Chapter 4: Results
4.3 The Clonality of the Strains
Figure 20: Macrorestriction patterns of the 45 E. coli strains. Dashed lines represent 80% and 90% pattern similarities
The 324 K. pneumoniae strains were also subjected to macrorestirction analysis. They were assigned into 45 pulsotypes, including 8 singletons (Figure 21).
Nine pulsotypes were identified with members 5, 6, 7, 8, 8, 20, 36 and 111, respectively. The clones identified by MLST analyses are shown in Table 20.
Table 20: The Klebsiella pneumoniae clones identified
Clone N % among K. pneumoniae
ST11 8 2.5
ST14 111 34.3
ST15 5 1.5
ST45 8 2.5
ST101 16 4.9
CC147 43 13.3
ST231 36 11.1
All clones 227 70.1
Sporadic isolates* 97 29.9
The 4 major clones© 206 63.6
*Defined as strains forming pulsotypes <5 members
© ST14, ST101, CC147 and ST231
The strains belonging to these clones represented over 70%, the 4 largest clones: ST14, ST101, CC147 (ST147 and single locus variants ST273 and ST392) and ST231, respectively) did more than 60% of the K. pneumoniae strains. Next, we investigated whether any of the characteristics of the strains could be linked to any of these clones.
89 Figure 21: Macrorestriction patterns of the 324 K. pneumoniae strains. Dashed lines represent 80% and 90% pattern similarities
The representation of the 4 major clones among K. pneumoniae submitted from different hospitals is shown in Figure 22. Members of clone ST101 were almost completely restricted to Mafraq hospital. It was Mafraq hospital and SKMC that carried all the 4 major clones. Although 3 of the 4 largest clones (ST14, C147 and ST231 were present in 5 out of the 6 hospitals, their representation in the hospitals was uneven. Over 80% of the strains from Al Ain hospital belonged to this clone and it was the dominating clone in Zayed Military hospital and SKMC, as well. Tawam hospital, with the considerable presence of ST14, characteristically yielded ST231, while this clone, although present in four other hospitals, was less significant there.
Figure 22: Representation of the four major clones among K. pneumoniae isolated from different hospitals
Mafraq (103)
S KMC (85)
T aw am (69)
A L Ain (46)
Z ayed Military (12)
R ahba (9) 0
2 0 4 0 6 0 8 0 1 0 0
H o s p ita ls (n u m b e r o fK . p n e u m o n ia e)
%among local K. pneumoniae S T 1 4 ( 1 1 1 )
C C 1 4 7 ( 4 3 ) S T 2 3 1 ( 3 6 ) C C 1 0 1 ( 1 6 )
The relation between clonality and carbapenem resistance mechanism is shown in Table 21. Clonal strains were more likely to express a carbapenemase than non- clonal ones (P<0.0001). There was no significant difference between clonal and sporadic strains regarding the expression of NDM (P=0.8764) and clonal and sporadic strains also carried OXA-48-like carbapenemases with almost equal frequencies.
However, while among sporadic strains OXA-48 was the most common carbapenemase (P<0.0001, compared to clonal strains), OXA-producing clonal isolates were more likely to express OXA-232 (P<0.0001). This was largely due to ST231 strains, i.e. nearly 90% of them expressed this carbapenemase making this feature highly characteristic to the entire clone. Among all K. pneumoniae strains producing OXA-48-like enzymes as a single carbapenemase, members of this clone were significantly the most likely to produce OXA-232 (P<0.0001). About a third of members of the ST14 clone also carried the gene of this oxacillinase, but among them the expression of NDM-1, as well as the co-expression of these two enzymes were equally common. This latter feature made this clone the most common one carrying two carbapenemase genes (P<0.0001). The two smaller clones, ST11 and ST15, also contained strains expressing either NDM-1 or OXA-48, but never both. While clone ST101 was almost uniformly made of OXA-48 producing isolates, a very high rate of non-carbapenem producers was noted in clone ST45.
There was a considerable difference between the efficacy of carbapenems against clonal and sporadic strains. In general, by comparing their respective MIC10, MIC50 and MIC90 values, clonal strains, as a group, exhibited higher level of resistance to carbapenems than their sporadic counterparts, as shown on Table 22.
