Study design, site and source of bacteria isolates
This was a cross sectional-laboratory-based study conducted at the Microbiology Laboratory and Molecular Biology Laboratory, College of Veterinary Medicine Animal Resources and Biosecurity (CoVAB) Makerere University. The study involved use of archived MDR Escherichia coli samples isolated between January and December, 2019 from clinical specimens in the Microbiology Laboratories of Mulago National Referral Hospital (MNRH), Mbale Regional Referral Hospital (MRRH), Mbarara Regional Referral Hospital (MBRRH) and Kampala International University Teaching Hospital (KIU-TH). The samples were transported in peptone water to the Microbiology Laboratory, CoVAB. Overnight cultures of E. coli were prepared by pipetting 1 ml of peptone water containing each isolate into 49 ml of Luria-Bertani (LB) broth. Glycerol stocks of each different isolate were made by adding 500 µl of the overnight LB culture to 500µL of 50% glycerol in a 2 ml screw top tube and mixed gently mix. The screw tubes were stored at -800C until further use.
Biochemical assays to confirm the identity of E. coli
To confirm the identity of each isolate, Microgen (Micro-biology International) kits for biochemical assays were employed using procedures described by the manufacturer (www.microgenbioproducts.com).
Screening for carbapenem susceptibility
This was achieved using the Kirby Bauer Disk Diffusion method and the results obtained were interpreted according to Clinical and Laboratory Standards Institute (CLSI) guidelines [19]. Ampicillin (AMP) 25 𝜇g, Amoxicillin/clavulanic acid (AMO) 20/10 𝜇g, trimethoprim-sulfamethoxazole (TMP/SMX) 1.25/23.75 𝜇g, Ciprofloxacin (CIP) 5 𝜇g, Cefuroxime (CXM) 30 µg, Temocillin (TEM) 30 µg, Piperacillin-tazobactum (TPZ) 110 µg, Cefoxitin (FOX) 30 µg, Cefipime (FEP) 30 µg, Ceftriaxone (CRO) 30 µg, Ceftazidime (CAZ) 30 µg, Cefotaxime (CTX) 30 µg, Ertapenem (ERT) 10 µg, Meropenem (MEM) 10 µg and Imipenem (IMI)10 µg (Oxoid United Kingdom) carbapenem antibiotics disks were used. The turbidity of overnight Escherichia coli broth was adjusted using peptone water to a standard uniform concentration of 0.5 McFarland. Each E. coli isolate was inoculated on Mueller Hinton agar (Oxoid, United Kingdom) plates. Three antibiotic discs were placed about 2.0 cm apart and from the edge of plates, then incubated at 37 °C for 24 hours. The diameter zones of growth inhibition were scored in millimeters. For quality control, E. coli ATCC 25922 was used as a susceptible strain and Klebsiella pneumoniae ATCC BAA-1705 as a positive control.
DNA extraction
Pure colonies of E. coli from different samples were selected and each sub-cultured in 5 ml of Luria-Bertani broth using sterile inoculating loop. The bacterial suspension was incubated in shaker incubator at 37ºC for 24hrs. Then, 1 ml of bacterial suspension was transferred into a 1.5 ml eppendorf tube, centrifuged at 10,000 rpm for 10 minutes. The supernatant was discarded and the pellet was re-suspended in 200 µl of Gram-negative bacteria lysis buffer provided in the Qiagen DNA extraction. Bacterial total genomic DNA was extracted following the Qiagen DNA extraction protocol and stored at -20oC until further use.
Molecular characterization of virulent genes and carbapenem resistance determinants
Molecular identification of carbapenem resistance determinants and virulent genes in Escherichia coli was carried out using multiplex PCR. Primers used for molecular characterization were obtained from Eurofins Genomics AT GmbH and PCR amplification was performed in a Bio-Rad PTC-200 Thermal Cycler (Bio-Rad, Hercules, CA, USA)
Multiplex PCR amplification of carbapenem resistance genes
The existence of carbapenem resistance genetic determinants was determined using primers targeting b𝑙𝑎VIM, 𝑏𝑙𝑎IMP, 𝑏𝑙𝑎KPC, 𝑏𝑙𝑎OXA-48, and 𝑏𝑙𝑎NDM that carbapenemase encoding genes, Table 1. For co-amplification of target genes, multiplex PCR was conducted by adapting methods used by Dallenne et al., [20]. Briefly, 2.5 µl of template DNA (100 ng/ µl) was added to 47.5 µl PCR mix containing 200 µM dNTPs (Biomatik, USA), 0.5 µM of each primer pair and 1X PCR Buffer (1.5 mM MgCl2, 10 mM Tris–HCl, pH 8.3/50 mM KCl) (Biomatik USA) and 1.2 µl of 1U Taq DNA Polymerase. Amplification was performed as follows; preliminary denaturation at 95 °C for 5 minutes; then denaturation at 95 °C for 30 seconds; annealing at 56 °C for 30 seconds and elongation at 72 °C for 1 minute; and a final elongation at 72 °C for 10 minutes. For quality assurance positive and negative control isolates were obtained as a kind donation from the Microbiology Laboratory, College of Health Science, Makerere University. Antibiotics susceptible DSMZ 9377 Klebsiella pneumoniae was used as a negative control for all genes. Klebsiella pneumonia Nr.8 for NDM-1, Klebsiella pneumoniae 714 for OXA-48, Klebsiella pneumoniae 211 (T) for KPC, P. aeruginosa for IMP (Positive control strains from the Institute of Microbiology, Giessen, Germany) and E. coli for the VIM gene, obtained from RESET research collaboration [21] were used as positive controls.
