Phenotypic colistin resistance in the strain collection.
Of the isolates tested (n=97), 54 (56%) were resistant to colistin by the standard broth microdilution testing and 43 (44%) were susceptible. Among the resistant isolates, 23 (43%) belonged to the bacterial species P. vulgaris, P. mirabilis, S. marcescens and H. alvei and thus possess intrinsic resistance. Whereas 31 (57%) belonged to Acinetobacter spp., E. cloacae, E. aerogenes, E. coli, K. pneumoniae, K. oxytoca and P. aeruginosa which display various mechanisms of acquired resistance. Among these latter non-intrinsically resistant isolates, three (10%) had a MIC value of 4mg/L, nine (29.0%) had a MIC of 8mg/L, eight (26%) had a MIC of 16mg/L, seven (23%) had a MIC value of 32mg/L, and four (13%) isolates displayed a MIC of ≥64mg/L (Figure 1). A total of 19 (20%) isolates had a high MIC value, considered in this study as ≥16mg/ml, which belonged to the species Acinetobacter spp. (n=1), E. aerogenes (n=1), E. cloacae (n=1), E. coli (n=4), K. oxytoca (n=2) and K. pneumoniae (n=10).
Rapid polymyxin NP Test shows highest concordance with susceptibility category and UMIC with MIC measurements.
Susceptibility category concordance (susceptible or resistant) between the broth dilution gold standard and the test protocols (Vitek 2®, Colistin MIC Test Strip, UMIC and Rapid Polymyxin NP Test) is shown in Table 1. The highest overall concordance was achieved with Rapid Polymyxin NP test (98.8%), followed by UMIC (97.9%), colistin E-test MIC strip (96.9%), and Vitek 2 (95.6%). We calculated the sensitivity and specificity for the susceptibility category according to the BDM reference standard in all isolates. The highest sensitivity was shown for the Rapid Polymyxin test (98.8%) with a 100% specificity.
Table 1. Susceptibility category (susceptible or resistant) concordance of different assays compared to reference broth microdilution method.
Method
|
BDM
|
Vitek 21
|
Rapid Polymyxin NP 2
|
UMIC
|
E-test
|
Specie
|
S/R
|
S/R
|
% concordance
|
S/R
|
% Concordance
|
S/R
|
% Concordance
|
S/R
|
% Concordance
|
Acinetobacter spp.
|
6/1
|
4/0
|
100
|
1/1
|
50
|
6/1
|
100
|
6/1
|
100
|
C. koseri
|
3/0
|
3/0
|
100
|
3/0
|
100
|
3/0
|
100
|
3/0
|
100
|
E. aerogenes
|
1/1
|
1/1
|
100
|
1/1
|
100
|
1/1
|
100
|
1/1
|
100
|
E. cloacae
|
2/3
|
3/2
|
80
|
2/3
|
100
|
2/3
|
100
|
3/2
|
80
|
E. coli
|
13/11
|
13/11
|
100
|
13/11
|
100
|
13/11
|
100
|
13/11
|
100
|
Hafnia spp.
|
0/15
|
1/14
|
93.3
|
0/15
|
100
|
0/15
|
100
|
0/15
|
100
|
K. oxytoca
|
1/2
|
1/2
|
100
|
1/2
|
100
|
1/2
|
100
|
1/2
|
100
|
K. pneumonaie
|
8/12
|
9/10
|
94.7
|
9/11
|
95
|
9/11
|
95
|
9/11
|
95
|
M. morganii
|
0/3
|
0/3
|
100
|
0/3
|
100
|
0/3
|
100
|
0/3
|
100
|
P. mirabilis
|
0/2
|
0/2
|
100
|
0/2
|
100
|
0/2
|
100
|
0/2
|
100
|
P. vulgaris
|
0/1
|
0/1
|
100
|
0/1
|
100
|
0/1
|
100
|
0/1
|
100
|
P. aeruginosa
|
9/1
|
8/0
|
87.5
|
NA
|
NA
|
10/0
|
90
|
10/0
|
90
|
S. marcescens
|
0/2
|
0/2
|
100
|
0/2
|
100
|
0/2
|
100
|
0/2
|
100
|
Total
|
43/54
|
43/48
|
95.6
|
30/52
|
98.8
|
45/52
|
97.9
|
46/51
|
96.9
|
BDM, Broth microdilution; S, susceptible; R, resistant; NA; not applicable
|
1A total of 91 isolates were tested by Vitek method. Isolates not tested by Vitek test, were excluded from the comparison analysis with broth microdilution.
|
2A total of 82 were tested by the Rapid Polymyxin NP method. Isolates not tested by Rapid Polymyxin NP test were excluded from the comparison analysis with gold standard.
|
Exploring subsets of bacterial isolates, the most robust test compared to the gold standard in Enterobacterales was the UMIC test, with a concordance of 98.7%. The Rapid Polymyxin test also achieved a high concordance level (98.5%), whereas the Vitek 2 and the E-test were concordant only in 96.2% of the tested isolates in both tests. In susceptibility testing for non-fermenting bacteria, the highest concordance to broth microdilution was the UMIC (94.1%) and E-test (94.4%) followed by Vitek (90.9%). The rapid polymyxin NP test is specifically designed to detect polymyxin resistance among Enterobacteriaceae(32). The susceptibility concordance with BDM was high for the subset of Enterobacterales isolates included in this study (98.5%). However, and as expected, the performance for non-fermenting bacteria was poor, reaching a concordance percentage of only 50%. No differences were observed in the capability to detect resistant isolates in non-fermenting and fermenting Enterobacterales between the different assays used in this study (Additional file 3).