92 Table 21: Rate of different carbapenem resistance mechanisms within the clones
Carbapenem resistance mechanisms
Frequency (%) among strains belonging to (size of the group) All K.
pneumoniae (324)
ST11 (8)
ST14 (111)
ST15 (5)
ST45 (8)
ST101 (16)
CC147 (43)
ST231 (36)
All clones
(227)
Sporadic (97)
NDM 23.1 50 30.6 60.0 0 0 34.9 0.0 24.7 19.6
NDM-1 22.8 50 30.6 40.0 0 0 34.9 0.0 24.2 19.6
NDM-4 0.0 0 0.0 0.0 0 0 0.0 0.0 0.0 0.0
NDM-5 0.0 0 0.0 0.0 0 0 0.0 0.0 0.0 0.0
NDM-7 0.3 0 0.0 20.0 0 0 0.0 0.0 0.4 0.0
OXA-48-like 46.3 37.5 32.4 40.0 12.5 93.75 37.2 97.2 47.6 43.3
OXA-48 15.4 37.5 0.0 40.0 12.5 93.75 2.3 0.0 9.7 28.9
OXA-162 0.9 0 0.9 0.0 0 0 0.0 0.0 0.4 2.1
OXA-181 7.4 0 0.9 0.0 0 0 32.6 11.1 8.4 5.2
OXA-232 22.2 0 30.6 0.0 0 0 2.3 86.1 29.1 6.2
OXA-244 0.3 0 0.0 0.0 0 0 0.0 0.0 0.0 1.0
NDM+OXA 17.0 0 36.9 0.0 0 0 14.0 0.0 20.7 8.2
NDM-1+OXA-48 1.5 0 1.8 0.0 0 0 2.3 0.0 1.3 2.1
NDM-1+OXA-162 0.3 0 0.0 0.0 0 0 0.0 0.0 0.0 1.0
NDM-1+OXA-181 0.9 0 1.8 0.0 0 0 0.0 0.0 0.9 1.0
NDM-1+OXA-232 12.0 0 33.3 0.0 0 0 2.3 0.0 16.7 1.0
NDM-5+OXA-181 2.2 0 0.0 0.0 0 0 9.3 0.0 1.8 3.1
No Carbapenemase 13.3 12.5 0.0 0.0 87.5 6.25 14.0 2.8 7.0 27.8
93 Table 22: Relation of the MIC levels of carbapenems to the alleles of OXA-48-like carbapenemases
Antibiotics MIC All clones (227) Sporadic (97)
4 major clones
ST14 (111) ST101 (16) ST147 (43) ST231(36) Ertapenem
MIC90 >64 >64 >64 >64 64 >64
MIC50 >64 >64 >64 >64 >64 >64
MIC10 >64 8 >64 >64 >64 >64
Imipenem
MIC90 128 128 128 16 128 32
MIC50 16 8 64 8 32 8
MIC10 4 1 8 2 1 4
Meropenem
MIC90 128 64 128 16 128 32
MIC50 32 4 64 16 32 16
MIC10 4 0.5 8 8 2 16
As can be seen, all major clones, where their respective numbers allowed such calculation, exhibited carbapenem resistance levels higher than sporadic isolates. The highest values were seen in the largest clone, i.e. clone ST14. This was also reflected in the proportion of isolates within each clone for which carbapenem treatment could still be an option: a significantly higher proportion of sporadic isolates exhibited MIC values ≤8 mg/L for imipenem and for meropenem (P=0.0032 and P<0.0001) than did clonal isolates. Alarmingly, while in the case of meropenem, these options were still there for more than half of the sporadic isolates, it was available for a much lower rate of isolates in the four major clones (Table 23).
Table 23: Rate of isolates with imipenem and meropenem MIC values that still allows the consideration of these drugs for treatment
Clones N
% with MIC values
MIC of IMI (mg/L) MIC of MER (mg/L)
≤4 ≤8 ≤4 ≤8
All clones 227 13.2 38.3 11.9 16.3
Sporadic 97 39.2 56.7 52.6 58.8
ST14 111 2.7 26.1 2.7 10.8
ST101 16 12.5 62.5 10.2 12.5
ST147 43 16.2 18.6 14.0 14.0
ST231 36 13.9 72.2 8.3 8.3
Certain other resistance-related features were also more common among some of the clones than among sporadic isolates. Generally, the rate of colistin resistance in clones was only slightly higher than among sporadic strains (P=0.4252). However, in the large ST14 clone, it significantly exceeded the rates seen among all K. pneumoniae outside of this clone (P=0.0032), as over a quarter of strains within this clone exhibited resistance to polymyxins (Table 24).
