Important Virulence Factors and Related Genes in Uropathogenic E. coli and their Relation to Fluoroquinolone Resistance.
Aim of Work
The present study was conducted to detect some important virulence factors of E. coli phenotypically: haemolytic activity, cytotoxic activity and haemagglutination and related virulence genes: hlyA, cnf-1 and papC, by using multiplex PCR. Study the relation between the bacterial resistance to fluoroquinolones and the virulence potential of E. coli.
MATERIALS AND METHODS
The study has been approved by the Research and Ethical Committee of Medical Microbiology and Immunology Department, Faculty of Medicine, Cairo University. Isolation and Identification of E. coli
Urine samples from non-hospitalized cases of urinary tract infections were collected from clinical laboratories of Kasr El Ainy hospital. Microscopic examination of a wet film of urine was carried out. Samples with pus cells [greater than or equal to] 10/ HPF were quantitatively inoculated on MacConkey's agar (Oxoid, UK) and then incubated at 37 [degrees]C for 24 hrs. Culture plates with bacterial count [greater than or equal to] [10.sup.5] CFU/ ml and growth of single morphotype of lactose fermenting colonies were selected for identification of E. coli by standard biochemical methods.
Phenotypic Detection of UPEC Virulence Factors
The E. coli isolates were tested for expression of haemolytic, cytotoxic activity and haemagglutination as described below:
1. Assessment of haemolytic activity
The cytolytic toxin secreted by most haemolytic E. coli isolates is known as alpha haemolysin. E. coli isolates were subcultured onto 5% sheep blood agar (Oxoid, UK) and incubated overnight at 37[degrees]C. Haemolysin production was detected by presence of a zone of complete haemolysis around the inoculation site (Raksha et al, 2003; EL-Mosallamy et al, 2015; Tabasi et al., 2015).
2. Assessment of cytotoxic activity
The cytotoxic effect of E. coli isolates was detected according to Ghadir et al. (2010), however, HEp-2 cells was used instead of Vero cells according to Mills et al. (2000).
Preparation of bacterial isolates
E. coli isolates were obtained from frozen stock and subcultured on blood agar (Oxoid, UK) then on MacConkey's agar (Oxoid, UK). Colonies were then inoculated in test tubes containing 10 ml of tryptic soy broth (TSB) (Oxoid, UK) and incubated for 18-20 h at 37[degrees] C and kept at 4[degrees] C and used for inoculum preparation. Bacteria were harvested by centrifugation at 3000 rpm (MSE Centaur 2, UK). The supernatant was discarded and the bacterial pellets were washed once with phosphate buffered saline (PBS), then resuspended in Eagle's minimum essential medium with Earl's balanced salts (EMEM) (Lonza, Belgium) and adjusted to a count1 * [10.sup.8] CFU/ ml. Cell line and growth conditions
HEp-2 cells were maintained in EMEM (Lonza, Belgium) supplemented with 2% fetal bovine serum (FBS) (Lonza, Belgium), 100 U/ml penicillin and 100 [micro]g/ml streptomycin at 37[degrees]C in 5 % C[O.sub.2] and subcultured every 3-4 days. HEp-2 cells suspended in MEM Earl's and supplemented with 10 % FBS were seeded in 24-well tissue culture plates and kept at 37[degrees]C with 5 % C[O.sub.2] for 24 hours to form a confluent monolayer. 100 [micro]l of the bacterial cell suspension prepared above was added to each well and incubated at 37[degrees]C in 5 % C[O.sub.2] for 3 hours. HEp-2 cell morphology was assessed by examination using an inverted microscope (Olympus bx 50, Japan) 1, 2 and 3 hours post inoculation.
