Table I
Calculations of Biofilm classification values.
| Biofilm formation ability | Range | Negative control | ||
|---|---|---|---|---|
| Strong (S) | Moderate (M) | Weak (W) | ||
| BF = AB– CW | ≥ 0.200 – 0.299 | 0.100 – 0.199 | < 0.100 | < 0.100 |
| BF = AB ÷ CW | 4.00 – 5.99 | 2.00 – 3.99 | < 2.00 – 1.00 | < 1.00 |
Table II
The sequence, annealing temperatures and the product size of the primers used to amplify genes encoding virulence factors and antimicrobial resistance.
| Gene/ primer | Sequence | Product size (bp) | Annealing temperature |
|---|---|---|---|
| kps MTII | FP: 5'–GCGCATTTGCTGATACTGTTG-3' RP: 5'–CATCCAGACGATAAGCATGAGCA-3' | 270 | 62 |
| traT | FP: 5'–GGTGTGGTGCGATGAGCACAG-3' RP: 5'–CACGGTTCAGCCATCCCTGAG-3' | 288 | 62 |
| sat | FP: 5'–TCAGAAGCTCAGCGAATCATTG-3' RP: 5'–CCATTATCACCAGTAAAACGCACC-3' | 931 | 59 |
| csgA | FP: 5'–GGCGGAAATGGTTCAGATGTTG-3' RP: 5'–CGTATTCATAAGCTTCTCCCGA-3' | 301 | 60 |
| vat | FP: 5'–AACGGTTGGTGGCAACAATCC-3' RP: 5'–AGCCCTGTAGAATGGCGAGTA-3' | 418 | 58 |
| iutA | FP: 5'–GGCTGGACATCATGGGAACTGG-3' RP: 5'–CGTCGGGAACGGGTAGAATCG-3' | 302 | 85 |
| blaKPC | FP: 5'–GATACCACGTTCCGTCTGG-3' RP: 5'–GCAGGTTCCGGTTTTGTCTC-3' | 246 | 50-60 |
| blaCTX-M | FP: 5'–TTTGCGATGTGCAGTACCAGTAA-3' RP: 5'–CGATATCGTTGGTGGTGCCATA-3' | 544 | 62 |
Table III
Antimicrobial resistance (Intermediate resistance*) profiles of uropathogenic E. coli in western Saudi Arabia.
| Sample origin | N | P (%) | AM | AC | PT | CEFT | CEFE | ERT | IMI | MER | AMI | G | CIP | NOR | NIT | TS |
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Male | 774 | 22 (2.84) | 12 | 1, (1*) | 1 | 7 | 7 | 0 | 0 | 0 | 0 | 2 | 6 | 19 | 2 | 9 |
| Female | 226 | 28 (12.4) | 18 | 1, (1*) | (1*) | 12 | 12 | 0 | 0 | 0 | 0 | 4 | 8 | 22 | (2*) | 13 |
| Total | 1,000 | 50 (5) | 30 | 4 | 2 | 19 | 19 | 0 | 0 | 0 | 0 | 6 | 14 | 41 | 4 | 22 |
| p | < 0.001 |
[i] N– total number of urine samples examined for E. coli, P– total number of samples positive for E. coli, % – percentage of positive samples,
p – p-values from chi-square (x2), * – strains with intermediate resistance
AM – ampicillin, AC – amoxicillin/clavulanic acid, PT – piperacillin/tazobactam, CEFT – ceftazidime, CEFE – cefepime, ERT – ertapenem,
IMI – imipenem, MER – meropenem, AMI – amikacin, G– gentamicin, CIP – ciprofloxacin, NOR – norfloxacin, NIT – nitrofurantoin,
TS – trimethoprim/sulfamethoxazole
Table IV
Multidrug-resistance patterns among uropathogenic E. coli.
