǀĞƌƐŝƚLJŽĨ hospital-associated carbapenemase-producing Enterobacteriaceae. Sci.

Transl. Med. 6͕;ϮϬϭϰͿ͘ ϱ͘ ^ŝŐƵŝĞƌ͕W͘/^ĮŶĚĞƌ͗ƚŚĞƌĞĨĞƌĞŶĐĞĐĞŶƚƌĞĨŽƌďĂĐƚĞƌŝĂůŝŶƐĞƌƟŽŶƐĞƋƵĞŶĐĞƐ͘ EƵĐůĞŝĐĐŝĚƐZĞƐ͘ 34͕ϯϮʹϯϲ;ϮϬϬϲͿ͘ ϲ͘ ĂƌĂƩŽůŝ͕ ͘ et al. /Ŷ ^ŝůŝĐŽ ĚĞƚĞĐƟŽŶ ĂŶĚ ƚLJƉŝŶŐ ŽĨ ƉůĂƐŵŝĚƐ ƵƐŝŶŐ ƉůĂƐŵŝĚĮŶĚĞƌ ĂŶĚ ƉůĂƐŵŝĚ ŵƵůƟůŽĐƵƐ ƐĞƋƵĞŶĐĞ ƚLJƉŝŶŐ͘ ŶƟŵŝĐƌŽď͘ ŐĞŶƚƐŚĞŵŽƚŚĞƌ͘ 58͕ϯϴϵϱʹϯϵϬϯ;ϮϬϭϰͿ͘ ϳ͘ >ĂƌƐĞŶ͕ D͘ s͘ et al. DƵůƟůŽĐƵƐ ƐĞƋƵĞŶĐĞ ƚLJƉŝŶŐ ŽĨ ƚŽƚĂůͲŐĞŶŽŵĞͲ ƐĞƋƵĞŶĐĞĚďĂĐƚĞƌŝĂ͘ :͘ůŝŶ͘DŝĐƌŽďŝŽů͘ 50͕ϭϯϱϱʹϭϯϲϭ;ϮϬϭϮͿ͘ ϴ͘ ĂƌǀĞƌ͕d͘:͘ et al.d͗dŚĞƌƚĞŵŝƐĐŽŵƉĂƌŝƐŽŶƚŽŽů͘ŝŽŝŶĨŽƌŵĂƟĐƐ 21͕ ϯϰϮϮʹϯϰϮϯ;ϮϬϬϱͿ͘ ϵ͘ ^ŚĂƉŝƌŽ͕:͘͘DŽůĞĐƵůĂƌŵŽĚĞůĨŽƌƚŚĞƚƌĂŶƐƉŽƐŝƟŽŶĂŶĚƌĞƉůŝĐĂƟŽŶŽĨ bacteriophage Mu and other transposable elements. Proc. Natl. Acad.

Sci. U. S. A. 76͕ϭϵϯϯʹϭϵϯϳ;ϭϵϳϵͿ͘ ϭϬ͘ ŚĂǀĚĂ͕<͘͘et al. ŽŵƉůĞƚĞƐĞƋƵĞŶĐĞŽĨĂďůĂ<WͲ,ĂƌďŽƌŝŶŐĐŽŝŶƚĞŐƌĂƚĞ ƉůĂƐŵŝĚ ŝƐŽůĂƚĞĚ ĨƌŽŵ ƐĐŚĞƌŝĐŚŝĂ ĐŽůŝ͘ ŶƟŵŝĐƌŽď͘ ŐĞŶƚƐ ŚĞŵŽƚŚĞƌ͘ 59͕ϮϵϱϲʹϮϵϱϵ;ϮϬϭϱͿ͘ ϭϭ͘ >ŝŶ͕͘>͕͘ZĂŵŝƌĞnj͕D͘^͕͘dƌĂŶ͕d͘ΘdŽůŵĂƐŬLJ͕D͘͘ĐŽŝŶƚĞŐƌĂƚĞͲůŝŬĞ ƉůĂƐŵŝĚ ƚŚĂƚ ĨĂĐŝůŝƚĂƚĞƐ ĚŝƐƐĞŵŝŶĂƟŽŶ ďLJ ĐŽŶũƵŐĂƟŽŶ ŽĨ ƚŚĞ ĞdžƚĞŶĚĞĚͲ ƐƉĞĐƚƌƵŵ ɴͲůĂĐƚĂŵĂƐĞ dyͲDͲϭϳ͘ ŶƟŵŝĐƌŽď͘ ŐĞŶƚƐ ŚĞŵŽƚŚĞƌ͘ 57͕ ϱϭϵϭʹϱϭϵϮ;ϮϬϭϯͿ͘

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: 123PDF page: 123PDF page: 123PDF page: 123

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