Genetic diversity, virulence factors and drug resistance of Pantoea strains isolated from samples of fresh fruits, vegetables and soil

Fig. 1.

Fig. 2.

Primers used in the study for molecular identification and assessment of virulence and drug resistance

	Primer	Target gene	Sequence (5′→3′)	Product size (bp)	Reference
For molecular identification	infB-01_FinfB-02_R	infB	ATYATGGGHCAYGTHGAYCAACKGAGTARTAACGCAGATCCA	1,124	(44)
	PANsp_atpD_FPANsp_atpD_F	atpD	GAGGGTAACGACTTCTACCACCTGTACGGAGGTGATTGAAC	330
	PANAG_infB_FPANAG_infB_R	infB	TGTCCGGCGTGCCGGCTGCCAACGCGAACGTCGTTGT	730
	PANAN_gyrB_FPANAN_gyrB_R	gyrB	GATGACGARGCCATGCTGCGATCTTGCGGTATTCGCCAC	423	(28)
	PANST_rpoB_FPANST rpoB_R	rpoB	CACCGGTGAACTGATTATCGGTCCTGAGGCATCAATGTGT	539
	16S_27F16S907	16S rRNA	AGAGTTTGATCMTGGCTCAGCCCCGTCAATTCMTTTRAGTTT	920
	p27_Fpl525_R	16S rRNA	AGAGTTTGATCMTGGCTCAGAAGGAGGTGWTCCARCC	1,450	(30)
For virulence gene detection	acrA_FacrA_R	acrA	CTCTCAGGCAGCTTAGCCCTAATGCAGAGGTTCAGTTTTGACTGTT	106	(31)
	acrB_FacrB_R	acrB	GGTCGATTCCGTTCTCCGTTACTACCTGGAAGTAAACGTCATTGGT	104
	hcp_Fhcp_R	hcp	TGTAAACCAGCGCCATCAGTACCGGTAATGCACAGCTGAA	1,301	(22)
	vgrG_FvgrG_R	vgrG	TGAATCCGCTTGCTTCCTGTATATCGCCCATGCGTTCCAT	1,011
For antimicrobial resistance gene detection	MOXM_FMOXM_RCITM_FCITM_R	MOX-1, MOX-2, CMY-1, CMY-8-CMY-11, LAT-1-LAT-4, CMY-2-CMY-7, BIL-1	GCTGCTCAAGGAGCACAGGATCACATTGACATAGGTGTGGTGCTGGCCAGAACTGACAGGCAAATTTCTCCTGAACGTGGCTGGC	520462
	DHAM_FDHAM_R	DHA-1, DHA-2	AACTTTCACAGGTGTGCTGGGTCCGTACGCATACTGGCTTTGC	405	(37)
	ACCM_FACCM_R	ACC	AACAGCCTCAGCAGCCGGTTATTCGCCGCAATCATCCCTAGC	346
	EBCM_FEBCM_R	MIR-1T, ACT-1	TCGGTAAAGCCGATGTTGCGGCTTCCACTGCGGCTGCCAGTT	302
	FOXM_FFOXM_R	FOX-1-FOX-5b	AACATGGGGTATCAGGGAGATGCAAAGCGCGTAACCGGATTGG	190
	SHV_FSHV_R	SHV	CACTCAAGGATGTATTGTG TTAGCGTTGCCAGTGCTCG	885	(26)
	TEM-1_FTEM-1 R	TEM	AAGCCATACCAAACGACGAGATTGTTGCCGGGAAGCTAGA	108	(27)

Results of biochemical and molecular identification of Pantoea strains isolated from food and soil