3. Assessment of haemagglutination
E. coli isolates were inoculated into nutrient broth and incubated at 37 [degrees]C for 48 hours for production of fimbriae. Five milliliter of group O venous blood was collected then washed three times in normal saline and a suspension of 3% RBCs in phosphate buffered saline (pH 7.4) was prepared (Lonza, Belgium). They were used immediately or within a week when stored at 3-5 [degrees]C. The slide haemagglutination test was carried out on a multiple-concavity slide by adding 50 [micro]l of the RBC suspension to 50 [micro]l of the broth culture and the slide was rocked at room temperature for 5 minutes. Presence of clumping was taken as positive for haemagglutination. At the same time, 50 [micro]l of 3% D-mannose (Fluka AG, Switzerland) was added to the red cells and broth culture in another set and rocked as above. Haemagglutination inhibited in presence of D-mannose was labeled as mannose sensitive haemagglutination (MSHA); indicating type1 fimbriae, if agglutination occurred even in presence of D-mannose, it was called mannose resistant haemagglutination (MRHA); indicating presence of P fimbriae (Uma et al., 2013). Molecular Detection of UPEC Virulence Genes E. coli isolates were tested for the presence of virulence genes including: hlyA, cnf-1 and papC using multiplex PCR.
1. DNA Extraction
DNA extraction was performed for all the clinical isolates using CinnaPure DNA extraction kit (Tehran, Iran) according to manufacturer's instructions.
Amplification was done according to Bashir et al. (2012) using 50 [micro]l reaction mixture for each isolate. Primers sequences are illustrated in Table (1).
The thermal cycler (Biometra T 3000) conditions were as follows: an initial denaturation step at 94[degrees]C for 5 min followed by 30 cycles of amplification (denaturation at 94[degrees]C for 1 min, annealing at 56[degrees]C for 1 min and extension at 72[degrees]C for 1 min) and final extension of 5 min at 72[degrees]C. 3. Agarose Gel electrophoresis
Amplified PCR products were visualized by 2% (w/v) agarose gel electrophoresis (stained with ethidium bromide; 0.5 [micro]g/ml) at 120 volts for 45 minutes. The bands were visualized under UV transilluminator (Biometra Ti 3) and photographed. 100 bp DNA ladder H3 RTU (GeneDire, Taiwan) (which gave 12 discrete bands at 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500 and 3000 bp) was used.
Fluoroquinolones susceptibility using Kirby Bauer Disk Diffusion method
Disk diffusion method was done using fluoroquinolones: ciprofloxacin (CIP 5 [micro]g), levofloxacin (LEV 5[micro]g), norfloxacin (NOR 10[micro]g), and ofloxacin (OFX 5[micro]g) (Oxoid, UK).
The E. coli colonies were suspended in sterile normal saline and adjusted to 0.5 MacFarland standard, then inoculated on Muller-Hinton agar plates and incubated with the previously mentioned antimicrobial disks at 35[degrees]C [+ or -] 2 for 18 hours. The interpretation of the diameters of the inhibition zones were done according to CLSI, (2014) (Table 3).
Data were statistically described in terms of frequencies (number of cases) and percentages and compared using Chi-square ([chi square]) test. Exact test was used instead when the expected frequency is less than 5, P values less than 0.05 was considered statistically significant. All statistical calculations were done using computer program SPSS (Statistical Package for the Social Science; SPSS Inc., Chicago, IL, USA) release 15 for Microsoft Windows.
The present study was performed on 50 E. coli isolates isolated from urine samples of patients with UTI. E. coli isolates were identified by their colony morphology on MacConkey's agar and standard biochemical reactions.
Phenotypic Detection of Virulence Factors of E. coli Isolates
1. Haemolytic activity
In the current study 15 (30%) isolates showed [beta]-haemolysis, while the remaining 35 (70%) isolates were non-haemolytic.
2. Cytotoxic assay
Upon visual assessment of the HEp-2 cell line one hour after infection with 1 x [10.sup.8] CFU/ ml of the tested isolates, no effect was seen on HEp-2 cells. Two hours post infection; it was found that out of 50 UPEC isolates, 46 (92%) isolates showed cytotoxic effect with mild detachment and lysis of HEp-2 cell line and almost complete detachment of the cell line 3 hours post infection, while only 4 (8%) isolates showed no effect on the cell line (Figures 1, 2 and 3).
