Clinical epidemiology
Twenty-five E. coli were isolated from haemoculture in the study period. The overall incidence density was 0.48 per 1,000 patient days and the incidence was 3.31 per 1,000 hospital admission. Six from 25 isolates (24%) proved to be resistant to third-generation cephalosporins. Phenotypic detection tests showed that all of them were ESBL producers.
Antimicrobial susceptibility
The isolates were resistant to ceftriaxone, cefotaxime, and ciprofloxacin but remained susceptible to colistin, fosfomycin, ceftazidime-avibactam, and carbapenems (Table 1). The Ec1, Ec2, and Ec3 showed resistance to ceftazidime, gentamicin, and tobramycin too. Moreover, the Ec3, Ec5 isolates showed resistance to tigecycline and Ec6 to amikacin.
Table 1
Antimicrobial susceptibility of the ESBL-producing E. coli isolates (MIC (mg/L)
Isolates | CIP | CRO | CAZ | CTX | CZA | GM | AK | TM | TGC | FOS | COL | ETP | MEM | IMI |
Ec1 | 256 | 256 | 32 | ≥ 256 | 1 | 16 | 2 | 8 | 0.25 | 0,25 | 0.125 | 0.125 | 0.064 | 0.125 |
Ec2 | 64 | 256 | 32 | ≥ 256 | 0,5 | 64 | 4 | 32 | 0.5 | 1 | 0.25 | 0.125 | 0.125 | 0.125 |
Ec3 | 64 | 256 | 32 | ≥ 256 | 1 | 64 | 2 | 8 | 1 | 4 | 0.125 | 0.125 | 0.064 | 0.125 |
Ec4 | 64 | 256 | 4 | 64 | 0.5 | 2 | 4 | 1 | 0.25 | 2 | 0.25 | 0.008 | 0.032 | 0.125 |
Ec5 | 64 | 256 | 4 | 64 | 0.25 | 1 | 4 | 2 | 1 | 4 | 0.25 | 0.008 | 0.032 | 0.125 |
Ec6 | 16 | 256 | 4 | ≥ 256 | 0.125 | 0.5 | 16 | 1 | 0.125 | 1 | 0.064 | 0.016 | 0.032 | 0.125 |
The used antibiotics: ciprofloxacin (CIP), ceftriaxone (CRO), ceftazidime (CAZ), cefotaxime (CTX), ceftazidime/avibactam (CZA), gentamicin (GM), amikacin (AK), tobramycin (TM), tigecycline (TGC), fosfomycin (FOS), colistin (COL), ertapenem (ETP), meropenem (MEM), imipenem (IMI). The Ec1, Ec3, Ec3 correspond to ST131 C2/H30Rx isolates, Ec4, Ec5 correspond to ST131 C1-M27 isolates and Ec6, correspond to ST1193.
Molecular characterization
The six ESBL-producing E. coli isolates could be assigned to two different sequence types (ST) by MLST. Of the six isolates, five (Ec1-Ec5) belonged to the globally dominant ST131 clone (O25:H4 serotype), which comprised two distinguished groups of strains with different characteristics. The first group contains two isolates with blaCTX−M−15 and one isolate with blaCTX−M−15 and blaTEM−1B while the two other isolates harbored blaCTX−M−27 ESBL-genes. The remained one isolate belonged to the ST1193 with blaCTX−M−27. Analysis of genetic relatedness between the strains was performed by cgMLST. The five ST131 isolates were compared to one C2/H30Rx (JJ2434) and two C1-M27 (H105, EC81009) international ST131 ESBL-producing E. coli isolates, to determine the subclade-specific relatedness of the isolates within the ST131 (Fig. 1). The international isolate JJ2434 and the three CTX-M-15-producing isolates (named the first group below) belonged ST131 C2/H30Rx subclade. The H105 and EC81009 isolates with the two CTX-M-27-producing isolates from this study belonged to the ST131 C1-M27 subclade. The Ec4 and Ec5 harboured exclusively the C1-M27 clade-specific M27PP1 prophage-like genomic island.
According to cgMLST (Fig. 2), all the three Hungarian ST131 E. coli isolates producing blaCTX−M−15 formed a close cluster (≤ 6 allele differences), while the C1-M27 isolates producing blaCTX−M−27 differed at 35 alleles from each other. The ST1193 isolates showed ≥ 2209 allele differences from any others.