Table 1
Carbapenem resistance genes and respective primers
Gene
|
Primer sequence (51-31)
|
Band size (Bp)
|
Reference
|
BlaKPC
|
F-ATG TCA CTG TAT CGC CGT CT
R-TTT TCA GAG CCT TAC TGC CC
|
538
|
[20]
|
BlaIMP
|
F-TGA GCA AGT TAT CTG TAT TC
R-TTA GTT GCT TGG TTT TGA TG
|
139
|
[20]
|
BlaVIM
|
F-GAT GGT GTT TGG TCG CAT A
R-CGA ATG CGC AGC ACC AG
|
390
|
[20]
|
BlaNDM
|
F-GGT TTG GCG ATC TGG TTT TC
R-CGG AAT GGC TCA TCA CGA TC
|
822
|
[20]
|
BlaOXA-48
|
F-TTG GTG GCA TCG ATT ATC GG
R- GAG CAC TTC TTT TGT GAT GGC
|
281
|
[20]
|
Multiplex PCR components and conditions for E. coli pathotyping
Virulent genes eae for EPEC; stx for STEC/EHEC; est for TS-ETEC; elt for TL-ETEC; aggR for EAEC; ipaH for EIEC were amplified by multiplex PCR to characterize the different pathogenic bacteria using primers outlined in Table 2 [8]. Five E. coli strains INCQS 00181 (CDC 055 – EPEC), INCQS 00171 (CDC EDL – 933 – EHEC) and INCQS 00170 (CDC EDL – 1284 – EIEC) from Centre for disease control and prevention belonging to the five categories of pathogenic E. coli were used as control [22]. Multiplex PCR reaction was performed using [23] modified method to enable the concurrent amplification of all target genes. A final PCR volume of 50 µl containing 5 µl of 100 ng DNA sample, 25 µl of 1X PCR Buffer mixed with MgCl2 (1.5 mM), 1.2 µl of 1U Taq DNA Polymerase and dNTPs (200 µM) plus 0.5 µM each primer pair for DEC pathotypes. Sterile distilled deionized water was used to top up to 50 µl. The PCR was performed under the following condition; An initial denaturation at 95 °C for 5 minutes then 30 amplification cycles at 95 °C for 30 second, 50 °C for 30 second, 72 °C for 1 minute, and a final extension at 72 °C for 30 minutes
Table 2
Genes and their prime sequences for molecular typing of E. coli (adopted from Dias et al., 2012)
Gene
|
Primer Sequence (5’-3)
|
Size of amplicon (Bp)
|
Annealing Temp (0C)
|
Reference
|
eae
|
CCCGAATTCGGCACAAGCATAAGC
|
881
|
50
|
[24]
|
CCCGGATCCGTCTCGCCAGTATTCG
|
stx
|
GAGCGAAATAATTTATATGTG
|
518
|
50
|
[25]
|
TGATGATGGCAATTCAGTAT
|
est
|
ATTTTTMTTTCTGTATTRTCTT
|
190
|
50
|
[26]
|
CACCCGGTACARGCAGGATT
|
elt
|
GGCGACAGATTATACCGTGC
|
450
|
50
|
[26]
|
CGGTCTCTATATTCCCTGTT
|
paH
|
GTTCCTTGACCGCCTTTCCGATACCGTC
|
619
|
50
|
[27]
|
GCCGGTCAGCCACCCTCTGAGAGTAC
|
aggR
|
GTATACACAAAAGAAGGAAGC
|
254
|
50
|
[28]
|
ACAGAATCGTCAGCATCAGC
|
PCR amplification of PAI Markers
Seven different PAI markers designated as PAI I536, II536, IV536, ICFT073, IICFT073, IJ96 and IIJ96 have been previously characterized in UPEC [24], Table 3. Thus, the multiplex PCR used in the detection of PAI Markers, contained 2.5 µl of template DNA (100 ng/µl), 1U Taq DNA polymerase (Biomatik) in 1X PCR buffer (Biomatik), 200 µM of each dNTP, 2.5 mM MgCl2, and 0.5𝜇M of each primer, Table 3. The program consisted of initial denaturation at 94 °C for 5 min, followed by 30 cycles of 94 °C for 1 minute, 55 °C for 1 minute and 72 °C for 1minute, with a final extension step at 72 °C for 10 minutes [24]. The positive control used in the PCR was J96 O4:K6.
