Background
Materials and methods
Reference E. coli strains and clinical E. coli isolates
Determination of the hemolytic activity of E. coli isolates
In vitro experimental model to explore the prevalence of both toxins, the cytotoxic necrotizing factor 1 (Cnf 1) and colibactin
Bacterial growth and preparation of the crude cell lysate containing cytotoxic necrotizing factor-1 (Cnf 1)
Cytotoxicity of the crude cell lysate for the phenotypic detection of Cnf 1
Cytotoxicity of bacterial cell suspension for the phenotypic detection of colibactin
Giemsa staining of the treated mammalian cells
Detection of cytotoxic necrotizing factor-1 (cnf 1) and colibactin encoding genes clbA and clbQ by PCR
PCR amplification and gel electrophoresis detection of the target genes
Test gene | Primers used and their sequences | Expected PCR product size (bp) | Refs. | |
---|---|---|---|---|
Cytotoxic necrotizing factor-1 | cnf-1 forward | 5′-GGGGGAAGTACAGAAGAATTA-3′ | 1112 | [2] |
cnf-1 reverse | 5′-TTGCCGTCCACTCTCACCAGT-3′ | |||
Colibactin A (phosphopantetheinyl transferase) | clbA forward | 5′-CTAGATTATCCGTGGCGATTC-3′ | 1002 | |
clbA reverse | 5′-CAGATACACAGATACCATTCA -3′ | |||
Colibactin Q (thioesterase enzyme) | clbQ forward | 5′-CTTGTATAGTTACACAACTATTTC-3′ | 821 | |
clbQ reverse | 5′-TTATCCTGTTAGCTTTCGTTC-3′ |
Sequencing of the PCR products of cnf 1, clbA and clbQ genes
In vivo experimental model to explore the possible pathologic effect of cytotoxic necrotizing factor 1 and colibactin
Rat ascending UTI animal infection model
Histopathological examination of the autopsied kidneys and bladders
Bioinformatics analysis to explore the prevalence of colibactin and Cnf 1 among the bacterial species
Antimicrobial sensitivity testing
Results
Recovery, identification and hemolytic activity testing of the E. coli clinical isolates
Phenotypic detection of Cnf 1 and colibactin production
Isolate code | Phenotypic profile of test hemolytic E. coli isolates on cultured mammalian cells | Genotypic profile of test hemolytic E. coli isolates | ||||
---|---|---|---|---|---|---|
Intact bacterial cells (megalocytosis) | Crude cell lysate (multinucleation/cytotoxicity) | |||||
Caco2 cells | Hela cells | Vero cells |
cnf 1
|
clb A
|
clb Q
| |
1β | − | − | − | − | − | − |
3β | − | − | − | − | − | − |
4β | + | +* | +* | + | + | + |
5β | − | − | − | + | − | − |
6β | − | − | − | + | + | + |
10β | − | +* | +* | + | + | + |
12β | − | − | − | − | − | − |
13β | − | + | + | − | − | − |
19β | − | − | − | − | − | − |
27β | + | +* | +* | + | + | + |
28β | + | + * | − | + | + | + |
29β | + | +* | − | + | + | + |
32β | − | + | + | – | – | – |
55β | − | + | + | – | – | – |
70β | + | + * | +* | + | + | + |
85β | − | − | − | − | − | − |
88β | − | − | − | − | − | − |
99β | + | + | + | − | + | + |
100β | − | + | + | − | − | − |
108β | − | + | + | − | − | − |
114β | + | +* | +* | + | + | + |
E. coli strain J96 | + | +* | +* | + | + | + |
E. coli strain 28C | + | +* | +* | + | + | + |
Percent out of the hemolytic isolates | 7 (33.33%) | 13 (61.9%) | 10 (47.6%) | |||
cnf-1+ clb A+ clb Q+ clb M+ hly+ | 8/21 (38.1%) | |||||
clb A
+
clb Q
+
clb M
+
hly
+
only
| 1/21 (4.7%) | |||||
cnf-1+ hly+ only | 1/21 (4.7%) |
Detection of cnf 1, clb A and clb Q encoding genes among the test isolates
Histopathology of the infected kidneys and bladders
Pathological changes | Test isolatesa | |||
---|---|---|---|---|
70β | 6β | 13β | 67 | |
Kidneys | ||||
Interstitial blood vessels | ||||
Degree of congestion | ++++ | ++ | + | + |
Inflammatory cells infiltrations | ||||
Degree of infiltration | ++++ | ++ | + | + |
Renal tubular epithelium | ||||
Edema/swelling | ++++ | ++ | + | + |
Vacuolar degeneration | ++++ | ++ | + | + |
