3.1 Klebsiella pneumoniae of human origin
Seventeen K. pneumoniae strains were isolated from 12 patients (Table 1). Long-lasting infections in four patients were revealed by repeated detection of K. pneumoniae over time.
Table 1
Carbapenem-resistant strains isolated from humans and pigs in the study period
source
|
ID code
|
MIC values (µg/ml)
|
MLST
|
bla genes
|
|
K. pneumoniae
|
human
|
NEF 48
|
16 ± 0
|
37
|
VIM-1, OXA-1, CTX-M-15, SHV-12
|
human
|
NEF 87
|
32 ± 0
|
258
|
KPC-2, OXA-9, SHV-187
|
human
|
NEF 88
|
13.33 ± 4.62
|
258
|
KPC-2, OXA-9, SHV-12
|
human
|
NEF 112
|
10.67 ± 4.62
|
37
|
VIM-1, OXA-1, CTX-M-15
|
human
|
NEF 116
|
85.33 ± 36.95
|
554
|
KPC-3, OXA-9, SHV-182
|
human
|
NEF 119
|
256 ± 0
|
554
|
KPC-3, OXA-9, SHV-182
|
human
|
NEF 123
|
16 ± 0
|
4525
|
VIM-1, OXA-1, CTX-M-15, SHV-12
|
human
|
NEF 130
|
> 256
|
554
|
KPC-3, OXA-9, SHV-182
|
human
|
NEF 131
|
> 256
|
554
|
KPC-3, OXA-9, SHV-182
|
human
|
NEF 145
|
> 256
|
554
|
KPC-3, OXA-9, SHV-182
|
human
|
NEF 196/2
|
64 ± 0
|
554
|
KPC-3, OXA-9, SHV-182
|
human
|
NEF 197
|
64 ± 0
|
554
|
KPC-3, OXA-9, SHV-182
|
human
|
NEF 198
|
2 ± 0
|
277
|
-
|
human
|
NEF 199
|
1.67 ± 0.58
|
277
|
SHV-27
|
human
|
NEF 261
|
0.027 ± 0.009
|
307
|
SHV-28, CTX-M-15
|
human
|
NEF 278
|
0.15 ± 0.1
|
307
|
OXA-1, SHV-106, CTX-M-15
|
human
|
NEF 297
|
53.33 ± 18.48
|
554
|
KPC-3, OXA-9, SHV-182
|
|
P. aeruginosa
|
human
|
NEF 23
|
21.33 ± 9.24
|
938
|
OXA-396, PAO
|
human
|
NEF 156
|
8 ± 0
|
395
|
OXA-488, PAO
|
pig
|
CRE 98
|
2 ± 0
|
274
|
OXA-486, PAO
|
pig
|
CRE 102
|
4.67 ± 3.06
|
938
|
OXA-396, PAO
|
pig
|
CRE 153
|
16 ± 0
|
782
|
OXA-50, PAO
|
pig
|
CRE 295
|
1.67 ± 0.58
|
885
|
OXA-50, PAO
|
WGS identified carbapenemase genes in 13 (76.5%) strains from 9 patients. Apparent prevalence of CP K. pneumoniae infected patients was 4.8% (9/187) CI95[2.6–8.9]. Sequence identity (≥ 98%) and coverage minimum sequence length (> 60%) were used as selection criteria for gene detections. The 13 CP K. pneumoniae isolates harboured blaKPC-3 (61.5%), blaVIM-1 (23.1%) and blaKPC-2 (15.4%), as well as other β-lactamase-producing (bla) genes (Tables 1 and 2). CP K. pneumoniae belonged to ST554 (61.5%), ST37 (15.4%), ST258 (15.4%) and ST4525 (7.7%). The blaKPC-2 and blaKPC-3 determinants were linked to specific ST-types, namely ST258 and ST554, respectively. The blaVIM-1 gene was found in ST37 and ST4525 (Table 2).
