New Sequence Type ST3449 in Multidrug-Resistant Pseudomonas aeruginosa Isolates from a Cystic Fibrosis Patient
Abstract
:1. Introduction
2. Results and Discussion
2.1. Isolation, Phenotypic Features and Antimicrobial Susceptibility of the CF P. aeruginosa Strains
2.2. Genome Analysis of the CF P. aeruginosa Isolates
2.2.1. Resistome Analysis
2.2.2. Detection of Virulence Related Genes in the CF P. aeruginosa Isolates
2.2.3. Analysis of Genes Related with P. aeruginosa Hypermutability
2.3. Biofilm Formation Capacities of CF P. aeruginosa Isolates
2.4. Virulence in Galleria mellonella
3. Materials and Methods
3.1. Bacterial Isolates and Antimicrobial Susceptibility Testing
3.2. DNA Extraction and Sequencing
3.3. Bioinformatics Analysis
3.4. Biofilm Formation Assay
3.5. Confocal Laser Scanning Microscopy (CLSM)
3.6. Galleria mellonella Killing Assays
3.7. Statistical Analyses
3.8. Nucleotide Accession Numbers
4. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Isolate * | Source | Colony Morphology | Pigmentation | Hemolysis |
---|---|---|---|---|
MS6000 | Sputum | Small, mucoid | Brown | + |
MS6002 | Sputum | Large, irregular | Blue-green opaque | + |
MS6003 | Sputum | Small, circular | Green brilliant | + |
MS6004 | Tracheal aspirate | Small, wrinkled | Green opaque | + |
MS6005 | Tracheal aspirate | Large, irregular | Brown | + |
Isolate | CMI (µg/mL) | Resistance Phenotype | ||||||||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
AZT | FEP | CAZ | C/A * | C/T * | PIP | TZP | IMP | MER | DOR | GEN ** | TOB | AK | CIP | LEV | COL | FOS *** | ||
MS6000 | 64 (R) | 16 (R) | 64 (R) | 4 (S) | 0.5 (S) | 256 (R) | 256 (R) | 1 (I) | 0.25 (S) | 0.25 (S) | 8 (I) | 4 (R) | 16 (S) | 0.5 (I) | 2 (R) | 2 (S) | 256 (R) | XDR |
MS6002 | 64 (R) | 8 (I) | 16 (R) | 2 (S) | 0.38 (S) | 64 (R) | 128 (R) | 32 (R) | 16 (R) | 8 (I) | 8 (I) | 0.5 (S) | 8 (S) | 1 (R) | 4 (R) | 8 (R) | 512 (R) | XDR |
MS6003 | 8 (I) | 16 (R) | 16 (R) | 1 (S) | 0.75 (S) | 128 (R) | 4 (I) | 8 (R) | 1 (S) | 2 (S) | 16 (R) | 1 (S) | 32 (R) | >32 (R) | >32 (R) | 4 (R) | 128 (S) | XDR |
MS6004 | 0.25 (I) | 0.5 (I) | 1 (I) | 0.125 (S) | 0.125 (S) | 0.5 (I) | 1 (I) | 1 (I) | 0.06 (S) | 0.125 (S) | 4 (S) | 1 (S) | 8 (S) | 2 (R) | 4 (R) | 2 (S) | 64 (S) | MDR |
MS6005 | >128 (R) | 128 (R) | 128 (R) | 3 (S) | 0.25 (S) | >256 (R) | 256 (R) | 4 (I) | 2 (S) | 2 (S) | 8 (I) | 4 (R) | 16 (S) | 4 (R) | 16 (R) | 2 (S) | 512 (R) | XDR |
Sample | Reads | Average Read Length (bp) | Contigs | Genome Length (bp) | Average Contig Length (bp) | N50 (bp) | ORF | GC Content | Depth | Species | Percent ID |
---|---|---|---|---|---|---|---|---|---|---|---|
MS6000 | 629064 | 278 | 119 | 6,326,892 | 576,053 | 262,138 | 5815 | 66.37 | 27X | P. aeruginosa | 99.29 |
