Bacterial isolates and screening for ESBLs phenotype
Two hundred and eleven nonduplicated K. pneumoniae isolates were collected from fresh feces of captive giant pandas from 2018 to 2019 at the Chengdu Research Base of Giant Panda Breeding (Panda Base) in Sichuan, China.. These isolates were identified as K. pneumoniae by Gram staining, 16s rDNA, and bacterial biochemical identification. All of the isolates were tested for ESBLs production by the CLSI-recommended confirmatory double-disc combination test (CLSI, 2019). All of the isolates were screened for ESBLs production using cefotaxime (CTX) and ceftazidime (CAZ) alone and in combination with clavulanic acid according to the double-disk synergy test method (DDST) (CLSI, 2016). Phenotypic presence of ESBLs in the isolates was determined by detecting diameter enhancement of the inhibition zone of the clavulanate disk and corresponding β-lactam antimicrobial disk. If the enhancement value was > 5 mm, the isolate was presumed to be an ESBLs producer(Yang et al., 2014a; Yang et al., 2014b).
Antimicrobial susceptibility testing
Antimicrobial susceptibility tests of three ESBLs-producing isolates and seven non-ESBLs-producing isolates were evaluated using the disk diffusion method to Penicillin 1 μg (PEN), Piperacillin 100 μg (PRL), Ampicillin 10 μg (AMP), Oxacillin 1 μg (OX), Amoxicillin 20 μg (AML), Moxalactam 30 μg (MOX), Ceftazidime 30 μg (CAZ), Cefixime 5 μg (CFM), Cefmatazole (CMZ), Cefepime 30 μg (FEP), cephalosporine (CTX), Cephalexin 30 μg (CL), Cefazolin 30 μg (KZ), Ceftriaxone 30 μg (CRO), Cefoxitin 30 μg (FOX), Piperacillin/Tazobactam 100/10 μg (TZP), Cefuroxime 30 μg (CXM), Cefaclor (CEC), Ampicillin/Sulbactam 10/10 μg (SAM), Cefoperazone 75 μg (CFP), Ceftizoxime 30 μg (ZOX), Aztreonam 30 μg (ATM), Meropenem 10 μg (MEM), Imipenem 10 μg (IPM), Kanamycin 30 μg (K), Streptomycin 10 μg (S), Ofloxacin 5 μg (OFX), Norfloxacin 10 μg (NOR), Ciprofloxacin (CIP); Gatifloxacin 5 μg (GTX), Chloramphenicol 30 μg (C), Vancomycin 30 μg (VA), Azithromycin 15 μg (AZM), Doxycycline (TE), Minocycline 30 μg (MH), Compound Sulfamethoxazole (SMZ) 23.75/1.25 μg, and Trimethoprim 5 μg (TMP) (Oxoid, Basingstoke, United Kingdom). The results were interpreted according to the Clinical and Laboratory Standards Institute (CLSI) breakpoints. Escherichia coli ATCC 25922 was used as a control for antimicrobial susceptibility testing.
Multi-locus Sequence Typing (MLST)
MLST was performed on three ESBLs-producing isolates and 10 non-ESBLs producing isolates by amplifying the seven standard housekeeping loci gapA, infB, mdh, pgi, phoE, rpoB, and tonB as described previously (Diancourt et al. 2005). Sequence types (STs) were assigned using the online database on the Pasteur Institute MLST website (http://bigsdb.pasteur.fr/klebsiella/klebsiella.html).
Molecular investigations of antimicrobial resistance
All three ESBLs-producing isolates and seven non-ESBLs-producing isolates were screened by PCR for the presence of ESBL genes (blaCTM, blaCTM-M-1, blaCTM-M-2, blaCTM-M-8, blaCTM-M-9, blaCTM-M-25, blaTEM, blaSHV, blaGES, blaPER, blaVEB, blaOXA-1, and blaOXA-2), Transposons (IS26, intI1, traA, IS26, intI1, traA, trbC, tnpU, and IS903), Aminoglycoside genes (aac(3)-I, aac(3)-II, aac(6')-I, aac(2'')-I, aac(6')-II, tnp513, ant(3'')-I, rmtA, rmtB, rmtC, rmtD, armA, aadA5, and npmA), and Quinolones genes (qnrA, aac(6')-Ib, qnrS, and qnrB). The PCR amplicons of blaCTX-M-1, blaTEM, and blaSHE from three ESBLs-producing K. pneumoniae isolates were sent to Shanghai Sangong Biotech (Shanghai, China), and sequence analysis was performed using the National Center for Biotechnology Information (NCBI) website with the BLASTn tool.
Tab. 1 Primers used for detection and sequencing of target genes in Klebsiella pneumoniae isolates.
