Chapter 4: Results
4.2 Antibiotic Resistance of the Strains
4.2.2 Impact of the Mechanism of Carbapenem Resistance on
Although the overall rate of resistance was high, variation between the different groups of CRE was noted. Comparing carbapenemase producing and non-producing CRE, members of the former group exhibited non-susceptibility at a significantly higher rate to imipenem, meropenem, amikacin (P<0.0001) and to chloramphenicol (P=0.0246), while not to colistin and tigecycline (Table 12). CPase producing strains significantly more frequently exhibited the XDR or PDR phenotype (P<0.0001) than did their counterparts not producing any enzymes hydrolyzing carbapenems.
73 Figure 17: Allele distribution of carbapenemases
NDM-1
NDM-4
NDM-5
NDM-7 0
20 40 60 80 100
% among NDM producers
OXA-48
OXA-162
OXA-181
OXA-232
OXA-244 0
20 40 60 80 100
% among OXA-48-like producers
NDM-1-OXA-48 NDM-1-OXA-162
NDM-1-OXA181
NDM-1-OXA-232 NDM-5-OXA-181 0
20 40 60 80 100
% among NDM-OXA producers
Of the carbapenems tested, non-susceptibility to ertapenem was 100%, irrespective of the mechanism of carbapenem resistance. On the other hand, the highest rate of strains exhibiting susceptibility to a carbapenem was observed with meropenem (>15%). It was noteworthy, however, that beyond carbapenemase non-producers, for which the meropenem susceptibility rate was nearly 40%, the only carbapenemase producing group exhibiting some rate of susceptibility (about 20%) was the OXA-48- like enzyme producing group. In this group, and in this group only, a low rate (10- 15%) of susceptibility was observed to 3rd generation cephalosporins, as well (Table 13).
Strains expressing two different carbapenemases were significantly more often non-susceptible to meropenem (P=0.0016) and were more likely to be XDR/PDR (P=0.0028) than single carbapenemase producers (Table 12).
The MIC90, -50, and -10 values for carbapenemase non-producing strains, and for those producing groups that were represented by a sufficiently high number to make such calculations, are shown in Table 14. As can be seen, these values were considerably lower for non-producing strains than in other groups, while they were the highest among the NDM+OXA double carbapenemase producers.
Table 14: Relation of the MIC levels of carbapenems to the resistance mechanisms
Antibiotics MIC
Carbapenemase produced (n) NDM
(98)
OXA (173)
NDM+OXA (56)
None (63) Ertapenem
MIC90 >64 >64 >64 >64 MIC50 >64 >64 >64 >64
MIC10 64 16 >64 2
Imipenem
MIC90 128 32 128 8
MIC50 64 8 64 2
MIC10 16 2 16 <0.25
Meropenem
MIC90 128 32 >128 8
MIC50 64 16 128 2
MIC10 16 0.5 128 <0.25
A similar comparison was also conducted for strains expressing various alleles of OXA-48-like enzyme, if they were represented by a sufficiently high number. The results are shown in Table 15.
Table 15: Relation of the MIC levels of carbapenems to the alleles of OXA-48-like carbapenemases
Antibiotics MIC
Carbapenemase produced (n) OXA-48
(64)
OXA-181 (30)
OXA-232 (73) Ertapenem
MIC90 >64 >64 >64
MIC50 64 >64 >64
MIC10 8 16 >64
Imipenem
MIC90 32 64 16
MIC50 8 16 8
MIC10 2 1 4
Meropenem
MIC90 16 32 32
MIC50 2 16 16
MIC10 0.5 <0.25 4
While ertapenem had a uniformly high MIC50 against strains producing any of the alleles, this high MIC was the most uniform against OXA-232 producers. In their case it took >64 mg/L to inhibit as little as 10% of the isolates. This allele was the least active against imipenem and meropenem, as well requiring 4 mg/L of both drugs to inhibit 10% of the isolates. On the other hand, to achieve the same rate of inhibition among OXA-48 and OXA-181 producers, ≤2 mg/L were sufficient.