| Isolate code | Origin | Antibiotic resistance patterns | No. of antimicrobial classes | ESBL† | KPC‡ |
|---|---|---|---|---|---|
| ** UPEC 2 | F | AM, CEFT, CEFE, TS | 3 | – | – |
| ** UPEC 6 | F | AM, CEFT, CEFE, CIP, NOR, TS | 4 | + | + |
| ** UPEC 12 | F | AM, CEFT, CEFE, CIP, NOR, TS | 4 | + | + |
| ** UPEC 13 | F | AM, CEFT, CEFE, CIP, NOR, TS | 4 | + | + |
| ** UPEC 17 | M | AM, CEFT, CEFE, CIP, TS | 4 | – | – |
| UPEC 20 | F | NOR | 1 | – | – |
| ** UPEC 25 | F | AM, AC, PT*, CIP, NOR, NIT* | 3 | – | – |
| ** UPEC 32 | F | AM, CEFT, CEFE, CIP, NOR, TS | 4 | + | + |
| ** UPEC 39 | M | AM, CEFT, CEFE, NIT | 3 | + | + |
| ** UPEC 45 | M | AM, CEFT, CEFE, CIP, NOR, TS | 4 | + | + |
| UPEC 88 | F | AM, NOR | 2 | – | – |
| ** UPEC 95 | F | AM, CEFT, CEFE, NOR, TS | 4 | + | + |
| ** UPEC 119 | F | AM, CEFT, CEFE, NOR | 3 | + | + |
| UPEC 127 | M | NOR | 1 | – | – |
| UPEC 138 | F | NOR | 1 | – | – |
| UPEC 145 | F | NOR | 1 | – | – |
| UPEC 151 | F | NOR | 1 | – | – |
| UPEC 156 | M | NOR | 1 | – | – |
| UPEC 168 | F | NOR | 1 | – | – |
| ** UPEC 192 | M | AM, CEFT, CEFE, NOR, TS | 4 | + | + |
| ** UPEC 213 | M | AM, CEFT, CEFE, CIP, NOR, TS | 4 | + | + |
| ** UPEC 225 | M | AM, G, CIP, NOR, TS | 4 | – | – |
| UPEC 226 | M | NOR | 1 | – | – |
| UPEC 230 | M | NOR | 1 | – | – |
| UPEC 242 | M | NOR | 1 | – | – |
| ** UPEC 243 | F | AM, G, CIP, NOR, TS, NIT* | 4 | – | – |
| ** UPEC 316 | F | AM, CEFT, CEFE, G, CIP, NOR, TS | 5 | + | + |
| ** UPEC 326 | F | AM, CEFT, CEFE, G, CIP, NOR, TS | 5 | + | + |
| UPEC 331 | M | NOR | 1 | – | – |
| ** UPEC 353 | F | AM, CEFT, CEFE, TS | 3 | + | – |
| ** UPEC 425 | M | AM, AC, PT, NOR, TS | 3 | – | – |
| UPEC 432 | M | NOR | 1 | – | – |
| ** UPEC 450 | M | AM, AC*, CEFT, CEFE, G, CIP, NOR, TS | 5 | + | + |
| ** UPEC 544 | F | AM, AC*, G, NOR, TS | 4 | – | – |
| ** UPEC 549 | M | AM, CEFT, CEFE, CIP, TS | 4 | + | + |
| ** UPEC 661 | F | AM, CEFT, CEFE, TS | 3 | + | + |
| UPEC 662 | F | AM | 1 | – | – |
| UPEC 666 | F | AM, CEFT, CEFE | 2 | + | + |
| UPEC 738 | M | AM | 1 | – | – |
| UPEC 829 | F | AM, TS | 2 | – | – |
| UPEC 950 | M | AM, TS | 2 | – | – |
| UPEC 1000 | M | AM, NIT | 2 | + | + |
[i] * – intermediate resistance
** – multidrug resistant – a single isolate is resistant against more than 3 antimicrobial classes
† – ESBL positive phenotypes detected by CHROMagar ESBL
‡ – KPC positive phenotypes detected by CHROMagar KPC
AM – ampicillin, AC – amoxicillin/clavulanic acid, PT – piperacillin/tazobactam, CEFT – ceftazidime, CEFE – cefepime,
G– gentamicin, CIP – ciprofloxacin, NOR – norfloxacin, NIT – nitrofurantoin, TS – trimethoprim/sulfamethoxazole
Table V
Prevalence of ESBL, KPC and α-hemolysin positive phenotypes of UPEC.
| Isolates origin | Total number of UPEC | ESBL+† (%) | ESBL+‡ (%) | KPC+ (%) | β-hemolysis (%) |
|---|---|---|---|---|---|
| Male | 22 | 7 (32) | 7 (32) | 7 (32) | 11 (50) |
| Female | 28 | 11 (39.3) | 11 (39.3) | 10 (36) | 16 (57.2) |
| Total | 50 | 18 (36) | 18 (36) | 17 (34) | 27 (54) |
Table VI
Semiquantitative classification of biofilm formation using two different formulas and the association of biofilm-forming ability and multidrug-resistance in uropathogenic E. coli.