On the other hand, an even more pronounced difference between clonal and sporadic strains was seen regarding tigecycline resistance (P<0.0001), the presence of 16S methylase (P<0.0001) and expressing the XDR or PDR phenotype (P<0.0001). In these respects, particularly the larger clones, i.e. ST14, CC147 and ST231, were noticeable. It should be noted, however, that some of the smaller clones, and even one of the larger one (ST101) did not exhibit high rates in resistance-related figures (Table 24).
Table 24: Rate of selected resistance features within the clonal groups
Group N
Representation (%) within the group Colistin
NS
Tigecycline NS
16S methylase production
XDR PDR All K.
pneumoniae 324 17.3 65.4 50.6 35.5 6.5
ST11 8 0.0 50.0 0.0 12.5 0.0
ST14 111 26.1 75.7 78.4 52.3 13.5
ST15 5 20.0 100.0 40.0 40.0 20.0
ST45 8 12.5 75.0 0.0 0.0 0.0
ST101 16 18.8 6.3 0.0 0.0 0.0
CC147 43 14.0 72.1 46.5 39.5 2.3
ST231 36 5.6 100.0 100.0 88.9 5.6
All clones 227 18.5 73.6 63.9 48.5 8.4
Sporadic 97 14.4 46.4 19.6 5.2 1.0
While the production of 16S methylases was a common feature among strains belonging to some of the clones (Table 24), the type produced was unevenly distributed (Table 25).
96 Table 25: Distribution of the various 16S methylase genes among K. pneumoniae clones
Groups
Frequency (%) of genes in groups
armA rmtB rmtC rmtF
Whole group
Among members with any 16S
methylase
Whole group
Among members with any 16S
methylase
Whole group
Among members
with any 16S methylase
Whole group
Among members
with any 16S methylase
All K. pneumoniae 33.0 65.2 1.5 3.0 0.6 1.2 17.6 34.8
ST11 0.0 NA 0.0 NA 0.0 NA 0.0 ND
ST14 78.4 100.0 0.0 0.0 0.0 0.0 0.0 0.0
ST15 0.0 0.0 20.0 50.0 0.0 0.0 20.0 50.0
ST45 0.0 NA 0.0 NA 0.0 NA 0.0 NA
ST101 0.0 NA 0.0 NA 0.0 NA 0.0 NA
CC147 7.0 15.0 9.3 20.0 2.3 5.0 39.5 85.0
ST231 5.6 5.6 0.0 0.0 0.0 0.0 97.2 97.2
All clones 40.5 63.4 2.2 3.4 0.4 0.7 23.3 36.6
Sporadic 15.5 78.9 0.0 0.0 1.0 5.3 4.1 21.1
NA – not applicable
As can be seen, while some of the clones did not carry any 16S methylase genes (e.g. ST11, ST45, ST101) others either carried multiple (ST15, CC147) or where characterized by a single type (armA in case of the largest clone, ST14) present in almost 80% of members of this clone.
Due to their size and resistance level exhibited by most clones their impact on the rate of some resistance-related features of the entire collection exceeded their numerical proportion. Clonal strains, particularly those in ST14, CC147 and ST231 were responsible for the overwhelming majority of XDR and PDR cases. Particularly the largest (N=111) clone, ST14 had a considerable impact. Beyond its size (representing nearly a third of the collection) it gave over 70% of all double carbapenemase producers, half of all XDR and nearly 70% of all PDR strains. (Table 26).
98 Table 26: The impact of clonal and sporadic strains on selected feature of the collection
Group
Representation (%) among all strains (N) with the same features Entire collection
(394)
Colistin NS (64)
Tigecycline NS (227)
16S methylase producer (171)
Double carbapenemase
producer (57)
XDR (116)
PDR (22)
All K. pneumoniae 82.2 87.5 93.4 95.9 96.5 99.1 95.5
ST11 2.0 0.0 1.8 0.0 0.0 0.9 0.0
ST14 28.2 45.3 37.0 50.9 71.9 50.0 68.2
ST15 1.3 1.6 2.2 1.2 0.0 1.7 4.5
ST45 2.0 1.6 2.6 0.0 0.0 0.0 0.0
ST101 4.1 4.7 0.4 0.0 0.0 0.0 0.0
CC147 10.9 9.4 13.7 11.7 10.5 14.7 4.5
ST231 9.1 3.1 15.9 21.1 0.0 27.6 9.1
All clones 57.6 65.6 73.6 84.8 82.5 94.8 86.4
Sporadic 24.6 21.9 19.8 11.1 14.0 4.3 4.5
4.4 The Emergence of CRE and the Change of the Rates of their Selected