TABLE 1.
Bacterial strains and plasmids used in this study
| Strain or isolate(s) or plasmid | Genotype or descriptiona | Source or reference |
|---|---|---|
| Strains or isolates | ||
| E. coli | ||
| DH10B | F−mcrA Δ(mrr-hsdRMS-mcrBC) φ80dlacZΔM15 ΔlacX74 deoR recA1 endA1 araD139 Δ(ara, leu)7697 galU galK λ−rpsL nupG | Life Technologies, Eragny, France |
| P. aeruginosa | ||
| KG2505 | P. aeruginosa reference strain that does not express the naturally chromosome-encoded AmpC β-lactamase and is deficient for the multidrug efflux system MexAB-OprM mexA::res-ΩSm ampC::res-ΩSm of PAO1; Smr | 11 |
| Isolate JES | Clinical isolates from a French patient hospitalized in Thailand; blaVEB-1 positive; Cazr | 9 |
| Isolates 1 to 31 | Clinical isolates from the Siriraj Hospital, Bangkok, Thailand; blaVEB-1 positive; Cazr | 3 |
| E. coli | ||
| Isolates 1 to 10 | Clinical isolates from the Siriraj Hospital, Bangkok, Thailand; blaVEB-1 positive; Cazr | 4 |
| E. cloacae | ||
| Isolates 11 to 14 | Clinical isolates from the Siriraj Hospital, Bangkok, Thailand; blaVEB-1 positive; Cazr | 4 |
| E. sakazakii | ||
| Isolate 15 | Clinical isolates from the Siriraj Hospital, Bangkok, Thailand; blaVEB-1 positive; Cazr | 4 |
| K. pneumoniae | ||
| Isolates 16 to 18 | Clinical isolates from the Siriraj Hospital, Bangkok, Thailand; blaVEB-1 positive; Cazr | 4 |
| Plasmids | ||
| pBK-CMV phagemid | Neor, Kanr | Stratagene, La Jolla, Calif. |
| pCR-Blunt II-TOPO | Kanr | Invitrogen, Cergy Pontoise, France |
| pBBR1MCS.3 | Low-copy-number broad-host-range cloning vector; Tetr | 5 |
| pDA-1 | pBK-CMV recombinant plasmid containing genomic HindIII fragment from P. aeruginosa 14, including blaVEB-1 | This work |
| pDA-2 | pBK-CMV recombinant plasmid containing genomic HindIII fragment from P. aeruginosa 1, including blaVEB-1 and IS1999 | This work |
| pDA-3 | pBK-CMV recombinant plasmid containing genomic HindIII fragment from P. aeruginosa JES, including blaVEB-1 and IS1999/IS2000 | This work |
| pInt-Veb | A 2.4-kb SpeI-XhoI fragment from plasmid pDA-1, containing | This work |
| blaVEB-1 inserted in the SpeI-XhoI site of pBBR1MCS.3 | ||
| pInt-1999-Veb | A 3.7-kb SpeI-XhoI fragment from pDA-2, containing blaVEB-1 and IS1999 inserted in the SpeI-XhoI site of pBBR1MCS.3 | This work |
| pInt-1999-2000-Veb | A 4.9-kb SpeI-XhoI fragment from pDA-3, containing blaVEB-1 and IS1999/IS2000 inserted in the SpeI-XhoI site of pBBR1MCS.3 | This work |
| pTOP1 | A 1.1-kb fragment resulting from PCR amplifications using VEBCAS-A/VEBCAS-B primers and genomic DNA of P. aeruginosa 1 as template; this fragment, including the veb-1 gene cassette, was inserted in the cloning site of pCR-BluntII-TOPO | This work |
| pTOP2 | A 2.5-kb fragment resulting from PCR amplifications using 5′-CS/VEBCAS-B primers and genomic DNA of P. aeruginosa 1 as the template; this fragment, including the veb-1 gene cassette and IS1999, was inserted in the cloning site of pCR-BluntII-TOPO | This work |
| pTOP3 | A 1.1-kb fragment resulting from PCR amplifications using VEBCAS-AStu/VEBCAS-B primers and genomic DNA of P. aeruginosa 1 as the template; this fragment, including a StuI restriction site in the veb-1 gene cassette upstream of blaVEB-1, was inserted in the cloning site of pCR-BluntII-TOPO | This work |
| pTOP4 | A 2.2-kb fragment resulting from PCR amplifications using MUTIS1999Stop/VEBCAS-B primers and genomic DNA of P. aeruginosa 1 as the template; this fragment includes a site-directed mutation generating after transcription a stopcodon within the IS1999 transposase sequence; this fragment, inserted in the cloning site of pCR-BluntII-TOPO, includes the two XmnI sites that are present in IS1999 and blaVEB-1 sequences and that are located on each side of the mutation | This work |
| pVeb | A 1.1-kb KpnI-ApaI fragment from pTOP1, containing the veb-1 gene cassette inserted in the KpnI-ApaI site of pBBR1MCS.3 | This work |
| p1999R-Veb | pInt-1999-Veb from which a 1.9-kb SpeI-StuI fragment has been deleted that includes intII and the left end of IS1999 | This work |
| p1999-Veb | A 2.6-kb KpnI-ApaI fragment from pTOP1, containing the veb-1 gene cassette and IS1999, inserted in the KpnI-ApaI site of pBBR1MCS.3 | This work |
| p1999L-Veb | A 2.1-kb KpnI-StuI fragment from pTOP3, containing the veb-1 gene cassette, inserted in the KpnI-StuI site of p1999-Veb, containing the left end of IS1999 | This work |
| pInt-1999*-Veb | A 1.5-kb XmnI fragment from pTOP4, containing parts of the mutated IS1999 and blaVEB-1 sequences inserted in the XmnI site of pInt-1999-Veb | This work |
| p1999R-2000R-Veb | pInt-1999-2000-Veb from which a 1.8 kb SpeI-PstI fragment has been deleted that includes intII and the right ends of IS1999/IS2000 | This work |
a
Superscript “r,” resistance. Abbreviations: Caz, ceftazidime; Kan, kanamycin; Neo, neomycin; Sm, streptomycin; Tet, tetracycline.