Background
Methods
Bacterial isolation and identification
Target | Primer sequence (5′ → 3′) | Amplicon size (bp) | Annealing tempreture | Ref. |
---|---|---|---|---|
ddl
faecalis
| ATCAAGTACAGTTAGTCTTTATTAG | 941 | 55 °C | [13] |
ACGATTCAAAGCTAACTGAATCAGT | ||||
ddl
faecium
| TTGAGGCAGACCAGATTGACG | 658 | 55 °C | [13] |
TATGACAGCGACTCCGATTCC | ||||
van A
| CATGAATAGAATAAAAGTTGCAATA | 1030 bp | 54 °C | [14] |
CCCCTTTAACGCTAATACGATCAA | ||||
van B
| GTGACAAACCGGAGGCGAGGA | 433 bp | 54 °C | [14] |
CCGCCATCCTCCTGCAAAAAA | ||||
van C
| GAAAGACAACAGGAAGACCGC | 796 bp | 54 °C | [15] |
ATCGCATCACAAGCACCAATC | ||||
aac(6′)-Ie-aph(2″)-Ia
| CAGGAATTTATCGAAAATGGTAGAAAAG | 369 bp | 55 °C | [16] |
CACAATCGACTAAAGAGTACCAATC | ||||
aph(3′)-IIIa
| GGCTAAAATGAGAATATCACCGG | 523 bp | 55 °C | [16] |
CTTTAAAAAATCATACAGCTCGCG | ||||
ant(4′)-Ia
| CAAACTGCTAAATCGGTAGAAGCC | 294 bp | 55 °C | [16] |
GGAAAGTTGACCAGACATTACGAACT | ||||
aph(2″)-Ic
| CCACAATGATAATGACTCAGTTCCC | 444 bp | 55 °C | [16] |
CCACAGCTTCCGATAGCAAGAG | ||||
aph(2″)-Ib
| CTTGGACGCTGAGATATATGAGCAC | 867 bp | 55 °C | [16] |
GTTTGTAGCAATTCAGAAACACCCTT | ||||
aph(2″)-Id
| GTGGTTTTTACAGGAATGCCATC | 641 bp | 55 °C | [16] |
CCCTCTTCATACCAATCCATATAACC | ||||
ant(3″)-III
| TGATTTGCTGGTTACGGTGAC | 284 bp | 55 °C | [17] |
CGCTATGTTCTCTTGCTTTTG | ||||
ant(6′)-Ia
| ACTGGCTTAATCAATTTGGG | 596 bp | 55 °C | [17] |
GCCTTTCCGCCACCTCACCG | ||||
PAI
| GACGCTCCCTTCTTTTGAC | 387 bp | 54 °C | [18] |
CCAGAGAAATTACTACCAT | ||||
sprE
| GGTAAACCAACCAAGTGAATC | 300 bp | 56 °C | [18] |
TTCTTCCGATTGACGCAAAA | ||||
ace
| CAGGCCAACATCAAGCAACA | 125 bp | 58 °C | [18] |
GCTTGCCTCGCCTTCTACAA | ||||
gelE
| CGAAGTTGGAAAAGGAGGC | 372 bp | 54 °C | [18] |
GGTGAAGAAGTTACTCTGA | ||||
hyl
| ACAGAAGAGCTGCAGGAAATG | 276 bp | 55 °C | [19] |
GACTGACGTCCAAGTTTCCAA | ||||
cylA
| ACTCGGGGATTGATAGGC | 688 bp | 56 °C | [19] |
GCTGCTAAAGCTGCGCTT | ||||
esp
| TTTGGGGCAACTGGAATAGT | 407 bp | 56 °C | [18] |
CCCAGCAAATAGTCCATCAT |
Antimicrobial susceptibility testing
DNA extraction
Detection of resistance and virulence genes
Phenotypic detection of virulence factors
Cytolysin activity
Gelatinase activity
Haemagglutination assay
Biofilm forming assay
Statistical analysis
Results
Patient demographics
Antimicrobial susceptibility
Antimicrobial agents | No. (%) ofresistantE. faecalis(n = 69) | No. (%) ofresistantE. faecium(n = 10) | No. (%) ofresistant otherEnterococcalspp. (n = 21) | No. (%) oftotalresistantisolates(n = 100) |
---|---|---|---|---|
Vancomycin | 0 (0) | 7 (70) | 14 (66.6) | 21 (21) |
Ampicillin | 0 (0) | 10 (100) | 12 (57.1) | 22 (22) |
Tetracycline | 62 (89.8) | 9 (90) | 15 (71.4) | 86 (86) |
Gentamicin | 31 (45) | 7 (70) | 12 (57.1) | 50 (50) |
Linezolid | 4 (5.8) | 0 | 1 (4.76) | 5 (5) |
