Why the discovery of adherent-invasive Escherichia coli molecular markers is so challenging?

Table 1 Phenotype, phylogroup and prevalence of virulence genes found to be more frequent in adherent-invasive Escherichia coli than non-adherent-invasive Escherichia coli strains in PCR-based and genomic studies

Virulence gene	Group of study (n)		Phylogroup (n)1					Prevalence (%)
	AIEC	non-AIEC	A	B1	B2	D	Others	AIEC	non-AIEC
malX[37]	49	1342	39	19	98	19	8	71	47
kpsMTII[37]	49	1342	39	19	98	19	8	71	52
pduC[31]	24	25	14	16	10	9	0	50	20
lpfA[31]	24	25	14	16	10	9	0	71	20
lpfA + gipA[39]	35	103	Undetermined					31	0
chuA[13]	15	37	11	5	18	18	0	93	59
ibeA[40]	19	57	Undetermined					37	3
colV[40]	19	57	Undetermined					42	16
vat[38]	22	37	9	8	29	12	1	59	30
pic[38]	22	37	9	8	29	12	1	41	16
papGII/III[38]	22	37	9	8	29	12	1	18	0
iss[38]	22	37	9	8	29	12	1	32	11

¹Based on house-keeping genes identified by triplex PCR[41] or by structure analysis included in the multilocus sequence typing analyses[42].

²Nineteen intestinal pathogenic Escherichia coli strains and 78 ExPEC strains isolated from animals and 37 human mucosal-associated non-AIEC strains. AIEC: Adherent-invasive Escherichia coli.

Table 2 Review of studies in which the prevalence of particular virulence genes has been examined according to the adherent-invasive Escherichia coli pathotype

Ref.	AIEC	Non-AIEC	Genes studied
Darfeuille-Michaud et al[1], 2004	26	0	afaD, eae, ipaC, tia
Martinez-Medina et al[11], 2009	22	38	afa/draBC, bfpA, cdtB,cnf1, eae, eltA, est, fimAvMT78, fimH, hlyA, ibeA, ipaH, iucD, neuC, papC, pCDV432, sfa/focDE, stx1, stx2
Martinez-Medina et al[26], 2009	27	59	afa/draBC, bfpA, bmaE, cdtB, cnf1, cvaC, eae, eltA, est, fimA, fimAvMT78, fimH, focG, gafD, hlyA, ibeA ,ipaH, iroN, iucD, kpsMII, kpsMIII, malX, neuC, papC, papGI, papGII, papGIII alleles,pCDV432, sat, sfa/focDE, sfaS, stx1, stx2, traT, usp
Martinez-Medina et al[37], 2011	49	134	afa/draBC, astA, bmaE, chuA, cnf, csgA, cvaB, cvaC, eaI, eitA, eitC, etsB, etsC, fimC, focG, fyuA, gafD, gimB, hlyA, hlyF, hra, ibeA, iha, ireA, iroN, irp2, iss, iucD, iutA, kpsMTII, malX, mat, neuC, nfaE, ompA, ompT, papC, papEF, papGI, papGII, papGII/III, papGIII, pic, pks, sat, sfa/foc, sfaS, sitA, sitD (chr.), sitD (epis.), tia, traT, tsh, vat
Chassaing et al[5], 2011	249		lpfA
Conte et al[16], 2014	27	0	afa/draBC, aggR, cnf1, cvaC, fimH, focG, fyuA, gafD, hlyA, ibeA, iutA, kpsMT1, kpsMT5, kpsMTII, kpsMTIII, nfaE, pAA, PAI1, papA, papC, papEF, papG alleles, sfa/focDE, traT
Vazeille et al[39], 2016	35	103	lpfA + gipA
Céspedes et al[13], 2017	15	37	afa/draBC, aufA, cdtB, chuA, cnf1, cvaC, eaaA, eatA, ecNA144, espC, espP, fhuD, fimAvMT78 , fimH, gipA, hlyA, ibeA, irp2, neuC, papC, pet, pic, ratA, sat, sepA, sfa/focDE, sigA, tsh, vat
Dogan et al[40], 2018	19	57	afaC, chuA, cnf1, colV, focG, fyuA, gsp, hcp, ibeA, iss, kpsMII, lpfA, malX, papC, pduC, pmt1, ratA, sfaDE, traC
Camprubí-Font et al[38], 2019	48	56	afa/draBC, bmaE, csgA, fimC, focG, gafD, hra, iha, mat, nfaE, papC, papEF, papGII/III, papGI, papGII, papGIII, sfa/foc, sfaS, tsh, chuA, eitA, eitC, fyuA, ireA, iroN, irp2, iucD, iutA, sitA, sitD, (epis.), sitD (chr.), iss, neuC, kpsMTII, ompA, ompT, traT, astA, cnf, sat, vat, hlyA, hlyF, ibeA, gimB, tia, malX, pic, pks, eaI, cvaB, cvaC, etsB, etsC, lpfA141, lpfA154, fimH, chiA, astA, cnf, sat, vat
Camprubí-Font et al[46], 2019	13	30	ompA, ompC, ompF

Genes associated with pathotype or origin of isolation are highlighted in bold.

¹Pathogenicity island described in a virulent uropathogen. AIEC: Adherent-invasive Escherichia coli; CD: Crohn’s disease patients; UC: Ulcerative colitis patients.

