Table 1.
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_F infB-02_R | infB | ATYATGGGHCAYGTHGAYCA ACKGAGTARTAACGCAGATCCA | 1,124 | (44) |
| PANsp_atpD_F PANsp_atpD_F | atpD | GAGGGTAACGACTTCTACCAC CTGTACGGAGGTGATTGAAC | 330 | ||
| PANAG_infB_F PANAG_infB_R | infB | TGTCCGGCGTGCCGGCTG CCAACGCGAACGTCGTTGT | 730 | ||
| PANAN_gyrB_F PANAN_gyrB_R | gyrB | GATGACGARGCCATGCTGC GATCTTGCGGTATTCGCCAC | 423 | (28) | |
| PANST_rpoB_F PANST rpoB_R | rpoB | CACCGGTGAACTGATTATCG GTCCTGAGGCATCAATGTGT | 539 | ||
| 16S_27F 16S907 | 16S rRNA | AGAGTTTGATCMTGGCTCAG CCCCGTCAATTCMTTTRAGTTT | 920 | ||
| p27_F pl525_R | 16S rRNA | AGAGTTTGATCMTGGCTCAG AAGGAGGTGWTCCARCC | 1,450 | (30) | |
| For virulence gene detection | acrA_F acrA_R | acrA | CTCTCAGGCAGCTTAGCCCTAA TGCAGAGGTTCAGTTTTGACTGTT | 106 | (31) |
| acrB_F acrB_R | acrB | GGTCGATTCCGTTCTCCGTTA CTACCTGGAAGTAAACGTCATTGGT | 104 | ||
| hcp_F hcp_R | hcp | TGTAAACCAGCGCCATCAGT ACCGGTAATGCACAGCTGAA | 1,301 | (22) | |
| vgrG_F vgrG_R | vgrG | TGAATCCGCTTGCTTCCTGT ATATCGCCCATGCGTTCCAT | 1,011 | ||
| For antimicrobial resistance gene detection | MOXM_F MOXM_R CITM_F CITM_R | MOX-1, MOX-2, CMY-1, CMY-8-CMY-11, LAT-1-LAT-4, CMY-2-CMY-7, BIL-1 | GCTGCTCAAGGAGCACAGGAT CACATTGACATAGGTGTGGTGC TGGCCAGAACTGACAGGCAAA TTTCTCCTGAACGTGGCTGGC | 520 462 | |
| DHAM_F DHAM_R | DHA-1, DHA-2 | AACTTTCACAGGTGTGCTGGGT CCGTACGCATACTGGCTTTGC | 405 | (37) | |
| ACCM_F ACCM_R | ACC | AACAGCCTCAGCAGCCGGTTA TTCGCCGCAATCATCCCTAGC | 346 | ||
| EBCM_F EBCM_R | MIR-1T, ACT-1 | TCGGTAAAGCCGATGTTGCGG CTTCCACTGCGGCTGCCAGTT | 302 | ||
| FOXM_F FOXM_R | FOX-1-FOX-5b | AACATGGGGTATCAGGGAGATG CAAAGCGCGTAACCGGATTGG | 190 | ||
| SHV_F SHV_R | SHV | CACTCAAGGATGTATTGTG TTAGCGTTGCCAGTGCTCG | 885 | (26) | |
| TEM-1_F TEM-1 R | TEM | AAGCCATACCAAACGACGAG ATTGTTGCCGGGAAGCTAGA | 108 | (27) |
1 F – forward; R – reverse; infB – translation initiation factor 2; atpD – adenosine triphosphate synthase δ subunit; gyrB – DNA gyrase subunit B; rpoB – RNA polymerase β subunit; rRNA – ribosomal RNA; acrA/B – anti–clustered regularly interspaced short palindromic repeat protein A/protein B; hcp – haemolysin-coregulated protein; vgrG – glycine-valine repeat sequence G; MOX – conferring resistance to moxalactam; CMY – conferring resistance to cephamycin; LAT – conferring resistance to latamoxef/moxalactam; BIL – patient Bilal; DHA –Dhahran Hospital; ACC – Ambler class C; MIR – Miriam Hospital; ACT – Ambler class C type; FOX – conferring resistance to cefoxitin; SHV – sulfhydryl variant reagent; TEM – patient Temoneira
Table 2.