Table 2
Resistomes of CP K. pneumoniae strains of human origin
Antimicrobial class
|
Mechanism (*)
|
|
Carbapenemase-producing K. pneumoniae
|
|
|
ST37
(NEF 48)
|
ST37
(NEF 112)
|
ST258
(NEF 87)
|
ST258
(NEF 88)
|
ST554
(NEF 116)
|
ST554
(NEF 119)
|
ST554
(NEF 130)
|
ST554
(NEF 131)
|
ST554
(NEF 145)
|
ST554
(NEF 196/2)
|
ST554
(NEF 197)
|
ST544
(NEF 297)
|
ST4525
(NEF 123)
|
Aminoglycosides
|
acetyltransferase
|
|
aac(6')-Ib
|
aac(6')-Ib
|
aac(6')-Ib
|
aac(6')-Ib
|
aac(6')-Ib
|
aac(6')-Ib
|
aac(6')-Ib
|
aac(6')-Ib
|
aac(6')-Ib
|
aac(6')-Ib
|
aac(6')-Ib
|
|
adenyltransferase
|
|
|
aadA2
|
aadA2
|
aadA2
|
|
aadA2
|
aadA2
|
aadA2
|
aadA2
|
aadA2
|
aadA2
|
|
nucleotidyltransferase
|
ant(3'')-Ia
|
ant(3'')-Ia
|
|
|
|
|
|
|
|
|
|
|
ant(3'')-Ia
|
phosphotranferase
|
aph(3')-XV
|
aph(3')-XV
|
|
|
|
|
|
|
|
|
|
|
aph(3')-XV
|
|
|
|
|
aph(3')-Ia
|
aph(3')-Ia
|
aph(3')-Ia
|
aph(3')-Ia
|
|
aph(3')-Ia
|
aph(3')-Ia
|
aph(3')-Ia
|
|
aph(3'')-Ib
|
aph(3'')-Ib
|
|
|
|
|
|
|
|
|
|
|
aph(3'')-Ib
|
aph(6)-Id
|
aph(6)-Id
|
|
|
|
|
|
|
|
|
|
|
aph(6)-Id
|
16 rRNA methylase
|
armA
|
armA
|
|
|
|
|
|
|
|
|
|
|
|
β-lactams
|
Class A
|
blaCTX−M−15
|
blaCTX−M−15
|
|
|
|
|
|
|
|
|
|
|
blaCTX−M−15
|
blaSHV−12
|
|
|
blaSHV−12
|
|
|
|
|
|
|
|
|
blaSHV−12
|
|
|
|
|
blaSHV−182
|
blaSHV−182
|
blaSHV−182
|
blaSHV−182
|
blaSHV−182
|
blaSHV−182
|
blaSHV−182
|
blaSHV−182
|
|
|
|
blaSHV−187
|
|
|
|
|
|
|
|
|
|
|
Class D
|
blaOXA−1
|
blaOXA−1
|
|
|
|
|
|
|
|
|
|
|
blaOXA−1
|
|
|
blaOXA−9
|
blaOXA−9
|
blaOXA−9
|
blaOXA−9
|
blaOXA−9
|
blaOXA−9
|
blaOXA−9
|
blaOXA−9
|
blaOXA−9
|
blaOXA−9
|
|
Carbapenems
|
Class A
|
|
|
blaKPC−2
|
blaKPC−2
|
|
|
|
|
|
|
|
|
|
|
|
|
|
blaKPC−3
|
blaKPC−3
|
blaKPC−3
|
blaKPC−3
|
blaKPC−3
|
blaKPC−3
|
blaKPC−3
|
blaKPC−3
|
|
Class B
|
blaVIM−1
|
blaVIM−1
|
|
|
|
|
|
|
|
|
|
|
blaVIM−1
|
Fluoroquinolones
|
Efflux
|
oqxA
|
oqxA
|
oqxA
|
oqxA
|
oqxA
|
oqxA
|