Table 3 Summary of the comparative genomics studies conducted in adherent-invasive Escherichia coli to date

Ref.	AIEC	Non-AIEC	Phylogroup	AIEC origin of isolation
Miquel et al[27], 2010	1	211	AIEC: B2; Commensals: 4A, 2B1, 1B2; ExPEC: 2B1, 6B2, 3D, 3E	From an I-CD patient
Nash et al[28], 2010	2	101	AIEC: B2; Commensals: 2A; ExPEC: 7B2, 1E	From I-CD patients
Dogan et al[31], 2014	24	25	14 strains from A phylogroup, 16 B1, 10 B2 and 9 D2	From I-CD patients and controls
Desilets et al[32], 2015	143	6	AIEC: A: 1; B1: 1; B2: 10; D: 1; F: 1. non-AIEC: A: 2; B1: 2; B2: 2	From CD and UC patients[47]
Zhang et al[35], 2015	13	11	AIEC: 1A, 1B1, 4B2, 1D, 5 Unknown. non-AIEC: 3A, 8 Unknown	From CD and UC patients and non-CD subjects
Deshpande et al[33], 2015	4	13071	All B2	From CD patients
O’Brien et al[34], 2015	11	30	All B2, ST95	From IBD patients and controls
Camprubí-Font et al[36], 2018	3	3	AIEC: 1 B1, 1 B2 and 1 D. Non-AIEC: 1 B1, 1 B2 and 1 D	From CD patients and controls

The strain collection examined according to pathotype and phylogroup is depicted. Adherent-invasive Escherichia coli origin of isolation and study observations are also presented.

¹Include commensals and ExPEC.

²Human AIEC: 1A, 1B1, 1B2 and 1D; Murine AIEC: 1B1 and 1 B2; Dog AIEC: 2 B2; Human non-AIEC A phylogroup.

³Apart from LF82, UM146 and NRG857c the other strains were only assessed for intramacrophage replication in J774 cells. AIEC: Adherent-invasive Escherichia coli; CD: Crohn’s disease; UC: Ulcerative colitis.

Table 4 Genetic elements more frequently found in strains from the adherent-invasive Escherichia coli pathotype and suggested as putative adherent-invasive Escherichia coli molecular markers

Marker	Group of study (n)		Prevalence (%)		Sensitivity (%)	Specificity (%)	Accuracy (%)
	AIEC	non-AIEC	AIEC	non-AIEC
pduC[31]1	24	25	50	20	50	80	65
lpfA[31]1	24	25	71	20	71	80	75
29 SNPs[33]2	4	1307	100	4	-	-	-
lpfA + gipA[39]	35	103	31	0	31	100	83
3 genomic regions[32]3	14	6	79	0	79	100	85
chuA[13]4	15	37	93	59	93	41	56
SNP algorithm[36]	22	29	-	-	82	86	84
pic + ampR[38]	22	27	86	33	86	67	75

¹This strain collection was mainly formed by strains from A and B1 phylogroup (14 A, 16 B1, 10 B2 and 9D).

²Only B2 strains were included. In this case, the non-AIEC group included commensal and ExPEC strains.

³Only present in B2 AIEC strains. The strains’ phylogroup were: AIEC: 1 A, 1 B1, 10 B2, 1 D and 1 F; non-AIEC: 2 A, 2 B1 and 2 B2.

⁴Strain collection with mainly B2 and D strains (11 A, 5 B1, 18 B2 and 18 D). AIEC: Adherent-invasive Escherichia coli.

Table 5 Comparison of the principal experimental conditions of the protocols used to assess bacterial invasion to intestinal epithelial cells and survival and replication inside macrophages

Invasion assays
MOI	Infection conditions	Incubation conditions	Ref.
10	30 min	3 h with amikacin 100 µg/mL	[13]
10	1 h	2 h with gentamicin 100 µg/mL	[70]
10 or 20	3 h	1 h with gentamicin 100 µg/mL	[1,9,11,14,16,31,34,47,56-60,63-65,71]
10	3 h	1 h with gentamicin 3 mg/mL	[72]
100	2 h	1 h with gentamicin 50 µg/mL	[54]
100	3 h	1 h with gentamicin 50 µg/mL	[62]
Survival and replication assays
10	20 min	Media replacement with gentamicin 100 µg/mL for 40 min and media replacement with gentamicin 50 µg/mL for 24 h	[16]
10	2 h	Media replacement with amikacin 100 µg/mL for 3 and 24 h	[13]
10	2 h	Media replacement with gentamicin 100 µg/mL for 1 h and media replacement with gentamicin 20 µg/mL for 24 h	[1,34,58]
10 or 100	Centrifugation 10 min at 1000 g and incubation 10 min	Media replacement with gentamicin 100 µg/mL for 40 min and media replacement with gentamicin 20 µg/mL for 24 h	[11,47,66]
10	Centrifugation 5 min at 500 g and incubation 30 min	Media replacement with gentamicin 100 µg/mL for 2 h and media replacement with gentamicin 15 µg/mL for 24 h	[70]
20	2 h	Media replacement with gentamicin 100 µg/mL for 1 h and media replacement with gentamicin 20 µg/mL for 24 h	[9,31]
20	2 h	Media replacement with gentamicin 100 µg/mL for 1 and 24 h	[14]
20	2 h	Media replacement with gentamicin 3 mg/mL for 1 and 24 h	[72]
100	Centrifugation 10 min at 1000 g and incubation 10 min	Media replacement with gentamicin 20 µg/mL for 1 and 24 h	[21,59]
100	2 h	Media replacement with gentamicin 50 µg/mL for 1 and 24 h	[54]