MS6002 | 876966 | 279 | 109 | 6,324,360 | 926,907 | 375,329 | 5830 | 66.38 | 38X | P. aeruginosa | 99.35 |
MS6003 | 877052 | 277 | 123 | 6,339,440 | 920,338 | 351,508 | 5836 | 66.35 | 38X | P. aeruginosa | 99.28 |
MS6004 | 915080 | 278 | 105 | 6,451,152 | 777,263 | 281,904 | 5953 | 66.37 | 39X | P. aeruginosa | 98.61 |
MS6005 | 773800 | 278 | 122 | 6,334,587 | 777,177 | 351,513 | 5826 | 66.35 | 33X | P. aeruginosa | 99.30 |
Average | 814392 | 278 | 116 | 6,355,286 | 795,548 | 324,478 | 5852 | 66.36 | 35X | - | 99.37 |
Protein/Gene | MS6000 | MS6002 | MS6003 | MS6004 | MS6005 |
---|---|---|---|---|---|
AmpC | T105A | T105A | T105A | T105A | T105A |
AmpR | +T at nt 860, stop TGA at 874 | +T at nt 860, stop TGA at 874 | +T at nt 860, stop TGA at 874 | +T at nt 860, stop TGA at 874 | +T at nt 860 stop TGA at 874 |
AmpD | G148A, D183Y | G148A, D183Y | D183Y, +G at 443 | G148A, D183Y | G148A, D183Y |
AmpDh2 | wt | wt | wt | wt | wt |
AmpDh3 | A219T | A219T | A219T | A219T | A219T |
mpl | Del. 14 bp (1120–1133) | Del. 14 bp (1120–1133) | wt | G133D | Del. 14 bp (1120–1133) |
PBP1a (ponA) | D329G, +CCG at nt 1845 | D329G, +CCG at nt 1845 | D329G, +CCG at nt 1845 | D329G, +CCG at nt 1845 | D329G, +CCG at nt 1845 |
PBP1b (mrcB) | L353Q | L353Q | L353Q | L353Q | L353Q |
PBP3a (pbpC) | Not present | Not present | Not present | A104P | Not present |
PBP7 (pbpG) | S250N | S250N | S250N | S250N | S250N |
MexA | wt | wt | wt | wt | |
MexB | L672R | L672R | L684P | L684P | L672R |
OprM | wt | wt | wt | wt | wt |
MexR | wt | wt | wt | wt | wt |
NalC | G71E, S209R | G71E, S209R | G71E, S209R | G71E, S209R | G71E, S209R |
NalD | wt | wt | wt | wt | wt |
OprD | D43N, S57E, S59R, E202Q, I210A, E230K, S240T, N262T, A267S, A281G, K296Q, Q301E, R310G, V359L, M372V, S373D, D374S, N375S, N376S, V377S, Del.G378, Del.Y379, K380Y, N381A, Y382G, G383L | Stop at nt 18 (TGG→TGA) W6 * | D43N, S57E, S59R, E202Q, I210A, E230K, S240T, N262T, A267S, A281G, K296Q, Q301E, R310G, V359L, M372V, S373D, D374S, N375S, N376S, V377S, Del.G378, Del.Y379, K380Y, N381A, Y382G, G383L | D43N, S57E, S59R, E202Q, I210A, E230K, S240T, N262T, A267S, A281G, K296Q, Q301E, R310G, V359L, M372V, S373D, D374S, N375S, N376S, V377S, Del.G378, Del.Y379, K380Y, N381A, Y382G, G383L | D43N, S57E, S59R, E202Q, I210A, E230K, S240T, N262T, A267S, A281G, K296Q, Q301E, R310G, V359L, M372V, S373D, D374S, N375S, N376S, V377S, Del.G378, Del.Y379, K380Y, N381A, Y382G, G383L |
MexX | K329Q, L331V, W358R | K329Q, L331V, W358R | K329Q, L331V, W358R | K329Q, L331V, W358R | K329Q, L331V, W358R |
MexY | T543A, A992T | T543A, A992T | T543A | T543A | T543A, A992T |
MexZ | wt | wt | wt | wt | wt |
AmgS | wt | wt | wt | wt | wt |
Fmt | I181V, E277K | I181V, E277K | I181V, E277K | I181V, E277K | I181V, E277K |
FolD | T189A | T189A | T189A | T189A | T189A |