oqxA
|
oqxA
|
|
oqxA
|
oqxA
|
oqxA
|
oqxA
|
oqxB
|
oqxB
|
oqxB
|
oqxB
|
oqxB
|
oqxB
|
oqxB
|
oqxB
|
|
oqxB
|
oqxB
|
oqxB
|
oqxB
|
Macrolides
|
phosphotranferase
|
mph(A)
|
mph(A)
|
mph(A)
|
mph(A)
|
mph(A)
|
mph(A)
|
mph(A)
|
mph(A)
|
|
mph(A)
|
mph(A)
|
mph(A)
|
mph(A)
|
mph(E)
|
mph(E)
|
|
|
|
|
|
|
|
|
|
|
|
ribosomal protection
|
msr(E)
|
msr(E)
|
|
|
|
|
|
|
|
|
|
|
|
Major facilitator
superfamily (MFS)
|
|
|
|
|
|
|
|
mdf(A)
|
mdf(A)
|
|
mdf(A)
|
mdf(A)
|
|
|
Phosphonic acids
|
thiol transferase
|
fosA6
|
fosA6
|
fosA6
|
fosA6
|
fosA6
|
fosA6
|
fosA6
|
fosA6
|
fosA6
|
fosA6
|
fosA6
|
fosA6
|
fosA6
|
Phenicols
|
acetyltransferase
|
catA1
|
catA1
|
catA1
|
catA1
|
|
|
catA1
|
catA1
|
catA1
|
catA1
|
catA1
|
catA1
|
|
catB2
|
catB2
|
|
|
|
|
|
|
|
|
|
|
catB2
|
catB3
|
catB3
|
|
|
|
|
|
|
|
|
|
|
catB3
|
Suphonamides
|
dihydropteroate synthase
|
sul1
|
sul1
|
sul1
|
sul1
|
sul1
|
sul1
|
sul1
|
sul1
|
sul1
|
sul1
|
sul1
|
sul1
|
sul1
|
sul2
|
sul2
|
|
|
|
|
|
|
|
|
|
|
sul2
|
Trimethoprim
|
dihydropholate reductase
|
dfrA5
|
dfrA5
|
|
|
|
|
|
|
|
|
|
|
|
|
|
dfrA12
|
dfrA12
|
dfrA12
|
dfrA12
|
dfrA12
|
dfrA12
|
|
dfrA12
|
dfrA12
|
dfrA12
|
|
dfrA14
|
dfrA14
|
|
|
|
|
|
|
|
|
|
|
dfrA14
|
No. of genes
|
|
22
|
22
|
13
|
13
|
13
|
11
|
15
|
15
|
8
|
15
|
15
|
|
17
|
(*) Comprehensive Antibiotic Data Base [34] |
ST4525 is a novel ST-type with the following allelic profile: gapA (2); infB (9); mdh (2); pgi (1); phoE(13); rpoB (214); tonB (16). This allelic profile differs from ST37 by only one nucleotide within rpoB gene: T at position 100 of allele rpoB_1 (ST37) is substituted by a C in allele rpoB_214 (ST4525) (Fig. 1).
In three K. pneumoniae isolates (3/17; 17.6%), ESBL genes belonging to blaSHV, blaTEM, blaoxa and blaCTX families were found. These isolates were characterized by MIC values lower than the clinical breakpoint (< 2 µg/ml) (Table 3).