Strain No.	Sample type	Biochemical identification	Molecular identification						Sequence match
			infB (Panyoea spp.)	infB (P. agglomerans)	gyrB (P. ananatis)	rpoB (P. stewartii)	atpD (Pantoea spp.)	16S (Eubacteria)
1S	strawberry	P. agglomerans	+	+	–	–	+	+	P. agglomerans
2S	raspberry	Pantoea spp.	+	+	–	–	+	+	P. agglomerans
3S	lettuce	Pantoea spp.	+	+	–	–	+	+	P. agglomerans
4S	spinach	P. agglomerans	+	+	–	–	+	+	P. agglomerans
5S	baby spinach	Pantoea spp.	+	+	–	–	+	+	P. agglomerans
6S	arugula	Pantoea spp.	+	+	–	–	+	+	P. agglomerans
7S	onion	P. agglomerans	+	+	–	–	+	+	P. agglomerans
8S	baby spinach	Pantoea spp.	–	–	–	–	+	+	P. eucalypti
9S	iceberg lettuce	Pantoea spp.	+	–	–	–	+	+	P. conspicua
10S	lam’s lettuce	Pantoea spp.	+	+	–	–	+	+	P. agglomerans
11S	soil	Pantoea spp.	+	+	–	–	+	+	P. agglomerans
12S	soil	Pantoea spp.	+	+	–	–	+	+	P. agglomerans
13S	soil	Pantoea spp.	–	+	–	–	+	+	P. agglomerans
14S	soil	Pantoea spp.	+	+	–	–	+	+	P. agglomerans
15S	soil	P. agglomerans	+	+	–	–	+	+	P. agglomerans
16S	soil	Pantoea spp.	+	+	–	–	+	+	P. agglomerans
17S	soil	Pantoea spp.	+	+	–	–	+	+	P. agglomerans
18S	soil	Pantoea spp.	+	+	–	–	+	+	P. agglomerans
19S	soil	Pantoea spp.	+	+	–	–	+	+	P. agglomerans
20S	soil	Pantoea spp.	+	+	–	–	+	+	P. agglomerans
21S	soil	P. agglomerans	+	+	–	–	+	+	P. agglomerans
22S	soil	Pantoea spp.	+	+	–	–	+	+	P. agglomerans
23S	soil	P. agglomerans	+	+	–	–	+	+	P. agglomerans
24S	soil	Pantoea spp.	+	+	–	–	+	+	P. agglomerans
25S	leek	P. ananatis	+	–	+	–	–	+	P. ananatis
26S	radish	P. agglomerans	+	+	–	–	+	+	P. agglomerans
27S	broccoli	Pantoea spp.	–	–	–	–	+	+	P. vagans
28S	soil	Pantoea spp.	+	+	–	–	+	+	P. agglomerans
29S	soil	Pantoea spp.	–	–	–	–	+	+	P. vagans
30S	leek	P. ananatis	+	–	+	–	–	+	Pantoea sp.

Occurrence of virulence and drug resistance genes in Pantoea strains

Species (strain No.)	Sample type	Virulence factor				Drug resistance gene
		acrA	acrB	vgrG	hcp	TEM	FOX	SHV	MOX	CIT	DHA	ACC	EBC
P. agglomerans (26S)	radish	–	–	+	+	+	+	–	–	–	–	–	–
P. agglomerans (1S)	raspberry	–	–	–	+	+	+	–	–	–	–	–	–
P. agglomerans (6S)	arugula	–	–	–	+	+	+	–	–	–	–	–	–
P. agglomerans (10S)	lamb’s lettuce	–	–	–	+	+	+	–	–	–	–	–	–
P. agglomerans (13S)	soil	–	–	–	+	+	+	–	–	–	–	–	–
P. agglomerans (17S)	soil	–	–	–	+	+	+	–	–	–	–	–	–
P. agglomerans (20S)	soil	–	–	–	+	+	+	–	–	–	–	–	–
P. agglomerans (21S)	soil	–	–	–	+	+	+	–	–	–	–	–	–
P. agglomerans (23S)	soil	–	–	–	+	+	+	–	–	–	–	–	–
P. agglomerans (28S)	soil	–	–	–	+	+	+	–	–	–	–	–	–
P. vagans (29S)	soil	–	–	–	+	+	+	–	–	–	–	–	–
P. agglomerans (22S)	soil	–	–	–	+	+	–	+	–	–	–	–	–
P. agglomerans (3S)	lettuce	–	–	–	+	–	–	–	–	–	–	–	–
P. agglomerans (12S)	soil	–	–	–	+	+	–	–	–	–	–	–	–
P. ananatis (25S)	leek	–	–	–	+	+	–	–	–	–	–	–	–
P. vagans (27S)	broccoli	–	–	–	+	+	–	–	–	–	–	–	–
P. ananatis (30S)	leek	–	–	–	+	+	–	–	–	–	–	–	–
P. agglomerans (4S)	spinach	–	–	–	–	+	+	–	–	–	–	–	–
P. agglomerans (5S)	baby spinach	–	–	–	–	+	+	–	–	–	–	–	–
P. eucalypti (8S)	baby spinach	–	–	–	–	+	+	–	–	–	–	–	–
P. agglomerans (11S)	soil	–	–	–	–	+	+	–	–	–	–	–	–
P. agglomerans (15S)	soil	–	–	–	–	+	+	–	–	–	–	–	–
P. agglomerans (18S)	soil	–	–	–	–	+	+	–	–	–	–	–	–
P. agglomerans (7S)	onion	–	–	–	–	+	–	+	–	–	–	–	–
P. conspicua (9S)	iceberg lettuce	–	–	–	–	+	–	+	–	–	–	–	–
P. agglomerans (2S)	raspberry	–	–	–	–	–	+	–	–	–	–	–	–
P. agglomerans (16S)	soil	–	–	–	–	–	+	–	–	–	–	–	–
P. agglomerans (19S)	soil	–	–	–	–	–	+	–	–	–	–	–	–
P. agglomerans (24S)	soil	–	–	–	–	–	+	–	–	–	–	–	–
P. agglomerans (14S)	soil	–	–	–	–	+	–	–	–	–	–	–	–