MIC concordance of the different assays compared to the gold standard is shown in Table 2. Concordance was established as the same MIC value or ±1 titre difference as that of the gold standard. All intrinsic species were excluded from the analysis since no MIC value was obtained from the reference standard method as they were automatically considered as resistant isolates. The highest concordance was achieved with UMIC test (80.4%), followed by Vitek 2 (72.5%). The lowest concordance was found for colistin MIC strip of 62.9%.
Table 2. MIC concordance of different assays compared to the reference broth microdilution method.
Method
|
BDM
|
VITEK
|
UMIC
|
E-test
|
Specie
|
No. Isolates tested
|
No. Isolates tested
|
No. of concordant isolates [%]1
|
No. Isolates tested
|
No. of concordant isolates [%]1
|
No. Isolates tested
|
No. of concordant isolates [%]1
|
Acinetobacter spp.
|
7
|
4
|
4 [100.0]
|
7
|
7 [100.0]
|
7
|
6 [85.7]
|
C. koseri
|
3
|
3
|
3 [100.0]
|
3
|
2 [66.7]
|
3
|
2 [66.7]
|
E. aerogenes
|
2
|
2
|
1 [50.0]
|
2
|
1 [50.0]
|
2
|
2 [100.0]
|
E. cloacae
|
5
|
5
|
3 [60.0]
|
5
|
5 [100.0]
|
5
|
3 [60.0]
|
E. coli
|
24
|
24
|
17 [70.8]
|
24
|
19 [79.2]
|
24
|
13 [54.2]
|
Hafnia spp.
|
15
|
15
|
14 [93.3]
|
15
|
14 [93.3]
|
15
|
13 [86.7]
|
K. oxytoca
|
3
|
3
|
1 [33.3]
|
3
|
3 [100.0]
|
3
|
1 [33.3]
|
K. pneumoniae
|
20
|
19
|
16 [84.2]
|
20
|
17 [85.0]
|
20
|
13 [65.0]
|
P. aeruginosa
|
10
|
8
|
7 [87.5]
|
10
|
10 [100.0]
|
10
|
8 [80.0]
|
Total
|
97
|
91
|
66 [72.5]
|
97
|
78 [80.4]
|
97
|
61 [62.9]
|
BDM, Broth microdilution method
1 Concordance was considered as the same MIC value or as one titre difference to that of the reference value
|
Additionally, for Enterobacterales species the most concordant test to the gold standard was UMIC (76.25%), followed by VITEK (69.62%) and E-test (58.75%). Similarly, the highest concordance for non-fermenting bacteria was found in the UMIC test (100%). The Vitek 2 test and the E-test achieved a concordance to the gold standard of 91.67% and 82.35%, respectively. Figure 2 shows the MIC distribution of the BDM vs. each method and the MIC distribution for all isolates.
Heterogenous molecular causes of colistin resistance.
MLST sequence type (ST) designation and core genome MLST (cgMLST) analysis were used to establish the diversity between isolates. The STs of isolates for which species MLST schemes exist was determined (Additional file 4). cgMLST could only be performed on species with more than two isolates. The diversity within E. coli and K. pneumoniae are shown in Figure 3. Isolates were genomically diverse in the cgMLST with the exception of isolates from the same patients (indicated with * in the figures): in some cases multiple isolates belonged to the same ST, such as K. pneumoniae ST512 (n=7), K. pneumoniae ST1825 (n=2), E. coli ST73 (n=3), and E. coli ST156 (n=2).
Genes encoding colistin resistance were identified first by comparing genome assemblies against known databases. This identified mcr-1 in four isolates (700099-17, NCTC-13846, 719645-16 and 705498-12) and mcr-2 in one isolate (KP-37), but these results did not explain all the phenotypic resistance.
Individual genomic analysis of the underlying colistin resistance mechanisms was performed, looking at genes previously described as being involved in colistin resistance. These genes were extracted from the genome and compared between sensitive and resistant isolates. This could only be performed on isolates within species with sufficient numbers of each, namely K. pneumoniae (n=20) and E. coli (n=24). The nucleotide sequences and derived protein sequences from phoPQ and pmrCAB in both species, and additionally mgrB in K. pneumoniae isolates were compared between resistant and susceptible isolates (Additional files 5, 6 and 7). Variations unique to the resistant isolates are described in Table 3.