Necrosis | ++++ | ++ | + | + |
Nuclear pyknosis | ++++ | + | − | − |
Thickening of bowman’s capsule | ++++ | − | − | − |
Renal glomeruli | ||||
Congestion of capillaries | ++++ | ++ | − | + |
Edema of glomerular tuft | ++++ | ++ | − | + |
Necrosis of glomerular tuft | ++++ | ++ | − | + |
Hyalinization of the glomerular tuft | ++++ | ++ | − | + |
Bladder | ||||
Lamina propria | ||||
Edema | ++++ | ++ | − | − |
Inflammatory cells infiltration | ++++ | ++ | + | + |
Transitional epithelium | ||||
Hyperplastic changes | ++++ | ++ | − | − |
Necrosis | ++++ | ++ | − | − |
Desquamation | ++++ | − | − | − |
Vacuolar degeneration | ++++ | ++ | + | + |
Bioinformatics analyses results
Analyses of cnf 1, clbA and clbQ isolated genes sequences
Analyses of the 54 kb colibactin pks genomic island and its encoded proteins, Cnf 1 and HlyA
Taxonomy | Number of hits | Number of Organisms | Relative percentage % |
---|---|---|---|
Bacteria | 100 | 28 | 100 |
Enterobacterales | 97 | 27 | 96.43 |
Enterobacteriaceae | 96 | 26 | 92.86 |
Escherichia | 41 | 15 | 53.57 |
Escherichia coli | 26 | 15 | 53.57 |
Citrobacter | 5 | 2 | 7.14 |
Citrobacter koseri | 4 | 2 | 7.14 |
Klebsiella | 48 | 8 | 28.57 |
Klebsiella pneumonia | 29 | 5 | 17.86 |
Klebsiella aerogenes | 8 | 2 | 7.14 |
Klebsiella variicola | 1 | 1 | 3.57 |
Enterobacter | 2 | 1 | 3.57 |
Enterobacter cloacae | 2 | 1 | 3.57 |
Obesumbacterium proteus | 1 | 1 | 3.57 |
Uncultured bacterium | 3 | 1 | 3.57 |
Taxonomy | Number of hits | Number of organisms | Relative percentage % |
---|---|---|---|
Bacteria | 540 | 185 | 100 |
Gammaproteobacteria | 538 | 184 | 99.46 |
Enterobacterales | 533 | 181 | 97.84 |
Enterobacteriaceae | 1 | 179 | 96.76 |
Escherichia | 1 | 175 | 94.59 |
Escherichia coli | 342 | 174 | 94.05 |
Shigella | 3 | 2 | 1.08 |
Salmonella enterica | 2 | 1 | 0.54 |
Yersinia pseudotuberculosis complex | 9 | 2 | 1.08 |
Photobacterium | 3 | 2 | 1.08 |
Moritella viscosa | 2 | 1 | 0.54 |
Carnobacterium maltaromaticum | 2 | 1 | 0.54 |
Bacterial species | Tested genes | ||||||||||||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
Colibactin PKS proteins (CLB) | Others | ||||||||||||||||||||
A | B | C | D | E | F | G | H | I | J | K | L | M | N | O | P | Q | R | ClbS | Cnf 1 | HlyA | |
Escherichia coli
| + | + | + | + | + | + | + | + | + | + | + | + | + | + | + | + | + | + | + | + | + |
Escherichia coli Nissle 1917 | − | − | − | − | − | − | − | − | − | − | − | + | + | + | − | − | − | − | − | − | − |
Klebsiella pneumonia
| + | + | + | + | + | + | + | + | + | + | + | + | + | + | + | + | + | + | + | − | − |
Klebsiella aerogenes
| + | + | + | + | + | + | + | + | + | + | + | + | + | + | + | − | − | + | − | − | − |
Klebsiella oxytoca
| + | + | − | − | − | − | − | − | − | − | + | − | − | + | + | + | − | − | + | − | − |
Erwinia oleae
| + | + | + | + | + | + | + | + | + | + | + | + | + | + | + | + | + | + | + | − | − |
Citrobacter koseri
| − | + | − | − | + | − | − | + | + | + | + | − | − | + | − | + | − | + | − | − | − |
Citrobacter braakii
| − | − | − | − | − | − | − | − | − | − | − | − | − | − | − | − | − | − | − | − | + |
Enterobacter aerogenes
| − | + | − | − | − | − | − | − | − | − | − | − | − | − | − | − | − | − | − | − | − |
Enterobacter hormaechei
| − | − | − | − | − | − | − | − | − | − | − | − | − | − | − | − | − | − | − | − | + |
Acinetobacter baumannii
| − | − | − | − | − | − | − | − | − | − | − | − | − | − | − | − | − | + | − | − | − |
Serratia marcescens
| − | − | + | + | + | + | + | + | + | + | − | + | + | + | + | + | + | + | + | − | − |
Frischella perrara
| − | − | − | + | + | + | − | − | + | − | − | + | − | − | − | − | + | − | − | − | − |
Gilliamella apicola