FusA2 | S176A, G695A | S176A, G695A | S176A, G695A | S176A, G695A | S176A, G695A |
NuoG | T484A | T484A | T484A | T484A | T484A |
MexC | S330A | S330A | S330A | S330A | S330A |
MexD | E257Q, S845A | E257Q, S845A | E257Q, S845A | E257Q, S845A | E257Q, S845A |
OprJ | M69V | M69V | M69V | M69V | M69V |
NfxB | wt | wt | wt | wt | wt |
MexT | Del. GCCGGCCA (240–247), F172I | Del. GCCGGCCA (240–247), F172I | Del. GCCGGCCA (240–247), F172I | Del. GCCGGCCA (240–247), F172I | Del. GCCGGCCA (240–247), F172I |
MexE | wt | wt | wt | wt | wt |
MexF | wt | wt | wt | wt | wt |
OprN | wt | wt | wt | wt | wt |
MexS | D249N | D249N | D249N | D249N | D249N |
GyrA | D87N | D87N | T83I | T83I | D87N |
GyrB | S618L | S618L | wt | wt | S618L |
ParC | V646L | V646L | E91K, V646L | E91K, V646L | E91K, V646L |
ParE | G285S | G285S | G285S | G285S | G285S |
CrpP | K4R, G7D | K4R, G7D | K4R, G7D | K4R, G7D | K4R, G7D |
PmrA | L71R | L71R | L71R | L71R | L71R |
PmrB | Y345H, V386M | Y345H, V386M | Y345H, V386M | Y345H, V386M | Y345H, V386M |
ParR | L153R, S170N | L153R, S170N | L153R, S170N | L153R, S170N | L153R, S170N |
ParS | H398R | H398R | Stop at nt 459 (TGG→TGA) W153 * | H398R | H398R |
ArnB | V302A, E376D | V302A, E376D | V302A, E376D | V302A, E376D | V302A, E376D |
ArnT | I509V | I509V | I509V | I509V | I509V |
ArnF | T106I | T106I | T106I | T106I | T106I |
PA3559 (ugd) | V101A | V101A | V101A | V101A | V101A |
OprP | A98V | A98V | A98V | A98V | A98V |
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Díaz-Ríos, C.; Hernández, M.; Abad, D.; Álvarez-Montes, L.; Varsaki, A.; Iturbe, D.; Calvo, J.; Ocampo-Sosa, A.A. New Sequence Type ST3449 in Multidrug-Resistant Pseudomonas aeruginosa Isolates from a Cystic Fibrosis Patient. Antibiotics 2021, 10, 491. https://doi.org/10.3390/antibiotics10050491
Díaz-Ríos C, Hernández M, Abad D, Álvarez-Montes L, Varsaki A, Iturbe D, Calvo J, Ocampo-Sosa AA. New Sequence Type ST3449 in Multidrug-Resistant Pseudomonas aeruginosa Isolates from a Cystic Fibrosis Patient. Antibiotics. 2021; 10(5):491. https://doi.org/10.3390/antibiotics10050491
Chicago/Turabian StyleDíaz-Ríos, Catalina, Marta Hernández, David Abad, Laura Álvarez-Montes, Athanasia Varsaki, David Iturbe, Jorge Calvo, and Alain A. Ocampo-Sosa. 2021. "New Sequence Type ST3449 in Multidrug-Resistant Pseudomonas aeruginosa Isolates from a Cystic Fibrosis Patient" Antibiotics 10, no. 5: 491. https://doi.org/10.3390/antibiotics10050491
APA StyleDíaz-Ríos, C., Hernández, M., Abad, D., Álvarez-Montes, L., Varsaki, A., Iturbe, D., Calvo, J., & Ocampo-Sosa, A. A. (2021). New Sequence Type ST3449 in Multidrug-Resistant Pseudomonas aeruginosa Isolates from a Cystic Fibrosis Patient. Antibiotics, 10(5), 491. https://doi.org/10.3390/antibiotics10050491