Ciprofloxacin | 47 (68.1) | 9 (90) | 17 (80.9) | 73 (73) |
Chloramphenicol | 16 (23.2) | 2 (20) | 3 (14.3) | 21 (21) |
Fosfomycin | 1 (1.44) | 0 (0) | 0 (0) | 1 (1) |
Quinupristin-Dalfopristin | 34 (34.8) | 6 (60) | 13 (61.9) | 53 (53) |
Streptomycin | 19 (27.5) | 5 (50) | 10 (47.6) | 34 (34) |
Distribution of vancomycin and aminoglycoside resistance genes
Genes | No. (%) of E. faecalis (n = 69) | No. (%) of E. faecium (n = 10) | No. (%) of Other species (n = 21) | No. (%) of Total(n = 100) |
---|---|---|---|---|
aac(6′)-Ie-aph(2″)-Ia
| 10 (14.5) | 1 (10) | 4 (19) | 15 (15) |
aph(3′)-IIIa
| 46 (66.6) | 7 (70) | 14 (66.7) | 67 (67) |
ant(4′)-Ia
| 3 (4.3) | 0 (0) | 1 (4.7) | 4 (4) |
aph(2″)-Ic
| 6 (8.7) | 0 (0) | 2 (9.5) | 8 (8) |
aph(2″)-Ib
| 4 (5.8) | 2 (20) | 1 (4.7) | 7 (7) |
aph(2″)-Id
| 5 (7.2) | 2 (20) | 1 (4.7) | 8 (8) |
ant(3″)-III
| 50 (72.5) | 9 (90) | 19 (90.5) | 78 (78) |
ant(6′)-Ia
| 41 (59.4) | 6 (60) | 15 (71.4) | 62 (62) |
vanA
| 0 | 10 (100) | 0 | 10 (10) |
vanB
| 0 | 0 | 0 | 0 |
vanC
| 0 | 0 | 2 (9.5) | 2 (2) |
No. of ARG | ARG combinations | No. (%) of ARGs in E. faecalis (n = 69) | No. (%) of ARGs in E. faecium (n = 10) | Total No. (%) (n = 79) |
---|---|---|---|---|
Without ARG | – | 1 (1.4) | – | 1 (1.2) |
1 ARG |
ant(6′)Ia
| 5 (7.2) | – | 16 (20.2) |
ant(3′)-IIIa
| 7 (10.1) | 2 (20) | ||
aph(3′)-IIIa
| 2 (2.8) | – | ||
2 ARGs |
aph(3′)-IIIa + ant(3′)-III
| 8 (11.5) | 2 (20) | 26 (32.9) |
ant(6′)Ia + ant(3′)-IIIa
| 4 (5.7) | – | ||
ant(6′)Ia + aph(3′)-IIIa
| 8 (11.5) | – | ||
ant(3′)-IIIa + aac (6′)-Ie
| 1 (1.4) | – | ||
ant(3′)-IIIa + aph(3′)-IIIa
| 2 (2.8) | – | ||
ant(6′)Ia + aac (6′)-Ie
| 1 (1.4) | – | ||
3 ARGs |
ant(3′)-IIIa + aph(2″)-Ic + ant(4′)Ia
| 1 (1.4) | – | 22 (27.8) |
ant(6′)Ia + ant(3′)-IIIa + aph(3′)-IIIa
| 10 (14.4) | 2 (20) | ||
ant(6′)Ia + aph(3′)-IIIa + aph(2″)-Ic
| 1 (1.4) | – | ||
ant(3′)-IIIa + aph(3′)-IIIa + ant(4′)Ia
| 1 (1.4) | – | ||
ant(3′)-IIIa + aph(3′)-IIIa + aph(2″)-Ic
| 1 (1.4) | – | ||
ant(3′)-IIIa + aph(3′)-IIIa + aac (6′)-Ie
| 1 (1.4) | – | ||
ant(6′)Ia + aac (6′)-Ie + aph(2″)-Ic
| 1 (1.4) | – | ||
ant(6′)Ia + ant(3′)-IIIa + aph(2″)-lb
| 1 (1.4) | 1 (10) | ||
ant(6′)Ia + aph(3′)-IIIa + aac (6′)-Ie
| – | 1 (10) | ||
ant(3′)-IIIa + aph(2″)-ld + aph(3′)-IIIa
| 1 (1.4) | – | ||
4 ARGs |
ant(6′)Ia + ant(3′)-IIIa + aph(2″)-ld + aph(3′)-IIIa
| 2 (2.8) | 1 (10) | 12 (15.1) |
ant(6′)Ia + ant(3′)-IIIa + aph(3′)-IIIa + aac (6′)-Ie
| 4 (5.7) | – | ||
ant(6′)Ia + ant(3′)-IIIa + aph(2″)-lb + aph(3′)-IIIa
| 1 (1.4) | – | ||
ant(3′)-IIIa + aph(2″)-ld + aph(3′)-IIIa + aph(2″)-Ic
| 1 (1.4) | – | ||
ant(6′)Ia + ant(3′)-IIIa + aph(2″)-ld + aac (6′)-Ie
| 1 (1.4) | – | ||
ant(6′)Ia + ant(3′)-IIIa + aph(3′)-IIIa + aph(2″)-Ic
| 1 (1.4) | – | ||
