Genomic Insights Into Nitrofurantoin Resistance Mechanisms and Epidemiology in Clinical Enterobacteriaceae

Figures & data

Table 1.  Phenotypic and genomic characteristics of nitrofurantoin resistance mechanisms of the Klebsiella pneumoniae isolates.

Isolate (clone)	MIC (mg/l)	Year isolated	Specimen type (ward)	Plasmid-/chromosomal-mediated oqxA/B	Chromosomal mutations
					nsfA	nsfB	ribE
Klebsiella pneumoniae^†
53_S27 (ST101)	128	2013	Urine (ICU)	oqxA, oqxB	R203C	R207L	R58H, D203E
52_S26 (ST101)	128	2013	Pus (surgical)	oqxA, oqxB
47_S22 (ST1478)	128	2013	Arterial line (ICU)	oqxA, oqxB	R59Q, T117I, E144A, R180H, E191D	G25S, V167I	R58H, M204V, A206S, V78I
38_S19 (ST101)	256	X^‡	X (ICU)	oqxB	R203C	R207L	R58H, D203E
36_S18 (ST101)	256	2013	Sputum	oqxB
35_S17(ST101)	256	2013	Urine (ICU)	oqxB
34_S16(ST101)	256	2013	Pus swab (Trachea)	oqxB
32_S15(ST101)	256	2013	Catheter tip (ICU)	oqxB
30_S14(ST101)	256	2013	Urine (ICU)	oqxB
29_S13(ST2017)	256	2012	Abdominal swab (ICU)	oqxB
21_S12(ST2017)	128	2012	Urine (ICU)	oqxA, oqxB
20_S11(ST2017)	256	2013	Tracheal fluid (ICU)	oqxA, oqxB
18_S10(ST101)	256	2013	Urine (neuronal)	oqxB
15_S8(ST101)	512	2013	Pus swab (leg) (neuronal)	oqxB
13_S6 (ST2016)	256	X	X (ICU)	oqxB
12_S5(ST101)	256	2013	Blood culture (ICU)	oqxB
3_S2(ST14)	64	X	Urine (surgical)	oqxB, oqxA	NM^§	NM	D203E
I(UNN45_S9) (ST323)	64	X	Urine (ICU)	oqxB, oqxA
J(UNN46_S10(ST101))	512	X	X	oqxB	R203C	R207L	R58H, D203E
D(UNN40_S4) (ST101)	256	X	Urine (ICU)	oqxB, oqxA
C(UNN39_S3) (ST101)	256	X	Urine (surgical)	oqxA, oqxB

^†K. pneumoniae ATCC 13883 (PRJNA244567) was used as a reference strain in the comparative genome analysis

^‡Missing data.

^§No unique mutation recorded in the conserved region of the gene.

ICU: Intensive care unit; MIC: Minimum inhibitory concentration; NM: No mutation.

Table 2.  Phenotypic and genomic characteristics of nitrofurantoin resistance mechanisms of the Enterobacter species isolates.

Isolate (clone)	MIC (mg/l)	Year isolated	Specimen type (ward)	Plasmid-/chromosomal-mediated oqxA/B	Chromosomal mutations
					nsfA	nsfB	ribE
Enterobacter species (unless otherwise stated in footnote, species is cloacae)^†
65_S32 (ST436)	256	X	CVP tip (ICU)	oqxB, oqxA	H16Y, Q186N, N190A	NM	N55H
63_S31^‡ (ST435)	128	X	Urine (surgical)	oqxA, oqxB	N6D, D19N, T55A, Q186R, M201L	A18P, S19A, M90L, E137Q	I26V
55_S28^§ (ST434)	128	X	ETA (ICU)	oqxB, oqxA	R9L, E28T, D32N, K54P, E94Q, L130I, Q186R, Q191H, H198N, M201L, G204D	A19T, D25E, A155G, L157M, L186V	V42I, Q58K, V77A, V125M, P154A, A173S, Q178H
49_S24^¶ (ST252)	128	X	Urine (ICU)	oqxB, oqxA	N6D, T55A, M201L	A18P, S19A, M90L, E137Q, V214L	I26V
43_S20 (ST433)^#	256	X		oqxB, oqxA	R9L, E24D, D32N, K54P, E137Q, E141D, L170I, Q191H, H198N, S213T	P18A, A19S, D25E	D34E, V42I, V125M, Q178H
16_S9^†† (ST54)	256	X		oqxA, oqxB	R9L, E28T, D32N, K54P, E94Q, L130I, Q186R, Q191H, H198N, M201L, G204D.	A19T, D25E, A155G, L157M, L186V	V42I, Q58K, V77A, V125M, P154A, A173S, Q178H
1_S1(ST108)	256	X		oqxB, oqxA	E24Q, E28D, A36G, K54P, L170I, N190H, H198N, M201L, G204D	D25E, N109A, T122C, L157M	V42I, T81I
H(UNN44_S8)^‡‡ (ST145)	128	X	Urine (ICU)	oqxA, oqxB	E24Q, E28D, A36G, K54P, E141Q, L170I, N190H, H198N, M201L, G204D	D25E, N109A, T122C, L157M	V42I
F(UNN42_S6) (ST121)^§§	256	X	Urine (surgical)	oqxA, oqxB	E24Q, E28D, A36G, K54P, A112E, L170I, N190H, H198N, M201L, G204D	D25E, N109A, T122C, L157M	V42I, T81I, V82I
A(UNN37_S1)^¶¶ (ST252)	512	X	Urine (pediatric)	oqxB, oqxA	N6D, T55A, M201L	A18P, S19A, M90L, E137Q, V214L	I26V

^†Unless otherwise stated in footnote, Enterobacter cloacae ATCC 13047 (CP001918.1) was used as a reference strain in the comparative genomics.

