Profile of antibiotic resistant genes (ARGs)
P. aeruginosa clinical isolates displayed different combinations of drug resistance genes such as efflux pump and outer membrane (MexAB-OprM, MexEF-OprN, OprJ, OpmB, OpmH) genes (Fig. 2) leading to resistance to various antibiotics. All 72 P. aeruginosa isolates carried OXA-50, FosA (fosfomycin-resistant) and different variants of Pseudomonas Derived Cephalosporinase (PDC)-beta-lactamase class C genes, predominantly PDC-2 (18/72, 25%) and PDC-7 (18/72, 25%), followed by PDC-3 (17/72, 24%), PDC-5 (15/72, 21%), PDC-1 (2/72, 3%) and PDC-8 (2/72, 3%). Other genes encoding antibiotic resistance were found in this study including genes for colistin (ArnA, 69/72, 96%), CARB-3-beta-lactamase (CARB-3, 43/72, 60%), acquired fluoroquinolone resistance (QnrVC1, n/N, 51%) and Vietnamese extended-spectrum beta-lactamase (Veb-1, 3/72, 4%). Among 72 strains, 48 (67%) isolates carried one of the three variants of IMP gene, among which IMP-15 was the most prevalent (34/72, 47%), followed by IMP-51 (12/72, 17%) and IMP-26 (2/72, 3%) (Table 1).
Table 1
Distribution of antibiotic resistance genes of P. aeruginosa (n = 72)
Carbapenem genes
|
IMP variants
|
Some important AMR genes
|
DIM-1
|
OXA-50
|
PDC (PDC-beta-lactamase class C)
|
CARB-3
|
Veb1
|
QnrVC1
|
FosA
|
ArnA
|
IMP
(n = 48)
|
IMP-15 (n = 34)
|
2/34
|
34/34
|
PDC-2 (n = 4); PDC-3 (n = 10); PDC-5 (n = 11); PDC-7 (n = 9)
|
32/34
|
-
|
31/34
|
30/34
|
33/34
|
IMP-26 (n = 2)
|
-
|
2/2
|
PDC-2; PDC-3
|
-
|
-
|
1/2
|
2/2
|
2/2
|
IMP-51 (n = 12)
|
-
|
12/12
|
PDC-2 (n = 12)
|
-
|
-
|
-
|
12/12
|
12/12
|
KPC-1 (n = 7)
|
-
|
7/7
|
PDC-7 (n = 7)
|
7/7
|
-
|
5/7
|
7/7
|
7/7
|
IMP, KPC negative isolates (n = 17)
|
-
|
17/17
|
PDC-1 (n = 2); PDC-2 (n = 1); PDC-3 (n = 6); PDC-5 (n = 4); PDC-7 (n = 2); PDC-8 (n = 2)
|
4/17
|
3/17
|
-
|
17/17
|
15/17
|
|
2/72 (2.77%)
|
72/72 (100%)
|
72/72 (100%)
|
43/72 (59.72%)
|
3/72 (4.16%)
|
37/72 (51.38%)
|
72/72 (100%)
|
69/72 (95.83%)
|
Notably, we detected the presence of DIM-1 gene (Dutch imipenemase 1) encoding carbapenemase for the first time in Vietnam in two P. aeruginosa isolates. The first DIM-1 P. aeruginosa isolate was from urine of posterior urethral stenosis patient in Better private care department, hospital C in mid-July 2012. The second DIM-1 was isolated from a pneumonia patient in early the period of 2013 in ICU, hospital A. Noticeably, both isolates with DIM-1 genes also carried the IMP-15 gene and KPC. Patients with DIM-1 positive isolates were hospitalized in two different hospitals, and these isolates belonged to ST1420 and ST3440 (Fig. 2). The seven KPC-1 positive P. aeruginosa belong to ST3151 were in hospital A from 2011 to 2013 in Nurseries (n = 3); ICU (n = 3); and Ophthalmology (n = 1). The first KPC-1 was in Oct-2011 from bronchial fluid of a Ventilator-associated pneumonia patient. The second and third isolates were from blood of a septic patient and in Corneal sample of a conjunctivitis patient in November, 2011 and the fourth was in pnemonia patient in December-2011. Two positive KPC-1 were in May 2012 isolated from bronchial fluid of pneumonia patients and the last case in January 2013.