ant(3′)-IIIa + aph(2″)-lb + aph(3′)-IIIa + ant(4′)Ia
| 1 (1.4) | – | ||
5 ARGs |
ant(6′)Ia + ant(3′)-IIIa + aph(2″)-lb + aph(3′)-IIIa + aac (6′)-Ie
| 1 (1.4) | – | 2 (2.5) |
ant(6′)Ia + ant(3′)-IIIa + aph(2″)-lb + aph(2″)-ld + aph(3′)-IIIa
|
–
| 1 (10) |
Distribution of enterococcal virulence related genes
virulencegenes | E. faecalis(n = 69) | E. faecium(n = 10) | P value | Total(n = 79) |
---|---|---|---|---|
PAI
| 31 (44.9) | 5 (50) | 0.488 | 36 (45.5) |
sprE
| 30 (43.4) | 3 (30) | 0.142 | 33 (41.7) |
ace
| 62 (89.8) | 8 (80) | 0.001* | 70 (88.6) |
gelE
| 17 (24.6) | 0 | 0.063 | 17 (21.5) |
hyl
| 2 (2.8) | 1 (10) | 0.337 | 3 (3.8) |
cylA
| 25 (36.2) | 0 | 0.017* | 25 (31.6) |
esp
| 50 (72.4) | 3 (30) | 0.012* | 53 (67.1) |
Genetic profile | No. (%) of E. faecalis(n = 69) | No. (%) of E. faecium(n = 10) |
---|---|---|
No virulence factor | – | – |
PAI
| – | 1 (10) |
Ace
| 2 (2.8) | 4 (40) |
PAI-ace
| 1 (1.4) | – |
sprE-ace
| 2 (2.8) | – |
ace-gelE
| 1 (1.4) | – |
ace-hyl
| 1 (1.4) | 1 (10) |
PAI-esp
| 1 (1.4) | 1 (10) |
ace-esp
| 4 (5.7) | – |
gelE-esp
| 2 (2.8) | – |
cylA-esp
| 1 (1.4) | – |
PAI-sprE-ace
| 4 (5.7) | 1 (10) |
sprE-ace-gelE
| 3 (4.3) | – |
PAI-ace-cylA
| 1 (1.4) | – |
ace-gelE-cylA
| 1 (1.4) | – |
ace-hyl-cylA
| 1 (1.4) | – |
PAI-sprE-esp
| 2 (2.8) | – |
PAI-ace-esp
| 5 (7.2) | – |
sprE-ace-esp
| 4 (5.7) | – |
ace-gelE-esp
| 2 (2.8) | – |
ace-cylA-esp
| 6 (8.6) | – |
sprE-ace-gelE-cylA
| 2 (2.8) | – |
PAI-sprE-ace-esp
| 9 (13) | 2 (20) |
sprE-ace-gelE-esp
| 1 (1.4) | – |
PAI-ace-cylA-esp
| 6 (8.6) | – |
sprE-gelE-cylA-esp
| 1 (1.4) | – |
ace-gelE-cylA-esp
| 4 (5.7) | – |
PAI-sprE-ace-cylA-esp
| 2 (2.8) | – |
Phenotypic detection of virulence factors
Virulencefactors | E. faecalis (n = 69) No. (%) | E. faecium (n = 10) No. (%) | Other spp.(n = 21)No. (%) | P value | Total(n = 100)No. (%) |
---|---|---|---|---|---|
Cytolytic activity | 32 (46.4) | 0 | 3 (14.3) | 0.001* | 35 (35) |
Gelatinase activity | 18 (26.1) | 0 | 7 (33.3) | 0.107 | 25 (25) |
Hemagglutination | 55 (79.7) | 7 (70) | 13 (61.9) | 0.245 | 75 (75) |
Biofilm formation | 62 (89.8) | 2 (20) | 10 (47.6) | 0.000* | 74 (74) |
Resistance genes | Van A(n = 10) | Van C (n = 2) |
aac(6′)-Ie-aph(2″)-Ia (n = 20)
|
aph(3′)-IIIa (n = 71)
|
ant(4′)-Ia (n = 6)
|
aph(2″)-Ic (n = 9)
|
aph(2″)-Ib (n = 5)
|
aph(2″)-Id (n = 7)
|
ant(3″)-III (n = 84)
|
ant(6′)-Ia (n = 57)
|
---|---|---|---|---|---|---|---|---|---|---|
Virulence factors | ||||||||||
Cytolytic activity (n = 35) | 8* | 1 | 14* | 20* | 4 | 8* | 4* | 6* | 34* | 32* |
Gelatinase activity (n = 25) | 8* | 2 | 18* | 16 | 6* | 9* | 4* | 7* | 25* | 25* |
Hemagglutination (n = 75) | 7 | 1 | 13 | 60* | 5 | 9 | 5 | 7 | 74* | 53* |
Biofilm formation (n = 74) | 10 | 1 | 17 | 66* | 6 | 8 | 5 | 7 | 74* | 56* |