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)a 2.5 μL 10× PCR buffer with 15 mM MgCl2b 2.5 μL 2 mM dNTPsc 1 μL 10 pmol of each primerd 2.5 μL DNA template 15.2 μL nuclease-free watere | 95°C/5 min | 94°C/45 s 55°C/1 min 72°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 MgCl2 2.5 μL 2 mM dNTPs 0.5 μL DNA template 12.8 μL nuclease-free water | 94°C/3 min | 94°C/3 min 94°C/30 s 58°C/30 s 72°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 MgCl2 5 μL 2 mM dNTPs 2 μL 10 pmol of each primer 5 μL DNA template 30.7 μL nuclease-free water | 94°C/2 min | 94°C/1 min 55°C/1 min 72°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 MgCl2 2.5 μL 2 mM dNTPs 0.5 μL 10 pmol of each primer 2 μL DNA template 12.34 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 vgrG1 ESβL genes1 | 25 μL | 0.2 μL Taq DNA Polymerase (5U/μL) 2.5 μL 10× PCR buffer with 15 mM MgCl2 2.5 μL 2 mM dNTPs 1 μL 10 pmol of each primer 2 μL DNA template 15.8 μL nuclease-free water | 95°C/5 min 96°C/1 min | 95°C/30 s 55°C/30 s 72°C/2 min 94°C/1 min 50°C/1 min 72°C/2 min | 25 35 | 72°C/10 min 72°C/10 min |
| AmpC genes2 | 25 μL | 0.2 μL Taq DNA Polymerase (5U/μL) 2.5 μL 10× PCR buffer with 15 mM MgCl2 2.5 μL 2 mM dNTPs 1 μL 10 pmol of each primer 2 μL DNA template 5.8 μL nuclease-free water | 94°C/3 min | 94°C/30 s 64°C/30 s 72°C/1 min | 25 | 72°C/10 min |
1 Reagents: Taq DNA Polymerase (Qiagen, Hilden, Germany), 10×PCR buffer containing 15 mM MgCl2 (Qiagen), dNTP Mix (deoxyribonucleoside triphosphate – Thermo Fisher Scientific, Vilnius, Lithuania), primers (Institute of Biochemistry and Biophysics, Warsaw, Poland), nuclease-free water (Qiagen). infB – translation initiation factor 2; atpD – adenosine triphosphate synthase δ subunit; gyrB – DNA gyrase subunit B; rpoB – RNA polymerase β subunit; rRNA – ribosomal RNA; acrA/B – anti–clustered regularly interspaced short palindromic repeat protein A/protein B; hcp – haemolysin-coregulated protein; vgrG – glycine-valine repeat sequence G; ESβL – extended-spectrum β-lactamase; AmpC – Ambler class C;
Table 3.
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. |
Fig. 1.
Phylogenetic tree based on the partial 16S rRNA gene sequences of Pantoea strains isolated from food and soil
Table 4.
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 | – | – | – | – | + | – | – | – | – | – | – | – |
1 acrA/B – anti–clustered regularly interspaced short palindromic repeat protein A/protein B; vgrG – glycine-valine repeat sequence G; hcp – haemolysin-coregulated protein; TEM – patient Temoneira; FOX – conferring resistance to cefoxitin; SHV – sulfhydryl variant reagent; MOX – conferring resistance to moxalactam; CIT – originally characterised in Citrobacter freundii; DHA – Dhahran Hospital; ACC – Ambler class C; EBC – originally characterised in Enterobacter cloacae complex
Fig. 2.
The activity of hydrolytic enzymes of Pantoea strains isolated from food and soil