| Isolate code | BF† | BF‡ | AMR |
|---|---|---|---|
| UPEC (2) | N (0.058) | W (1.167) | 3 |
| UPEC (6) | M (0.185) | N (0.420) | 4 |
| UPEC (12) | S (0.343) | W (1.988) | 4 |
| UPEC (13) | S (0.365) | M (2.051) | 4 |
| UPEC (17) | N (0.066) | W (1.190) | 4 |
| UPEC (20) | S (0.313) | W (1.902) | 1 |
| UPEC (25) | M (0.121) | W (1.348) | 2 |
| UPEC (32) | N (0.067) | W (1.193) | 4 |
| UPEC (39) | W (0.39) | M (2.123) | 2 |
| UPEC (45) | N (0.086) | W (1.247) | 4 |
| UPEC (88) | N (0.066) | W (1.190) | 2 |
| UPEC (95) | N (0.086) | W (1.247) | 4 |
| UPEC (119) | N (0.046) | W (1.132) | 4 |
| UPEC (127) | S (0.339) | W (1.976) | 1 |
| UPEC (138) | S (0.239) | W (1.688) | 1 |
| UPEC (145) | M (0.151) | W (1.435) | 1 |
| UPEC (151) | M (0.135) | W (1.389) | 1 |
| UPEC (156) | W (0.057) | W (1.164) | 1 |
| UPEC (168) | W (0.031) | W (1.089) | 1 |
| UPEC (192) | W (0.034) | W (1.097) | 4 |
| UPEC (213) | W (0.074) | W (1.213) | 4 |
| UPEC (225) | M (0.187) | W (1.538) | 4 |
| UPEC (226) | M (0.121) | W (1.348) | 1 |
| UPEC (230) | N (0.068) | W (1.178) | 1 |
| UPEC (242) | N (–0.015) | N (0.960) | 1 |
| UPEC (243) | N (0.062) | W (1.162) | 4 |
| UPEC (316) | N (0.016) | W (1.041) | 5 |
| UPEC (326) | N (–0.027) | N (0.929) | 5 |
| UPEC (331) | N (0.004) | W (1.010) | 1 |
| UPEC (353) | N (0.031) | W (1.081) | 3 |
| UPEC (425) | N (–0.017) | N (0.955) | 3 |
| UPEC (432) | N (0.027) | W (1.070) | 1 |
| UPEC (450) | N (0.054) | W (1.141) | 5 |
| UPEC (544) | N (0.061) | W (1.159) | 4 |
| UPEC (549) | S (0.216) | W (1.565) | 4 |
| UPEC (601) | N (0.024) | W (1.062) | 0 |
| UPEC (654) | N (0.013) | W (1.034) | 0 |
| UPEC (661) | N (0.088) | W (1.230) | 3 |
| UPEC (662) | M (0.125) | W (1.327) | 1 |
| UPEC (666) | N (0.068) | W (1.178) | 2 |
| UPEC (704) | S (0.328) | W (1.858) | 0 |
| UPEC (738) | N (–0.017) | N (0.955) | 1 |
| UPEC (829) | N (–0.027) | N (0.929) | 2 |
| UPEC (842) | N (0.074) | W (1.193) | 0 |
| UPEC (848) | S (0.379) | W (1.992) | 0 |
| UPEC (882) | N (0.09) | W (1.235) | 0 |
| UPEC (900) | N (0.068) | W (1.190) | 0 |
| UPEC (928) | N (0.074) | W (1.193) | 0 |
| UPEC (950) | M (0.121) | W (1.348) | 2 |
| UPEC (1000) | M (0.121) | W(1.338) | 2 |
| r | –0.0948 | –0.1475 | NS |
[i] All values were measured at OD620nm.
† – biofilm formation was determined by applying formula (BF = AB– CW)
‡ – biofilm formation was determined by applying formula (BF = AB/ CW)
AMR – antimicrobial resistance (showing resistance to the number of antimicrobial classes)
AB – stained attached bacteria
CW – stained control wells
S – strong
M – moderate
W – weak
N – negative
r – the value from Pearson correlation coefficient, no significant relationship between biofilm-forming ability and multidrug- resistant in all 50 UPEC isolates
NS – not significant, no significant association between biofilm-form- ing ability and multidrug-resistance to antimicrobial agents
Table VII
Prevalence of genes encoding virulence factors and antimicrobial resistance in uropathogenic E. coli.
| Isolates origin | Total number of UPEC | Genes encoding for virulence factors | |||||
|---|---|---|---|---|---|---|---|
| kpsMTII (%) | iutA (%) | traT (%) | csgA (%) | Sat (%) | Vat (%) | ||
| Male | 22 | 22 (100) | 22 (100) | 22 (100) | 22 (100) | 22 (100) | 9 (41) |
| Female | 28 | 28 (100) | 28 (100) | 28 (100) | 28 (100) | 27 (97) | 10 (36) |
| Total | 50 | 50 (100) | 50 (100) | 50 (100) | 50 (100) | 49 (98) | 19 (38) |
| Genes encoding for antimicrobial resistance | |||||||
| blaCTX-M (%) | blaKPC (%) | ||||||
| Male | 22 | 8 (37) | 7 (32) | ||||
| Female | 28 | 10 (36) | 5 (18) | ||||
| Total | 50 | 18 (36) | 12 (24) | ||||
Fig. 1A
Agarose gel electrophoresis shows positive results of kpsMTII virulence gene at (270 bp) in UPEC.
M– DNA marker, C– negative control
Fig. 1B
Agarose gel electrophoresis shows positive results of csgA virulence gene at (301 bp) in UPEC.
M– DNA marker, C– negative control
Fig. 1C
Agarose gel electrophoresis shows some positive results of vat virulence gene at (418 bp) in UPEC.
M– DNA marker, C– negative control