Table 3
Resistomes of ESBL K. pneumoniae strains of human origin
Antimicrobial class
|
Mechanism *
|
ESBL K. pneumoniae
|
|
|
ST277
(NEF 199)
|
ST307
(NEF 261)
|
ST307
(NEF 278)
|
Aminoglycosides,
non-specific
|
acetyltransferase
|
|
aac(3')-IIa
|
aac(3')-IIa
|
|
|
aac(6')-Ib-cr_1
|
adenyltransferase
|
|
aadA1_4
|
|
nucleotidyltransferase
|
|
ant(3’’)-Ia
|
|
phosphotranferase
|
|
aph(3’’)-Ib_5
|
aph(3’’)-Ib_5
|
|
|
aph(6)-Id
|
β-lactams
|
Class A
|
|
|
blaCTX−M−15
|
blaSHV−27
|
|
|
|
blaSHV−106
|
blaSHV−106
|
|
|
blaTEM−1B
|
|
blaTEM−102
|
|
Class D
|
|
|
blaOXA−1
|
Fluoroquinolones
|
efflux
|
oqxA
|
|
oqxA
|
oqxB
|
|
oqxB
|
|
|
qnrB1
|
Macrolides
|
phosphotranferase
|
|
mph(E)
|
|
Major facilitator
superfamily (MFS)
|
|
mdf(A)
|
mdf(A)
|
mdf(A)
|
Phosphonic acids
|
thiol transferase
|
|
fosA6
|
|
|
fosA7
|
|
Suphonamides
|
dihydropteroate synthase
|
|
|
sul2
|
Tetracyclines
|
efflux
|
|
|
tet(A)_6
|
Trimethoprim
|
dihydropholate reductase
|
|
|
dfrA14_5
|
N° of genes
|
|
4
|
10
|
15
|
(*) Comprehensive Antibiotic Data Base [34] |
ST258 blaKPC-2 isolates carried other bla genes, namely blaOXA-9, blaSHV-12, and blaSHV-187. Their resistome consisted of 13 genes conferring resistance to nine antimicrobial classes. ST554 blaKPC-3 isolates carried blaOXA-9 and blaSHV-182, with resistomes consisting of 11–15 genes. One ST37 blaVIM-1 strain carried blaOXA-1 and blaCTX-M-15, whereas the other ST37 blaVIM-1 additioanlly carried blaSHV-12. Their resistome consisted of 22 AMR genes. (Table 2)
The novel ST4525 blaVIM−1 (NEF123) was characterized by a resistome of 17 genes, which included blaCTX−M−15, blaSHV−12, blaOXA−1 and genes for resistance to aminoglycosides, fluoroquinolones, macrolides, fosfomycin, chloramphenicol, sulphonamides, and trimethoprim (Table 2).
Among the ESBL ST307 isolates, one strain harboured blaSHV−106 and blaTEM−102, and the other carried blaSHV−106, blaTEM−1B, blaCTX−M−15 and blaOXA−1. One ST277 strain carried blaSHV−27, whereas the other ST227 did not harbour any bla genes. Their resistomes contained from 4 to 15 genes and showed a great variety (Table 3).
3.2 Pseudomonas aeruginosa of human and porcine origin
Two CR P. aeruginosa strains were isolated from patients. Apparent prevalence was 1.1% (2/187) (95% CI: 0.3%-3.8%) (Table 1). The NEF23 strain belonged to ST938 and its resistome consisted of five genes, including blaOXA−396 and blaPAO, while the NEF156 strain belonged to ST395 and its resistome consisted of six genes, including blaOXA−488 and blaPAO (Table 4).
