Isolation and identification
In this work, four Citrobacter spp. isolates resistant to cephalosporins were cultivated by selective medium plates. Among these strains, isolate FF141 was identified as Citrobacter cronae, isolate FF371, isolate FF414, and isolate FF423 were identified as Citrobacter braakii. Of these, three C. braakii isolates were further confirmed to be ESBL-producer (Table 1).
Table 1
Isolate
|
TZP
|
CTX
|
CAZ
|
CPO
|
IPM
|
MEM
|
AMK
|
ATM
|
CIP
|
CHL
|
FLR
|
GEN
|
TOB
|
FOS
|
TGC
|
COL
|
FF141
|
64
|
32
|
64
|
1
|
0.25
|
0.0075
|
8
|
64
|
0.25
|
64
|
16
|
4
|
1
|
0.25
|
0.25
|
2
|
FF371
|
128
|
> 128
|
64
|
128
|
0.25
|
0.003
|
32
|
128
|
16
|
128
|
128
|
> 128
|
64
|
256
|
0.25
|
2
|
FF414
|
2
|
> 128
|
64
|
128
|
0.25
|
0.003
|
16
|
128
|
16
|
128
|
128
|
> 128
|
64
|
256
|
0.25
|
2
|
FF423
|
2
|
> 128
|
64
|
> 128
|
0.25
|
0.003
|
16
|
128
|
16
|
128
|
> 128
|
> 128
|
64
|
256
|
0.25
|
2
|
TZP: Piperacillin/Tazobactam; CTX: Cefotaxime; CAZ: Ceftazidime; CPO: Cefpirome; IMP: Imipenem; MEM: Meropenem; AMK: Amikacin; ATM: Aztreonam; CIP: Ciprofloxacin; CHL: Chloramphenicol; FLR: Florfenicol; GEN: Gentamicin; TOB: Tobramycin; FOS: Fosfomycin; TGC: Tigecycline; COL: Colistin |
Assessment of antibiotic susceptibility and MLST analysis
Four Citrobacter isolates are all MDR with resistance to multiple antimicrobials (Table 1). The total resistance rate was observed for cefotaxime, ceftazidime, aztreonam, and chloramphenicol (100%). All the isolates were susceptibility to. Imipenem, meropenem, amikacin, tigecycline, and colistin. Interestingly, only FF371 was resistant to piperacillin-tazobactam. Among these Citrobacter isolates, we found two sequence types (STs), which were ST350 (FF371, FF414, and FF423) and ST370 (FF141).
Resistant and virulence determinants of Citrobacter isolates
The acquired resistance genes detected in four Citrobacter isolates are summarized in Fig. 1A. The following genes were identified in three C. braakii isolates: the phenicol resistance gene floR; the trimethoprim resistance gene dfrA17; the tetracycline resistance gene tet(A); the disinfectant resistance gene qaeE; the quinolone resistance gene qnrS1; the fluoroquinolone and aminoglycoside resistance gene aac(6')-Ib-cr, the aminoglycoside resistance genes aph(6)-Id, aac(6')-Ib3, aph(3'')-Ib, aac(3)-IId, and aadA5; the macrolide resistance gene mph(A); the β-lactam resistance genes blaCTX−M−55, blaCMY−82, and blaOXA−1; the rifampicin resistance gene ARR-3; sulphonamide resistance genes sul1, and sul2. Of note, C. braakii FF414 and FF423 encoded the fosfomycin resistance gene fosA3, while C. cronae FF141 and C. braakii FF371 were negative. Among four isolates, FF141 encoded fewer resistance genes than other isolates, which carried blaCMY−98, qnrB34, tet(A), dfrA12, and aadA2. Plasmids of incompatibility (Inc) group pKPC-CAV1321, was identified in FF141, and IncR was detected in other Citrobacter braakii isolates. The antimicrobial genes dfrA12, tet(A), and aadA2 genes were co-harbored by IncR plasmid in three C. braakii isolates.
Virulence gene analysis showed that the presence of genes encoding efflux pump protein (arcB), the transport of siderophores (fepC), enterobactin (entB and entE), outer membrane protein A (ompA), extracellular nucleation factors (csgB, csgD, csgE, and csgF), and type VI secretion system-related proteins (hcp/tssD) in all isolates (Fig. 1B). Moreover, three C. braakii isolates also carried genes that involved polymer synthesis (tviB-tviE), and cell surface localization of the CPS (vexA-vexE).
Comparative genomics analysis of Citrobacter isolates
Roary matrix-based gene sequence analysis generated 60,923 total genes and a large core-genome of 1,578 gene clusters of 277 whole genomes. The whole-genome phylogeny (Fig. 2) revealed a population structure. Genetic diversity was observed in our bacterial collection, which clustered into five main clades. Interestingly, C. braakii isolates have close relatedness with a clinical isolate from Viet Nam and an environmental strain, while C. cronae FF141showed a high similarity with a clinical isolate from Nigeria and a clinical isolate from India (Fig. 2 & Table S1).