Article Figures & Data
Figures
- Fig 1
Distribution of STs among the XDR, MDR, modR, and multiS isolates studied. The number of isolates of each ST is shown in parentheses.
- Fig 2
Results for double-disk (imipenem-ceftazidime) AmpC induction test in wild-type PAO1, the ampR knockout mutant PAΔR, and PAΔR complemented with wild-type ampR (pUCPARWT) or with the G154R mutant (pUCPARG154R).
Tables
- Table 1
Primers used in this work for amplification or cloning of resistance determinants
Primer Sequence (5′→3′)a PCR product size (bp) Target Reference or source ampCF GCGCGCAGGGCGTTCAG 1,467 ampC 37 ampCR2 CGGAGGGGCGGGGAAGC This work ampDF GTACGCCTGCTGGACGATG 910 ampD 24 ampDR GAGGGCAGATCCTCGACCAG ampRF GTCGACCCAGTGCCTTCAGG 1,391 ampR 24 ampRR CTCGAGAGCGAGATCGTTGC AmpRCLF-ERI TCGAATTCGTCGACCAGTGCCTTCAGG 1,391 ampR This work AmpRCLR-BHI TCGGATCCCTCGAGAGCGAGATCGTTGC dacBF CGACCATTCGGCGATATGAC 1,721 dacB 38 dacBR CGCGTAATCCGAAGATCCATC oprDF CGCCGACAAGAAGAACTAGC 1,412 oprD 17 oprDR GTCGATTACAGGATCGACAG gyrA1 TTATGCCATGAGCGAGCTGGGCAACGACT 364 gyrA 41 gyrA2 AACCGTTGACCAGCAGGTTGGGAATCTT gyrB3 AGCTCGCAGACCAAGGACAAG 600 gyrB 41 gyrB4 GGGCTGGGCGATGTAGATGTA parC1 ATGAGCGAACTGGGGCTGGAT 208 parC 41 parC2 ATGGCGGCGAAGGACTTGGGA parE1 CGGCGTTCGTCTCGGGCGTGGTGAAGGA 592 parE 41 parE2 TCGAGGGCGTAGTAGATGTCCTTGCCGA INT-Ri CGCAGTGGCGGTTTTCAT Variable Class 1 Integrons 17 qacE-R CAAGAAAAAGCCAGCCTTTC mexRINT GGATGATGCCGTTCACCTG 1,016 nalB 18 mexR20 CCAGTAAGCGGATAC nalC1 TCAACCCTAACGAGAAACGCT 814 nalC 33 nalC2 TCCACCTCACCGAACTGC nalD1 GCGGCTAAAATCGGTACACT 789 nalD 53 nalD2 ACGTCCAGGTGGATCTTGG mexZF ATTGGATGTGCATGGGTG 1,000 mexZ 52 mexZR TGGAGATCGAAGGCAGC PA5471F GATCTACCGTTTCAATCACATGGAT 1,299 PA5471 This work PA5471R GGCCACCTCCTCGATTACCT ↵a Sites for restriction endonucleases are underlined.
- Table 2
Susceptibility profiles and resistance mechanisms of the studied isolates
Profile PFGE ST IDa MIC (μg/ml) and clinical category (CLSI breakpoint)b Resistance mechanism(s)c ATM CAZ FEP PTZ IMP MER GEN TOB AMK CIP COL ampCd oprD gyrA parC Integrons XDR 3 111 238 64 R 128 R 128 R 32 S 64 R 64 R 64 R 64 R 128 R 32 R 2 S N W339Xe T83I S87L (1) aacA7-blaVIM-2-aac(6′)-II (2) aac(6′)-Ib-blaOXA46 1 175 12 16 I 16 I 16 I 32 S 16 R 16 R 64 R 16 R 4 S 32 R 0.5 S P Q142Xf T83I, D87N S87W 1 175 27 32 R 64 R 128 R 64 S 8 I 64 R 64 R 64 R 2 S 32 R 2 S P Q142X T83I, D87N S87W 2 175 43 16 I 64 R 32 R 128 R 32 R 16 R 64 R 64 R 8 S 32 R 1 S P Q142X T83I, D87N S87W 1 175 93 32 R 64 R 32 R 128 R 16 R 16 R 64 R 64 R 4 S 32 R 0.25 S P Q142X T83I, D87N S87W 1 175 123 32 R 32 R 32 R 128 R 16 R 32 R 64 R 32 R 4 S 32 R 0.12 S P Q142X T83I, D87N S87W 1 175 147 16 I 32 R 32 R 64 S 32 R 16 R 64 R 64 R 4 S 32 R 4 I P Q142X T83I, D87N S87W 1 175 179 16 I 32 R 32 R 128 R 32 R 16 R 64 R 32 R 4 S 32 R 1 S P Q142X T83I, D87N S87W 1 175 207 16 I 32 R 16 I 64 S 16 R 16 R 