Table 4
Resistomes of P. aeruginosa of human and porcine origin
Antimicrobial class
|
Mechanism *
|
P. aeruginosa
|
|
|
ST-938
(NEF 23)
|
ST-938
(CRE 102)
|
ST-274
(CRE 98)
|
ST-782
(CRE 153)
|
ST-885
(CRE 295)
|
ST-395
(NEF 156)
|
Aminoglycosides, non-specific
|
phosphotransferase
|
aph(3')-IIb
|
aph(3')-IIb
|
aph(3')-IIb
|
aph(3')-IIb
|
aph(3')-IIb
|
aph(3')-IIb
|
β-lactams
|
Class D
|
|
|
|
blaOXA−50
|
blaOXA−50
|
|
blaOXA−396
|
blaOXA−396
|
|
|
|
|
|
|
bla OXA−486
|
|
|
|
|
|
|
|
|
blaOXA−488
|
PAO genes
|
blaPAO
|
blaPAO
|
blaPAO
|
blaPAO
|
blaPAO
|
blaPAO
|
Fluoroquinolones (Ciprofloxacin)
|
phosphorylase
|
|
|
crpP
|
crpP
|
crpP
|
crpP
|
Phosphonic acids
|
thiol transferase
|
fosA4
|
fosA4
|
fosA4
|
fosA4
|
fosA4
|
fosA4
|
Phenicols
|
acetyltransferase
|
catB7
|
catB7
|
catB7
|
catB7
|
catB7
|
catB7
|
N° of genes
|
|
5
|
5
|
6
|
6
|
6
|
6
|
(*) Comprehensive Antibiotic Data Base [34] |
In pigs, four CR P. aeruginosa strains were detected. Specifically, one ST938 isolate (CRE102) was identical to the NEF23 from a 83-old male patient hospitalized one month after pig slaughter for an episode of AKI due to sepsis, on previous chronic kidney disease. Moreover, based on SNP calling, the two genomes differed by only 41 SNPs suggesting the close genetic relationship among the porcine and the human isolates (Fig. 3, Table S3). No direct epidemiological link was found.
The remaining P. aeruginosa isolates of porcine origin belonged to ST274, ST782 and ST885 and all of them harboured the blaPAO gene. In addition, blaOXA−50 (ST782 and ST885) and blaOXA−486 (ST274) were identified together with other AMR genes (Tables 1 and 4).
3.3 Single Nucleotide Polymorphisms (SNPs) calling
Results on de novo assembly statistics are reported in table S1. SNP calling confirmed results of MLST typing with a higher level of discriminatory power (Figs. 2 and 3). Regarding K. pneumoniae, within the same ST types pairwise SNP differences were 41 (ST37), 51 (ST277), 55 (ST258), ranging from 29 and 89 (ST554) and 1071 (ST307). Isolate NEF123 (ST4525) showed 292 and 296 SNPs differences to NEF112 and NEF48 both belonging to ST37, demonstrating the close genetic relationship of the two ST-types (Fig. 2, table S2). Not surprisingly, between genomes of different ST-types the genetic distance increased with pairwise SNPs differences ranging from 905 to 32805.
Regarding Pseudomonas, CRE102 and NEF23 (ST938) of porcine and human origin respectively showed pairwise SNPs distances of 41 SNPs confirming their close genetic relationship (Fig. 3, table S3). Not surprisingly, comparing genomes of different ST-types (ST274, ST782, ST885, ST395) the genetic distance increased with pairwise SNPs differences ranging from 17359 to 20981.
3.4 Localisation of carbapenemase-encoding genes
Within the meropenem-resistant K. pneumoniae isolates, the carbapenemase-encoding genes blaKPC−2, blaKPC−3 or blaVIM−1were detected. To evaluate their mobilisation potential, their genetic environments were investigated. blaKPC−2 and blaKPC−3 were included in a complete Tn4401 (Figs. 4 and 5). Tn4401 was previously described as a transposon belonging to the Tn3 family ad involved in blaKPC mobilisation [25].
bla VIM−1 was located close to other AMR determinant genes (Fig. 6). Although the contig was too short to confirm the localisation of the gene in a class 1 integron, the sequence showed 99% identity in comparison to K. pneumoniae genomes (CP032178.1, MF344563.1) in which the sequence was included in a class 1 integron within a transposon (data not shown). Moreover, this gene was already detected within class 1 integrons as part of a Tn402 transposon [26].
Regarding P. aeruginosa, blaOXA and blaPAO showed genetic environments which did not show any specific sequence of mobilizable genetic elements (MGEs) confirming previous data (Figs. 7 and 8) [27].