^‡Enterobacter asburiae: E. asburiae L1 (CP007546.1) was used as a reference strain for the comparative genomics to find amino acid mutations.

^§Enterobacter kobei: E. kobei strain 35730 (JZYS01000016.1) or E. cloacae ATCC 13047 (CP001918.1) served as reference strains for amino acid mutations.

^¶E. asburiae: E. asburiae L1 (CP007546.1) was used as a reference strain for the comparative genomics to find amino acid mutations.

^#E. cloacae complex ‘Hoffman cluster IV’.

^††E. kobei: E. kobei strain 35730 (JZYS01000016.1) or E. cloacae ATCC 13047 (CP001918.1) served as reference strains for amino acid mutations.

^‡‡E. cloacae complex ‘Hoffman cluster III’.

^§§Enterobacter species.

^¶¶E. asburiae: E. asburiae L1 (CP007546.1) was used as a reference strain for the comparative genomics to find amino acid mutations.

CVP: Central venous puncture; ETA: Endotracheal aspirate; ICU: Intensive care unit; NM: No mutation.

Table 3.  Phenotypic and genomic characteristics of nitrofurantoin resistance mechanisms of the Citrobacter freundii, Escherichia coli and Klebsiella michiganensis isolates.

Isolate (clone)	MIC (mg/l)	Year isolated	Specimen type (ward)	Plasmid-/chromosomal-mediated oqxA/B	Chromosomal mutations
					nsfA	nsfB	ribE
C. freundii^†
51_S25 (NK^‡)	64	X	Sputum (ICU)	–	Insertion of LGLAEQLLLGVVDTAMI between positions 94 and 95, D185N, T186S, S190A	T19N, A169S	E75D
48_S23 (ST63)	64	X	Catheter tip  (neuronal)	–
14_S7 (ST62)	32	X	Sputum (ICU)	–	NM	NM	NM
E. coli^§
10_S4 (ST167)	256	X	Urine (outpatient)	–	Deletions of WVF at positions 77–79, T117I, D187G	G66D, V93A, A174E	Q212E
K. michiganensis^¶
69_S35 (ST170)	256	X	Urine (outpatient)	oqxB, oqxA	M176V	N100D, D112E	Y53H

^†C. freundii ATCC 8090 = MTCC 1658 (PRJNA177199) was used as a reference strain for the comparative genomics to find amino acid mutations.

^‡MLST unknown.

^§E. coli ATCC 25922 (CP009072) was used as a subject in the comparative genomics analysis to find the amino acid mutations.

^¶K. michiganensis KCTC 1686 (CP003218.1) was used as a reference strain for this comparative genomic analysis to find amino acid mutations.

ICU: Intensive care unit; MLST: Multi-locus sequence typing; NM: No mutation.

Figure 1.  Evolutionary relationship of nitrofurantoin-resistant Klebsiella pneumoniae strains from different parts of the world.

Phylogenomic tree of (A)Klebsiella pneumoniae drawn with MEGA 7 and (B)K. pneumoniae and associated metadata drawn with Phandango. The isolates clustered according to clones and country of origin, although the genomic phylogeny shows a closer clustering of strains of different clones and countries. Strains of ST101 were not all of the same clade while many strains from the same hospitals and wards were of the same clade.

Figure 2.  Phylogenomic tree of Escherichia coli nitrofurantoin-resistant strains from Belgium, USA and South Africa.

Clustering of the strains into clades were mainly country- and clone-specific.

MLST: Multi-locus sequence typing; NFT: Nitrofurantoin.

Figure 3.  Phylogenomic tree of nitrofurantoin resistance Enterobacter species from South Africa, USA, Malaysia and Ghana.

Clustering of the strains into clades were mainly country- and clone-specific except for 65_S32 of ST436 from South Africa and NXHI01.1 of ST455 (Ghana), and between 1_S1 of ST108 and the Enterobacter spp. strain UNN42_S6.

MLST: Multi-locus sequence typing; NFT: Nitrofurantoin.

Figure 4.  Phylogenomic tree of Citrobacter freundii nitrofurantoin intermediate resistant strains from South Africa.

Clustering of the strains into clades were mainly country- and clone-specific.

MLST: Multi-locus sequence typing; NFT: Nitrofurantoin.

Figure 5.  Geometric mean of nitrofurantoin, nitrofurantoin-carbonyl cyanide m-hydrophenylhydrazine, nitrofurantoin-tannic acid and nitrofurantoin-efflux pump inhibitor minimum inhibitory concentrations.

TZ and TA significantly reduced the geometric mean MIC of NFT (p < 0.001) than the remaining efflux-inhibiting agents.

CCCP: Carbonyl cyanide m-hydrophenylhydrazine; CPZ: Chlorpromazine; EPI: Efflux pump inhibitor; MIC: Minimum inhibitory concntration; NFT: Nitrofurantoin; TA: Tannic acid; TZ: Thioridazine; VER: Verapamil.