For a more detailed picture, we compared the MIC distribution among the major groups of strains representing different carbapenem resistant mechanisms. The results are shown in Figure 18.
As can be seen, with a few exceptions of some non-carbapenemase producing isolates, ertapenem had high MIC rates (>32 mg/L), against most isolates, irrespective of the resistance mechanisms (Figure 18 A). On the other hand, the distribution of imipenem and meropenem was more even for OXA-48 like producers with MIC values peaking at 8 mg/L for imipenem, and at 16 mg/L for meropenem. For the majority of non-carbapenemase producers the MIC values for both imipenem and meropenem was below 8 mg/L. Contrary to this, against NDM, and particularly against NDM+OXA- 48-like double carbapenemase producers, both antibiotic showed high MIC values with peaks at 64-128 mg/L (Figure 18 B, C).
Among OXA-48-like carbapenemase producers some of the alleles were present in sufficiently high numbers allowing a similar comparison (Figure 19).
Although the ertapenem MIC of all alleles peaked at >64 mg/L, only 50% of OXA-48 producers fall into this category. On the other hand, for OXA-181 and OXA-232 producers it was 80.0% and 90.4%, respectively. The distribution of imipenem MIC exhibited a different pattern. The values for OXA-48, and particularly those for OXA-
232 producers peaked at 8 mg/L, while imipenem had a higher MIC (16 mg/L) against OXA-181 producers. The pattern of meropenem distribution was partly similar.
Against this antibiotic also OXA-181 producers were the most active ones showing an MIC peak, represented by 50% of the strains, at 32 mg/L, followed by OXA-232 producers by a similar rate at 16 mg/L. However, not uncommonly, all allele type producers, most frequently that of OXA-48, were present with meropenem MIC values
≤2 mg/L.
Figure 18: MIC distribution of different carbapenems against strains of different carbapenem resistance mechanisms
< 0.1 25
0 .25 0 .5 1 2 4 8 1 6 3 2 6 4 > 64
0 2 0 4 0 6 0 8 0 1 0 0
%
N D M ( n = 9 8 ) O X A ( n = 1 7 3 ) N D M - O X A ( n = 5 6 )
N o C a r b a p e n e m a s e ( n = 6 3 )
A E r t a p e n e m
< 0.2 5 0 .5 1 2 4 8 1 6 3 2 6 4 1 28
> 12 8 0
2 0 4 0 6 0 8 0 1 0 0
%
B I m ip e n e m
< 0.2 5 0 .5 1 2 4 8 1 6 3 2 6 4 1 28
> 12 8 0
2 0 4 0 6 0 8 0 1 0 0
%
C M e r o p e n e m
M I C ( m g / L )
< 0.1 25
0 .25 0 .5 1 2 4 8 1 6 3 2 6 4 > 64
0 2 0 4 0 6 0 8 0 1 0 0
O X A - 4 8 ( n = 6 4 ) O X A - 1 8 1 ( n = 3 0 ) O X A - 2 3 2 ( n = 7 3 )
A E r t a p e n e m
< 0.2 5 0 .5 1 2 4 8 1 6 3 2 6 4 1 28
> 12 8 0
2 0 4 0 6 0 8 0 1 0 0
%
B I m i p e n e m
< 0.2 5 0 .5 1 2 4 8 1 6 3 2 6 4 1 28
> 12 8 0
2 0 4 0 6 0 8 0 1 0 0
%
M I C ( m g / L )
C M e r o p e n e m
Figure 19: MIC distribution of different carbapenems against strains producing different OXA-48-like carbapenemase alleles
%
Clinical data indicate that if meropenem or imipenem MIC are ≤4, or even ≤8 mg/L, in the absence of alternatives and using optimal dosing, particularly if applied in combination with other drugs, these antibiotics still can be considered as a treatment option (Rodriguez-Bano, Gutierrez-Gutierrez et al. 2018). Therefore, we calculated the rate of strains belonging to this group and exhibiting various carbapenem resistance mechanisms. The results are summarized in Table 16.