Target
|
Forward primer (5'-3′)
|
Reverse primer (5′-3′)
|
Product size (bp)
|
Annealing temperature (°C)
|
β-Lactamases and ESBLs-encoding genes
|
CTX-M
|
ATGTGCAGYACCAGTAARGT
|
TGGGTRAARTARGTSACCAGA
|
593
|
52
|
CTX-M-1
|
AAAAATCACTGCGCCAGTTC
|
AGCTTATTCATCGCCACGTT
|
415
|
52
|
CTX-M-2
|
CGACGCTACCCCTGCTATT
|
CCAGCGTCAGATTTTTCAGG
|
552
|
52
|
CTM-M-8
|
TCGCGTTAAGCGGATGATGC
|
AACCCACGATGTGGGTAGC
|
666
|
52
|
CTM-M-9
|
ATGGTGACAAAGAGAGTGCA
|
CCCTTCGGCGATGATTCTC
|
869
|
52
|
CTX-M-25
|
GCACGATGACATTCGGG
|
AACCCACGATGTGGGTAGC
|
327
|
52
|
TEM
|
CATTTCCGTGTCGCCCTTATTC
|
CGTTCATCCATAGTTGCCTGAC
|
800
|
52
|
SHV
|
AGCCGCTTGAGCAAATTAAAC
|
ATCCCGCAGATAAATCACCAC
|
713
|
52
|
GES
|
AGTCGGCTAGACCGGAAAG
|
TTTGTCCGTGCTCAGGAT
|
399
|
52
|
PER
|
GCTCCGATAATGAAAGCGT
|
TTCGGCTTGACTCGGCTGA
|
520
|
52
|
VEB
|
CATTTCCCGATGCAAAGCGT
|
CGAAGTTTCTTTGGACTCTG
|
648
|
52
|
Transposons gene
|
IS26
|
ATGAACCCATTCAAAGGCCGGCAT
|
TATGCAGCTTTGCTGTTACGACGG
|
387
|
55
|
intI1
|
CCGAGGATGCGAACCACTTC
|
CCGCCACTGCGCCGTTACCA
|
373
|
53
|
traA
|
CTGCTGTGGCGGCGTTCTTC
|
AGTAACCGGCGACCGACATACC
|
246
|
53
|
trbC
|
CGGYATWCCGSCSACRCTGCG
|
GCCACCTGYSBGCAGTCMCC
|
255
|
53
|
tnpU
|
GCAAGGAGAAGCGACGAGTGTG
|
TACATGGCGGTCTCGGCTATCG
|
367
|
55
|
IS903
|
GCAATACGCACGCTTTCAGGC
|
ACTGCACGGTTACGGTCTGCA
|
521
|
55
|
Aminoglycoside gene
|
aac(3)-I
|
ACCTACTCCCAACATCAGCC
|
ATATAGATCTCACTACGCGC
|
169
|
55
|
aac(3)-II
|
ACTGTGATGGGATACGCGTC
|
CTCCGTCAGCGTTTCAGCTA
|
237
|
52
|
aac(6')-I
|
ATGAGTGGCTAAATCGATC
|
CCCGCTTTCTCGTAGCA
|
394
|
55
|
aac(2'')-I
|
GCGAAATCTGCCGCTCTGG
|
GCTGTTACAACGGACTGGCCG
|
319
|
55
|
aac(6')-II
|
TTCATGTCCGCGAGCACCCC
|
ACTCTTCCGCCATCGCTCTGGG
|
178
|
54
|
tnp513
|
CGAGTCAACCTCACACGCTTCC
|
TGCTCAATGACCTTCGGATCTTCG
|
269
|
55
|
ant(3'')-I
|
TGATTTGCTGGTTACGGTGAC
|
CGCTATGTTCTCTTGCTTTTG
|
284
|
55
|
rmtA
|
CTAGCGTCCATCCTTTCCTC
|
TTTGCTTCCATGCCCTTGCC
|
634
|
55
|
rmtB
|
CCCAAACAGACCGTAGAGGC
|
CTCAAACTCGGCGGGCAAGC
|
565
|
55
|
rmtC
|
CGAAGAAGTAACAGCCAAAG
|
ATCCCAACATCTCTCCCACT
|
800
|
55
|
rmtD
|
CGGCACGCGATTGGGAAGC
|
CGGAAACGATGCGACGAT
|
750
|
55
|
armA
|
AGGTTGTTTCCATTTCTGAG
|
TCTCTTCCATTCCCTTCTCC
|
590
|
55
|
aadA5
|
ATGGGTGAATTYTTYCCTGCACAA
|
TCAACGCAAGATTCTCTCATTCGT
|
789
|
55
|
npmA
|
CGGGATCCAAGCACTTTCATACTGACG
|
CGGAATTCCAATTTTGTTCTTATTAGC
|
334
|
54
|
Quinolones gene
|
qnrA
|
AGAGGATTTCTCACGCCAGG
|
TGCCAGGCACAGATCTTGAC
|
578
|
53
|
aac(6')-Ib
|
TGACCTTGCGATGCTCTATG
|
TTAGGCATCACTGCGTGTT
|
497
|
53
|
qnrS
|
GCAAGTTCATTGAACAGGGT
|
TCTAAACCGTCGAGTTCGGCG
|
191
|
53
|
qnrB
|
GGMATHGAAATTCGCCACTG
|
TTTGCYGYYCGCCAGTCGAA
|
263
|
55
|