None of the analysed K. pneumoniae isolates were carriers of mcr genes. A key finding within K. pneumoniae isolates was the presence of mutations in mgrB causing amino acid substitutions, premature stop codons, or termination resulting from insertion sequences ISEcp1 (IS138 family) and ISkpn26 (IS5 family). This applied to all resistant isolates with MIC values of ≥16mg/L, whereas sensitive isolates have intact versions of mgrB. Further mutations leading to amino acid substitutions were found in other genes: the isolate with the highest MIC (≥64 mg/L) possesses additional amino acid substitutions in the PhoQ, PmrA, and PmrB proteins compared to those from sensitive isolates. Similarly, two isolates with MIC of 32 mg/L have amino acid substitutions in the PmrA and/or PmrC protein in addition to MgrB. Two resistant isolates with MICs of 4mg/ml and 8 mg/L have an unaltered mgrB gene but possesses non-synonymous mutations in the pmrA and/or pmrC genes.
Only four of the resistant E. coli isolates, with MICs between 4-8 mg/ml were carriers of the mcr-1.1 gene. Gene comparisons within E. coli showed that those with a MIC of 16mg/ml have amino acid changes in the PmrB protein sequence, whereas the isolates with a lower MIC (4-8mg/ml) possess PmrB identical to those in sensitive isolates. In one isolate with a MIC of 16mg/ml, the pmrA and pmrB genes were not found within the genome assembly and therefore could not be analysed for mutations. Two isolates with a MIC of 8 mg/ml that did not harbour mcr genes had an unaltered PmrB protein sequence compared to the susceptible isolates but had amino acid substitutions in the protein sequences of PmrA or PmrC.
All the A. bereziniae isolates (n=4) were isolated from the same patient, and were subjected to analysis of pmrAB and phoPQ genes, as well as lpxA, lpxD, lpxC genes since mutations in the latter genes produce a total loss of lipid A leading to colistin resistance in Acinetobacter spp. (5). The single resistant isolate (MIC 64mg/ml) had a mutation causing amino acid change Q242R in PmrB. No mutations were found in the other genes assessed.
Table 3. Mutations in associated colistin-resistance proteins in E. coli, K. pneumoniae and A. berziniae resistant isolates.
Species
|
Isolate
|
MIC (mg/l)1
|
Amino acid change
|
Plasmid mediated resistance
|
MgrB
|
PmrB
|
PmrA
|
PmrC
|
PhoP
|
PhoQ
|
mcr
|
K. pneumoniae
|
404507-16
|
≥64
|
D31N
|
L213M
|
A41T
|
|
|
S288N
|
|
4002006-2
|
32
|
K3*
|
|
|
|
|
|
|
16003084
|
32
|
C28S
|
|
A217V
|
G25S
|
|
|
|
20038016
|
32
|
C39Y
|
|
|
|
|
|
|
808927-16
|
32
|
L8*
|
|
|
R152H
|
|
|
|
D477N
|
26048671
|
32
|
I41* (ISKpn26)3
|
|
|
|
|
|
|
800138-16
|
32
|
I41* (ISEcp1)3
|
|
|
|
|
|
|
401433-14
|
16
|
Q30*
|
|
|
|
|
|
|
802208-17
|
16
|
L4*
|
|
D149E
|
|
|
|
|
187701876
|
16
|
C39G
|
|
|
|
|
|
|
19852760
|
8
|
|
|
A217V
|
G25S
|
|
|
|
809156-16
|
4
|
|
|
D149E
|
|
|
|
|
E. coli
|
721296-16
|
16
|
|
P97L
|
|
C27Y
|
|
|
|
700455-17
|
16
|
|
L197D
|
|
N12D
|
|
|
|
709006-16
|
16
|
|
E169K
|
|
|
|
|
|
705963-16
|
16
|
|
NF ²
|
NF ²
|
NF ²
|
|
|
|
700099-17
|
8
|
|
|
|
|
|
|
mcr 1.1
|
706090-16
|
8
|
|
|
R81H
|
Q479E
|
|
|
|
NCTC-13846
|
8
|
|
|
|
|
|
L467M
|
mcr 1.1
|
KP-37-MCR-2-18
|
8
|
|
|
|
C27Y
|
|
|
mcr 2
|
L74I
|
707671-17
|
8
|
|
|
|
C27Y
|
|
|
|
719645-16
|
8
|
|
|
|
|
|
|
mcr 1.1
|
705498-12
|
4
|
|
|
|
|
|
|
mcr 1.1
|
A. bereziniae
|
502814-14
|
≥64
|
|
Q242R
|
|
|
|
|
|
1MIC values obtained by the reference broth microdilution method
|
2NF, pmrAB not found in assembly
|
3Nucleotide sequence interrupted by insertion sequence
|