| − | − | − | + | + | + | − | − | + | − | − | + | − | − | − | − | − | − | − | − | − |
Shigella sonnei
| − | − | − | − | − | − | − | − | − | − | − | − | − | − | − | − | − | − | + | − | − |
Shigella boydii
| − | − | − | − | − | − | − | − | − | − | − | − | − | − | − | − | − | − | − | + | − |
Salmonella enterica
| − | − | − | − | − | − | − | − | − | − | − | − | − | − | − | − | − | − | − | + | − |
Mixta theicola
| − | − | − | − | − | − | − | − | − | − | − | − | − | − | − | − | − | − | + | − | − |
Photobacterium damselae
| − | − | − | − | − | − | − | − | − | − | − | − | − | − | − | − | − | − | − | + | − |
Carnobacterium maltaromaticum
| − | − | − | − | − | − | − | − | − | − | − | − | − | − | − | − | − | − | − | + | − |
Moritella viscosa
| − | − | − | − | − | − | − | − | − | − | − | − | − | − | − | − | − | − | − | + | − |
Yersinia pseudotuberculosis
| − | − | − | − | − | − | − | − | − | − | − | − | − | − | − | − | − | − | − | + | − |
Morganella morganii
| − | − | − | − | − | − | − | − | − | − | − | − | − | − | − | − | − | − | − | − | + |
Vibrio parahaemolyticus
| − | − | − | − | − | − | − | − | − | − | − | − | − | − | − | − | − | − | − | − | + |
Proteus columbae
| − | − | − | − | − | − | − | − | − | − | − | − | − | − | − | − | − | − | − | − | + |
Proteus penneri
| − | − | − | − | − | − | − | − | − | − | − | − | − | − | − | − | − | − | − | − | + |
Distribution of colibactin pks genomic island and cytotoxic necrotizing factor 1 among the bacterial species
Relatedness and function of colibactin pks proteins among different bacterial species
Prevalence of different pks encoded proteins, Cnf 1 and HlyA among different bacterial species
Antimicrobial sensitivity of E. coli test isolates
Isolate code | Resistance profile | Genetic pattern | ||||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
SXT 25 | CRO 30 | AMC 30 | CAZ 30 | LEVO 30 | CIP 5 | OFL 5 | GAT 5 | F 300 | NA 30 | DO 30 | cnf-1 |
clb A
|
clb Q
| |
ATCC 25299 | S | S | S | S | S | S | S | S | S | S | S | + | − | − |
Hemolytic toxin harboring E. coli isolates (hly+cnf1+clbA+clbQ+) | ||||||||||||||
4β | R | R | I | R | S | S | S | S | S | S | S | + | + | + |
5β | R | R | R | R | R | R | R | R | S | R | R | + | − | − |
6β | S | S | S | S | S | S | S | S | S | S | S | + | + | + |
10β | R | S | R | S | R | R | R | R | S | R | R | + | + | + |
27β | S | R | S | I | S | S | S | S | S | S | S | + | + | + |
28β | R | S | S | R | S | S | S | S | S | R | R | + | + | + |
29β | S | S | S | S | S | S | S | S | S | S | S | + | + | + |
70β | S | S | S | R | S | S | S | S | S | S | S | + | + | + |
99β | R | S | S | I | S | S | S | S | S | S | S | − | + | + |
114β | R | R | S | S | S | S | S | S | S | S | S | + | + | + |
Percent sensitive | 40% | 60% | 70% | 40% | 80% | 80% | 80% | 80% | 100% | 70% | 70% | |||
Percent intermediate | 0 | 0 | 10% | 20% | 0 | 0 | 0 | 0 | 0 | 0 | 0 | |||
Non hemolytic non-toxin harboring E. coli isolates (hly−cnf1−clbA−clbQ−) | ||||||||||||||
20 | R | S | I | R | S | S | S | S | S | S | R | − | − | − |
45 | R | R | R | R | I | I | S | S | S | S | R | − | − | − |
52 | R | R | R | R | R | R | I | I | S | S | R | − | − | − |
58 | R | R | R | R | R | R | R | R | S | S | R | − | − | − |
63 | R | R | R | R | R | R | R | R | S | S | R | − | − | − |
67 | R | R | R | R | R | R | R | R | S | I | R | − | − | − |
69 | R | R | R | R | R | R | R | R | S | I | R | − | − | − |
74 | R | R | R | R | R | R | R | R | S | R | R | − | − | − |
93 | R | S | S | R | S | S | S | S | S | S | S | − | − | − |
120 | R | R | R | R | R | R | R | R | R | I | R | − | − | − |
Percent resistance | 100% | 80% | 80% | 100% | 70% | 70% | 60% | 60% | 10% | 10% | 100% | |||
Percent intermediate | 0% | 0% | 10% | 0% | 10% | 10% | 10% | 10% | 0% | 30% | 0% |