Overall, more than a third of the strains fall in the category when meropenem treatment can still be an option, i.e. with MIC ≤8 mg/L. However, this rate depended very much on the mechanism of resistance to carbapenems. While almost all non- carbapenemase producers fall into this category, that was significantly lower among carbepenemase producers (P<0.0001). Comparing strains expressing either NDM or OXA-48-like enzymes to double carbapenemase producing isolates, the rates of strains with MIC values ≤4 or ≤8 mg/L, either for imipenem or meropenem, were always significantly lower in the latter group than among those isolates expressing a single carbapenemase (P values varying between 0.0003 and <0.0001). Furthermore, among carbapenemase producers, expression of NDM was more likely to cause a meropenem MIC of ≤8 mg/L than the production of OXA-type carbapenemase (P=<0.0001).
Regarding the allele types of OXA-48-like enzymes, of those present in high numbers, OXA-48 producers were the most, and those expressing OXA-181 and OXA-232 were the least likely to fall in the category where meropenem (or imipenem) treatment could still be considered (P<0.0001 OXA-181+OXA-232 vs. other OXA-48-like alleles).
It was noteworthy that while some of the strains with the XDR, and even with the PDR resistance patterns fall into these categories, they were more likely to have imipenem and ertapenem MIC values >4 or >8 mg/L than their counterparts exhibiting
wider susceptibility ranges. The P values, when comparing XDR/PDR strains vs.
isolates with broader susceptibility ranges varied between 0.0054 and <0.0001.
Table 16: Rate of isolates with imipenem and meropenem MIC values that still allows the consideration of these drugs for treatment
Resistance
mechanisms N
% with MIC values
MIC of IMI (mg/L) MIC of MER (mg/L)
≤4 ≤8 ≤4 ≤8
All 394 24.9 45.7 28.9 35.0
NDM 98 0.0 6.1 2.0 7.1
NDM-1 88 0.0 4.5 2.3 8.0
NDM-4 1 0.0 0.0 0.0 0.0
NDM-5 8 0.0 25.0 0.0 0.0
NDM-7 1 0.0 0.0 0.0 0.0
OXA 173 24.3 64.2 32.4 38.7
OXA-48 64 37.5 67.2 56.3 59.4
OXA-162 3 33.3 100.0 100.0 100.0
OXA-181 30 16.7 30.0 20.0 23.3
OXA-232 73 12.3 72.6 11.0 21.9
OXA-244 3 100.0 100.0 100.0 100.0
VIM 3 0.0 0.0 66.7 66.7
VIM-4 2 0.0 0.0 100.0 100.0
VIM-55 1 0.0 0.0 0.0 0.0
NDM-OXA 56 0.0 5.4 0.0 3.6
NDM-1-OXA-48 5 0.0 0.0 0.0 0.0
NDM-1-OXA-162 1 0.0 100.0 0.0 100.0
NDM-1-OXA-181 4 0.0 0.0 0.0 0.0
NDM-1-OXA-232 39 0.0 5.1 0.0 2.6
NDM-5-OXA-181 7 0.0 0.0 0.0 0.0
NDM-1-VIM-55 1 0.0 0.0 0.0 0.0
All
carbapenemase producer
331 12.7 36.3 18.1 23.6
No
carbapenemase 63 88.9 95.2 85.7 95.2
MBL 158 0.0 5.7 2.5 7.0
XDR 116 3.4 31.0 1.7 5.2
PDR 22 9.1 40.9 9.1 13.6
Non XDR/PDR 256 30.5 47.3 33.6 41.0
Isolates exhibiting susceptibilities in >1 classes