The isolates of each group belonged to ST131 harboured very similar resistance gene sets. The characteristics of resistome and plasmid replicon types of the isolates are shown in Table 2. Although, there are some differences between the two ST131 subclades in terms of resistance genes like the qnrB (FQ R) and aac(3)-IIa (aminoglycoside R) which were present only in C2/H30Rx isolates. The ST1193 isolate harboured L416F mutation in parE, and also had no qnrB and aac(3)-IIa genes. Three plasmid families were detected: IncF, IncQ, and Col-like. The IncF replicons were present in every isolate, but only the C1-M27 ST131 isolates (EC4, EC5) carried the Group1 replicons (IncFII_1, IncFIA_2, IncFIB_20). The coverage of the Group1 (F1:A1:B20) reference plasmid by the reads of Ec4 and Ec5 was 96%, and 72%, respectively (Table 3). None of the C2/H30Rx isolates (EC1-EC3) co-harboured the two reference replicons of Group2 (IncFII_2, IncFIA_1). The Ec2 did not carry any IncFIA replicon but carried the IncFII_2 replicon. The coverage of the Group2 (F2:A1:B-) reference plasmid by the reads of Ec1, Ec2, and Ec3 was 70%, 47%, and 70%, respectively. The reads of Ec1 and Ec3 covered pU1 plasmid to a higher breadth compared to Group2 reference (≥ 91% vs 70%). The ST1193 isolates harbored IncFII_4-like, IncF1A_1, IncFIB_10, and the repCol156, and repCol(BS512) replicons. The Col(pHAD28), Col156, and Col8282 replicons were at least present in one of the ST131 isolates.
Table 2
Resistome and plasmid replicons of the ESBL-producing ExPEC strains
Isolate | Ec1 | Ec2 | Ec3 | Ec4 | Ec5 | Ec6 |
β-lactam | blaCTX-M-15 | + | + | + | | | |
blaCTX-M-27 | | | | + | + | + |
blaTEM-1B | | + | | | | |
Fluoroquinolone | gyrA* (S83L) | + | + | + | + | + | + |
gyrA* (D87N) | + | + | + | + | + | + |
parC* (S80I) | + | + | + | + | + | + |
parC* (E84V) | + | + | + | + | + | |
parE* (I529L) | + | + | + | + | + | |
parE* (L416F) | | | | | | + |
qnrB19‡ | + | + | + | | | |
MLS | mph(A) | + | | + | + | + | + |
mdf(A) | + | + | + | + | + | + |
Sulphonamide | sul1 | + | + | + | + | + | + |
sul2 | + | + | + | + | + | + |
Tetracycline | tet(A) | + | + | + | + | + | + |
Trimethoprim | dfrA17 | + | + | + | + | + | + |
dfrA1 | | + | | | | |
Aminoglycoside | ant(3'')-Ia | | + | + | | | |
aadA5 | + | | + | + | + | + |
aph(3'')-Ib | + | + | + | + | + | + |
aph(6)-Id | + | + | + | + | + | + |
aac(3)-IIa | + | + | + | | | |
Plasmid replicon type | IncFIB IncFIA IncFII Col156 Col(pHAD28) | IncFIB IncFII IncQ1 Col8282 Col(pHAD28) | IncFIB IncFIA IncFII Col156 Col(pHAD28) | IncFIB IncFIA IncFII Col156 Col8282 Col(pHAD28 | IncFIB IncFIA IncFII | IncFIB IncFIA IncFII Col156 Col(BS512) |
Acquired antibiotic resistance genes and mutations of the isolates. The Ec1, Ec2, Ec3 correspond to ST131 C2/H30Rx isolates, Ec4, Ec5 correspond to ST131 C1-M27 isolates and Ec6, correspond to ST1193. The + indicates the presence of a gene, * the mutations in quinolone resistance determining region, and ‡ the plasmid-mediated quinolone resistance.
Table 3
The coverage and identity of the ST131 isolates and the references plasmids
Isolates | ST131 subclade | Reference plasmid sequence | Coverage | Identity |
Ec1 | C2/H30Rx | Group2 | 70% | 97.89% |
pU1 | 91% | 99.29% |
Ec2 | C2/H30Rx | Group2 | 47% | 99.20% |
pU1 | 52% | 96.67% |
Ec3 | C2/H30Rx | Group2 | 70% | 97.21% |
pU1 | 92% | 99.87% |
Ec4 | C1-M27 | Group1 | 96% | 99.93% |
Ec5 | C1-M27 | Group1 | 72% | 99.83% |
Group1 plasmid refers as CTX-M-27-encoding IncF[F1:A2:B20] plasmids, Group2 plasmid as CTX-M-15-encoding IncF[F2:A1:B-] plasmids, and pU1 as IncF[F1:A1:B16] plasmid. The Ec1, Ec3, Ec3 correspond to ST131 C2/H30Rx isolates, Ec4, Ec5 correspond to ST131 C1-M27 isolates.