Concordance of minimum inhibition concentration (MIC) with CARG’s profile of P. aeruginosa
The results of the imipenem minimum inhibition concentration (MIC) test showed that the isolates without IMP-15, IMP26, IMP-51, DIM-1 or KPC-1 genes had the lowest MICs (8–16 mg/L). Isolates carrying only IMP-51 gene had an MIC of 16–32 mg/L, and isolates carrying only IMP-15 gene had MICs of 32–64 mg/L. Noticeably, isolates with both IMP-15 and DIM-1 genes were extremely resistant to imipenem, with MICs > 128 mg/L. Similarly, isolates carrying KPC-1 gene had MIC > 128 mg/L. We also observed that isolates carrying IMP-15 + DIM-1; or only one gene among IMP-26, IMP-51, and KPC-1 genes were resistant to other five antibiotics (ciprofloxacin (CIP), ceftazidime (CAZ), gentamicin (GEN), amikacin (AK), aztreonam (AZT) (Table 2). However, most of the isolates carrying IMP-15 gene (18/32) were still susceptible to AZT, and isolates carrying both IMP-26 + DIM-1 genes were amikacin susceptible. Among 17 isolates not harbouring acquired carbapenemase genes (IMP, DIM-1 and KPC-1), 16 isolates remained susceptible to ciprofloxacin (0.125-1 mg/L); amikacin (2–4 mg/L) and gentamicin (1–2 mg/L), 12 to aztreonam (8 mg/L) and 10 to ceftazidime (4 mg/L). Finally, we observed 8 of 72 isolates resistant to colistin with MICs ranging from 4 to 16 mg/L (Table 2).
Table 2
Antimicrobial susceptibility by minimum inhibitory concentration of P. aeruginosa (n = 72)
P. aeruginosa strains (n = 72)
|
MIC (mg/L)
|
IMP
|
CIP
|
CAZ
|
AK
|
GEN
|
ATZ
|
CS
|
IMP-15 (n = 32)
|
R (32–64)
|
R (4–16)
|
R (64–256)
|
S (8; n = 1); R (> 256; n = 31)
|
R (> 128)
|
S (2–8; n = 18); I (16; n = 13); R (32; n = 1)
|
S (0.25-2; n = 28)
R (4–16; n = 4)
|
IMP-15 + DIM-1 (n = 2)
|
R (> 128)
|
R (16)
|
R (> 256)
|
S (16)
|
R (> 128)
|
R (128)
|
S (0.25-2; n = 2)
|
IMP-26 (n = 2)
|
R (> 128)
|
R (32)
|
R (> 256)
|
R (> 256)
|
R (> 128)
|
R (32)
|
S (0.5; n = 1); R (4; n = 1)
|
IMP-51 (n = 12)
|
R (16–32)
|
R (8–16)
|
R (256)
|
S (8–16; n = 6); R (64)
|
R (32–64)
|
I (1; n = 2); R (32–128; n = 5)
|
S (0.25-2; n = 11); R (4, n = 1)
|
KPC-1 (n = 7)
|
R (> 128)
|
R (8)
|
R (32)
|
R (> 256)
|
R (> 128)
|
R (> 128)
|
S (0.125-2; n = 6); R (4, n = 1)
|
IMP, DIM KPC negative (n = 17)
|
R (8–16; n = 17)
|
S (0.125-1; n = 16); R (64; n = 1)
|
S (4; n = 10); R (32–128; n = 7);
|
S (2–4; n = 16); R (> 256; n = 1
|
S (1–2; n = 16); R (32; n = 1)
|
S (8; n = 12); I (16; n = 3); R (> 128; n = 2)
|
S (0.25-2; n = 15); R (4, n = 1)
|
Note: S: Sensitive; I: Intermidiate; R: Resistant |
Genotypic relationship of P. aeruginosa isolates.
The phylogenetic tree placed the 72 P. aeruginosa isolates in nine genotype groups (Fig. 3). Each group had different characteristics of antibiotic resistance genes and sequence type. Interestingly, isolates in group I were 10 isolates from hospital B from between 2012–2014 belonging to ST360 with 8/10 isolates harbouring IMP-15 and most of the isolates carrying many different resistance encoding genes: OXA-50, CARB-3, PDC (PDC-beta-lactamase class C) and gene encoding quinolone resistant (QnrVC1). Group V isolates belonged to ST3151 and harboured one or a combination of two carbapenemase genes including IMP-15, KPC-1, OXA-50, QnrVC1, CARB-3 and PCD genes. Group IX which contained only carbapenemase - ST235 isolates that originated from all three hospitals between 2012 and 2014 (Fig. 2).
Multi-locus sequence typing of P. aeruginosa isolates showed that 72 isolates were classified into eighteen sequence types, in which the predominant ST was ST3151 (n = 14) followed by ST235 (n = 13), ST360 (n = 10), ST310 (n = 7) and ST357 (n = 7). Remaining 13 singleton included ST179; ST244; ST245; ST277; ST 313; ST654; ST773; ST856; ST1308; ST1420, ST2166, ST3440 and ST3361. Interestingly, all ST360 (10/10) were detected from patients in hospital B. ST3151 and ST310 were found significantly more often in hospital A compared to hospital C (13/15 vs 2/15 and 5/7 vs 1/7, respectively) (Fig. 2, Fig. 3).