64 R 8 I 0.5 S 32 R 0.12 S P Q142X T83I, D87N S87W MDR 3 111 19 4 S 64 R 16 I 32 S 64 R 64 R 64 R 16 R 8 S 16 R 0.5 S N W339X T83I S87L aacA7-blaVIM-2-aac(6′)-II 3 111 81 8 S 4 S 8 S 8 S 64 R 16 R 64 R 64 R 16 S 32 R 1 S N W339X T83I S87L aac(6′)-Ib-blaOXA46 1 175 67 8 S 8 S 8 S 16 S 8 I 4 S 64 R 16 R 8 S 32 R 0.25 S P Q142X T83I, D87N S87W 2 175 75 16 I 8 S 16 I 16 S 2 S 1 S 64 R 32 R 4 S 32 R 0.06 S N WT T83I, D87N S87W 1 175 245 8 S 8 S 4 S 64 S 32 R 8 I 32 R 2 S 0.25 S 32 R 0.125 S P Q142X T83I, D87N S87W 1 175 258 2 S 8 S 16 I 8 S 32 R 2 S 64 R 64 R 8 S 32 R 0.5 S P Q142X T83I, D87N S87W 13 244 205 16 I 8 S 16 I 16 S 2 S 8 I 4 S 1 S 4 S 32 R 0.5 S N T83I S87L 14 262 60 32 R 8 S 32 R 32 S 64 R 64 R 4 S 1 S 16 S 1 S 1 S N nt673Ins(T) 15 274 148 64 R 128 R 128 R 256 R 2 S 1 S 16 R 4 S 16 S 0.25 S 1 S P 20 381 187 16 I 128 R 64 R 32 S 1 S 0.25 S 2 S 0.5 S 2 S 32 R 0.03 S P T83I, D87N S87L 25 560 89 16 I 4 S 8 S 16 S 8 I 0.25 S 2 S 1 S 16 S 0.5 S 1 S N nt1113Ins(AT) 26 699 37 4 S 2 S 32 R 4 S 8 I 0.5 S 4 S 1 S 8 S 4 R 2 S N WT T83I WT 29 1111 59 16 I 64 R 32 R 128 R 32 R 8 I 4 S 0.5 S 4 S 0.12 S 0.5 S P MPg 29 1111 62 16 I 16 I 16 I 64 S 16 R 32 R 2 S 0.25 S 2 S 8 R 1 S N nt457Ins(A) T83I WT 39 1193 20 128 R 128 R 64 R 256 R 2 S 2 S 1 S 1 S 8 S 0.06 S 0.5 S P ModR 4 17 191 4 S 2 S 16 I 8 S 0.5 S 0.5 S 4 S 1 S 8 S 32 R 0.25 S N T83I S87L 5 111 131 0,25 S 1 S 4 S 1 S 0.12 S 0.06 S 8 I 16 R 32 I 8 R 0.12 S N T83I S87W aacA4-blaPSE-1-aadA2 6 162 17 8 S 4 S 4 S 8 S 32 R 8 I 1 S 0.25 S 2 S 0.12 S 0.25 S N nt482Δ17 7 179 54 4 S 2 S 16 I 8 S 0.5 S 0.25 S 1 S 1 S 4 S 0.12 S 2 S N 8 233 97 16 I 2 S 4 S 16 S 16 R 8 I 2 S 0.5 S 4 S 0.06 S 0.25 S N nt1163Δ2 10 244 16 2 S 2 S 4 S 8 S 2 S 4 S 0.25 S 0.12 S 2 S 32 R 0.25 S N T83I S87L 12 244 125 16 I 4 S 8 S 16 S 1 S 2 S 1 S 0.5 S 2 S 0.5 S 1 S N 17 319 48 4 S 4 S 2 S 4 S 4 S 1 S 0.5 S 0.25 S 4 S 0.25 S 8 I N 18 377 25 8 S 4 S 4 S 16 S 32 R 8 I 1 S 1 S 2 S 0.5 S 4 I N nt433Δ14 N/D 385 181 4 S 4 S 16 I 4 S 2 S 0.5 S 2 S 0.5 S 4 S 0.12 S 0.25 S N 22 446 63 8 S 16 I 8 S 64 S 16 R 4 S 2 S 0.5 S 4 S 0.06 S 1 S P MP 23 446 199 16 I 32 R 16 I 64 S 4 S 0.5 S 2 S 0.5 S 2 S 0.06 S 0.5 S P 27 1056 186 8 S 2 S 16 I 4 S 4 S 0.12 S 4 S 1 S 8 S 2 I 0,5 S N T83I WT 31 1114 120 4 S 4 S 2 S 4 S 64 R 2 S 1 S 0.5 S 2 S 0.12 S 0.125 S P MP 34 1177 132 32 R 32 R 16 I 32 S 0,5 S 0.5 S 2 S 0.5 S 4 S 0.25 S 0.125 S P ↵a ID, strain identification number.
↵b ATM, aztreonam; CAZ, ceftazidime; FEP, cefepime; PTZ, piperacillin-tazobactam; IMP, imipenem; MER, meropenem; GEN, gentamicin; TOB, tobramycin; AMK, amikacin; CIP, ciprofloxacin; COL, colistin.
↵c Sequencing results are in comparison to those of reference wild-type strain PAO1 (www.pseudomonas.com). All isolates showed wild-type gyrB sequence, and a single isolate (ST381) presented a mutation in parE (E459K). WT, wild type; nt, nucleotide.