Table 3
Oligonucleotides used to amplify PAI markers harboring virulent genetic determinants
PAI markers
|
Primer sequence (5’-3’)
|
Amplicon size (bp)
|
Virulent factors expressed by genes harboured by PAI markers
|
Reference
|
PAI I536
|
TAA TGC CGG AGA TTC ATT GTC
|
1800
|
𝛼-Haemolysin, CS12 fimbriae, and F17-like fimbrial adhesin
|
[24]
|
AGG ATT TGT CTC AGG GCT TT
|
PAI II536
|
CAT GTC CAA AGC TCG AGC C
|
1000
|
𝛼-Haemolysin and P-related fimbriae
|
[24]
|
CTA CGT CAG GCT GGC TTT G
|
PAI IV536
|
AAG GAT TCG CTG TTA CCG GAC
|
300
|
Yersiniabactin siderophore system
|
[24]
|
TCG TCG GGC AGC GTT TCT TCT
|
PAI ICFT073
|
GGA CAT CCT GTT ACA GCG CGC A
|
930
|
𝛼-Haemolysin, P-fimbriae, and aerobactin
|
[24]
|
TCG CCA CCA ATC ACA GC GAA C
|
PAI IICFT073
|
ATG GAT GTT GTA TCG CG
|
400
|
P-fimbriae and iron-regulated genes
|
[24]
|
ACG AGC ATG TGG ATC TGC
|
PAI IJ96
|
TCG TGC TCA GGT CCG GAA TTT
|
400
|
𝛼-Haemolysin and P-fimbriae
|
[24]
|
TGG CAT CCC ACA TTA TCG
|
PAI IIJ96
|
GGA TCC ATG AAA ACA TGG TTA ATG GG
|
2300
|
𝛼-Haemolysin, Prs-fimbriae, and cytotoxic necrotizing factor 1
|
[24]
|
GAT ATT TTT GTT GCC ATT GGT TAC C
|
Phylogenetic Classification.
Phylogenetic classification exhibited that the E. coli strains belonged to four groups (A, B1, B2, or D) based on the presence of the chuA and yjaA genes and the DNA fragment (TSPE4.C2). Thus, a multiplex PCR was run to determine the phylogenetic classes of the E. coli strains using primers targeting chuA, yjaA and TSPE4.C2 DNA sequences, Table 4. The PCR amplification was conducted by adapting [13] methods. Briefly, the PCR contained 2.5 µl of template DNA, 1U Taq DNA polymerase (Biomatik, USA) in 1x PCR buffer (Biomatik), 200 µM dNTP, 2.5 mM MgCl2, and 0.8 𝜇M of each primer, Table 1. Amplification was conducted using the following PCR conditions; initial denaturation at 94 °C for 5 minutes, then 30 cycles performed at 94 °C for 5 seconds, 54 °C for 10 seconds, 72 °C for 30 second with a final extension step at 72 °C for 5 minutes. Phylogenic groups and subgroups were assigned depending on chuA, yjaA, and TspE4.C2 gene combinations [13, 14], Table 5.
Table 4
Primers used in phylogenetic analysis of E. coli
Gene
|
Primer sequence (5’-3’)
|
Amplicon size (bp)
|
Protein expressed
|
Reference
|
chuA
|
GAC GAA CCA ACG GTC AGG AT
|
279
|
Hemetransport in enterohemorrhagic O157:H7 E. coli
|
[13]
|
TGC CGC CAG TAC CAA AGA CA
|
yjaA
|
TGA AGT GTC AGG AGA CGC TG
|
211
|
Protein function unknown
|
[13]
|
ATG GAG AAT GCG TTC CTC AAC
|
TSPE4.C2
|
GAG TAA TGT CGG GGC ATT CA
|
152
|
Putative DNA fragment (TSPE4.C2) in E. coli
|
[13]
|
CGC GCC AAC AAA GTA TTA CG
|
Table 5
chuA, yjaA, and TspE4.C2 gene combinations for assigning of phylogenetic groups and subgroups of Escherichia coli
TSECP4C2
|
yjaA
|
ChuA
|
Phylogenetic group
|
Phylogenetic subgroup
|
Negative
|
Negative
|
Negative
|
A
|
A0
|
Negative
|
Positive
|
Negative
|
A
|
A1
|
Positive
|
Negative
|
Negative
|
B1
|
B1
|
Negative
|
Positive
|
Positive
|
B2
|
B22
|
Positive
|
Positive
|
Positive
|
B2
|
B23
|
Negative
|
Negative
|
Positive
|
D
|
D1
|
Positive
|
Negative
|
Positive
|
D
|
D2
|
Data analysis
Data analysis was done using the SPSS version 25 (SPSS Inc., Chicago, IL). Statistical differences were computed by chi-square and Spearman’s correlation. A p value ≤ 0.05 indicated substantial statistical difference.