Reaction mixtures and conditions of PCR reactions performed in the study

Gene(s)	Total volume	PCR reaction mixture	Initial denaturation (temp./duration)	Denaturation, annealing, extension (temp./duration)	Number of cycles	Final extension (temp./duration)
		Reagents
infB1	25 μL	0.3 μL Taq DNA Polymerase (5U/μL)a2.5 μL 10× PCR buffer with 15 mM MgCl2b2.5 μL 2 mM dNTPsc1 μL 10 pmol of each primerd2.5 μL DNA template15.2 μL nuclease-free watere	95°C/5 min	94°C/45 s55°C/1 min72°C/1.5 min	30	72°C/7 min
atpD, infB, gyrB, rpoB, 16S rRNA2	25 μL	0.2 μL Taq DNA Polymerase (5U/μL)2.5 μL 10× PCR buffer with 15 mM MgCl22.5 μL 2 mM dNTPs0.5 μL DNA template12.8 μL nuclease-free water	94°C/3 min	94°C/3 min94°C/30 s58°C/30 s72°C/2 min	30	72°C/10 min
16S rRNA1	50 μL	0.3 μL Taq DNA Polymerase (5U/μL)5 μL 10× PCR buffer with 15 mM MgCl25 μL 2 mM dNTPs2 μL 10 pmol of each primer5 μL DNA template30.7 μL nuclease-free water	94°C/2 min	94°C/1 min55°C/1 min72°C/3 min	25	72°C/10 min
acrA or acrB1	20 μL	0.16 μL Taq DNA Polymerase (5U/μL)2.5 μL 10× PCR buffer with 15 mM MgCl22.5 μL 2 mM dNTPs0.5 μL 10 pmol of each primer2 μL DNA template12.34 μL nuclease-free water	95°C/5 min	95°C/30 s 56°C/30 s(acrA) and 55°C/30 s (acrB) 72°C/2 min	30	72°C/10 min
hcp or vgrG1ESβLgenes1	25 μL	0.2 μL Taq DNA Polymerase (5U/μL)2.5 μL 10× PCR buffer with 15 mM MgCl22.5 μL 2 mM dNTPs1 μL 10 pmol of each primer2 μL DNA template15.8 μL nuclease-free water	95°C/5 min96°C/1 min	95°C/30 s55°C/30 s72°C/2 min94°C/1 min50°C/1 min72°C/2 min	2535	72°C/10 min72°C/10 min
AmpCgenes2	25 μL	0.2 μL Taq DNA Polymerase (5U/μL)2.5 μL 10× PCR buffer with 15 mM MgCl22.5 μL 2 mM dNTPs1 μL 10 pmol of each primer2 μL DNA template5.8 μL nuclease-free water	94°C/3 min	94°C/30 s64°C/30 s72°C/1 min	25	72°C/10 min