↵d Strains were considered positive for ampC overexpression when the corresponding mRNA level was at least 10-fold higher than that of PAO1, negative if lower than 5-fold, and borderline if between 5- and 10-fold (7). P, positive; N, negative.
↵e TGG→TAG (premature stop codon) (boldface indicates the mutated nucleotide).
↵f CAA→TAA (premature stop codon) (boldface indicates the mutated nucleotide).
↵g MP, multiple polymorphisms of unknown effect compared to PAO1 sequence.
- Table 3
Sequence analysis of genes involved in MexAB-OprM and MexXY-OprM regulation
ST (resistance profile) IDa Expression levelb Presence of polymorphism(s) in sequencec MexAB-OprM regulator MexXY-OprM regulator mexB mexY nalB nalC nalD mexZ PA5471 175 (XDR/MDR) Severald V V WT (G71E), A186T WT G195E (L88P), D161G, H182Q, (V243A), V266 M 244 (MDR) 205 P P WT WT T11N WT (L88P), G157D, D161G, H182Q, (V243A) 262 (MDR) 60 P P T69P (G71E), (S209R) WT WT (L88P), D161G, H182Q, (V243A) 560 (MDR) 89 N P nt283Δ11 (C40R), (L88P), (S112N), (D119E), (I237V), (V243A), P244L 699 (MDR) 37 N P nt207Δ10 WT 1111 (MDR) 62 P N I68T, (V126E), V132A (G71E), (S209R) WT 179 (modR) 54 N P nt630Δ21 (L88P), D161G, H182Q, (V243A) 244 (modR) 125 P P WT WT WT nt292Δ10 (L88P), G157D, D161G, H182Q, (V243A) 377 (modR) 25 P N (V126E) nt239Δ10 WT 1056 (modR) 186 P P WT (G71E), (S209R) WT nt278Δ2 (C40R), (L88P), (S112N), (D119E), (I237V), (V243A) ↵a ID, strain identification number.
↵b Previously defined breakpoints were used (7). Strains were considered positive for mexY overexpression when the corresponding mRNA level was at least 10-fold higher than that of PAO1, negative if lower than 5-fold, and borderline if between 5- and 10-fold. Strains were considered positive for mexB overexpression when the corresponding mRNA level was at least 3-fold higher than that of PAO1, negative if lower than 2-fold, and borderline if between 2- and 3-fold. V, variable; P, positive; N, negative.
↵c Sequencing results are in comparison (www.pseudomonas.com) to those of reference wild-type strain PAO1; polymorphisms present in reference wild-type strain PA14 are shown in parentheses. WT, wild type; nt, nucleotide.
↵d Regulatory genes were sequenced in all ST175 isolates overexpressing mexB (n = 7) or mexY (n = 11), as well as 2 additional isolates not showing overexpression of either of the efflux pumps. The same sequences were documented in all cases, and therefore, they are shown only once.
- Table 4
Sequencing of genes involved in AmpC hyperproduction
Profile ST IDa ampCb Presence of polymorphism(s) in sequencec ampC dacB ampD ampR XDR 175 12 P WT WT G148A, D183Y G154R 175 27 P WT WT G148A, D183Y G154R 175 43 P WT WT T139 M, G148A, D183Y WT 175 93 P WT WT G148A, D183Y G154R 175 123 P WT WT G148A, D183Y G154R 175 147 P WT P59S G148A, D183Y G154R 175 179 P WT WT G148A, D183Y G154R 175 207 P WT WT G148A, D183Y G154R MDR 175 67 P WT WT G148A, D183Y G154R 175 75 N WT WT G148A, D183Y WT 175 245 P WT WT G148A, D183Y G154R 175 258 P WT WT G148A, D183Y G154R 274 148 P (T105A), (G391A) A394P R11L, P41L, G148A WT 381 187 P (T105A), G229S, G248S WT G148A, D183Y (G283E), (M288R) 1111 59 P (T105A) T239S G156S WT 1193 20 P (T105A), L176R, D233E Deletion from nt 853 A134V WT ModR 446 63 P (T105A), (V205L),(G391A) WT WT (E114A), (G283E), (M288R) 446 199 P (T105A), (V205L), (G391A) WT WT (E114A), (G283E), (M288R) 1114 120 P (T105A), (V205L),V356I, (G391A) Q33H, Q37H, L469 M WT A51T, (E114A) 1177 132 P (T105A), L176R Q212Xd G148, D183Y WT ↵a ID, strain identification number.
↵b Strains were considered positive for ampC overexpression when the corresponding mRNA level was at least 10-fold higher than that of PAO1, negative if lower than 5-fold, and borderline if between 5 and 10-fold (7). P, positive; N, negative.
↵c Sequencing results are in comparison (www.pseudomonas.com) to those of reference wild-type strain PAO1; polymorphisms present in reference wild-type strain PA14 are shown in parentheses. WT, wild type; nt, nucleotide.
↵d CAG→TAG (premature stop codon) (boldface indicates the mutated nucleotide).