3. Haemagglutination assay
Out of the 50 E. coli isolates, 9 (18%) isolates showed haemagglutination (HA) of group O RBCs, while 41 (82%) were non-haemagglutinating. Out of the 9 isolates positive for HA, 6 isolates were MRHA (6/50, 12%) and 3 isolates were MSHA (3/50, 6%).
Molecular Detection of E. coli Virulence Genes
Virulence genes including hlyA, cnf-1 and papC of 50 UPEC were detected by the multiplex PCR. Amplicon sizes of hlyA, cnf-1 and papC were 1177, 498, 328 (bp), respectively (Figure 4 and 5).
Twenty five isolates were positive for one or more of the targeted 3 genes, while the remaining isolates were negative for the targeted genes. It was found that one isolate was positive for the three targeted virulence genes, 10 isolates showed the presence of both cnf-1 and hlyA, 2 isolates showed the presence of both cnf-1 and papC, 2 isolates showed the presence of both hlyA and papC, while 8 isolates showed the presence of papC and 2 isolates showed the presence of cnf-1 gene (Table 4).
It was found that the most prevalent gene was cnf-1 which was detected in 15 (30%) isolates, while 13 (26%) isolates were positive for hlyA and 13 (26%) isolates were positive for papC.
Virulence Factors in Relation to Virulence Genes
Haemolytic activity in relation to hlyA gene
It was found that hlyA gene was detected in higher percentage in [beta]- haemolytic isolates (61.5%) than non- haemolytic isolates (38.5%) with statistically significant difference (P-value = 0.011).
Cytotoxicity in relation to cnf-1 and hlyA
Out of 46 isolates showing cytotoxicity by phenotypic methods, 17 (37%) isolates were positive for the cytotoxic genes; 11 isolates were positive for both cnf-1 and hlyA gene, while 4 isolates were only positive for cnf-1 gene and 2 isolates were only positive for hlyA gene. The 4 cytotoxicity negative isolates expressed neither of the 2 genes.
Haemagglutination (Ha) in relation to papC gene
Out of the 9 isolates showing haemagglutination by phenotypic methods; only 3 (33%) isolates were positive for papC gene (2 isolates were MRHA and one isolate was MSHA). On the other hand, 10 (24.4%) out of the 41 non-haemagglutinating isolates were positive for papC gene. Although, haemagglutination positive isolates showed a higher percentage of papC gene than the non- haemagglutinating isolates there was no statistically significant difference between them (P- value = 0.679).
Results of Fluoroquinolones Sensitivity of the E. coli Isolates
In vitro susceptibility pattern of E. coli isolates showed that 30 (60%) isolates of E. coli were resistant to all fluoroquinolones, while 20 (40%) isolates were sensitive (Figure 6).
Association of Virulence Factors Expression with Resistance to Fluoroquinolones among E. coli Isolates
Haemolytic activity in relation to fluoroquinolones resistance
Phenotypic analysis showed that out of the 15 isolates with [beta]-haemolysis; 10 (33.3%) were fluoroquinolone resistant and 5 (25%) were fluoroquinolones sensitive isolates with no statistically significant difference between the two groups (P- value = 0.380).
Cytotoxicity in relation to fluoroquinolones resistance
Out of the 46 isolates showing cytotoxicity to HEp-2 cells; 27 (90%) isolates were fluoroquinolone resistant and 19 (95%) isolates were fluoroquinolones sensitive with no statistically significant difference between the two groups (P- value = 0.641).
Haemagglutination in relation to fluoroquinolones resistance
Out of the 9 haemagglutinating isolates; 5 (16.6%) isolates were fluoroquinolones resistant, (3 MRHA and 2 MSHA), while 4 (20%) isolates were fluoroquinolones sensitive, (3 MRHA and 1 MSHA), with no statistically significant difference between the two groups (P- value = 0.456).
Association of Virulence Genes with Resistance to Fluoroquinolones among E. coli Isolates
Out of the 30 fluoroquinolones resistant isolates; 16 (53.3%) isolates harboured the virulence genes with variable distribution. It was found that hlyA, cnf-1 and papC genes were detected in the following percentages: 27%, 30% and 30%, respectively. On the other hand, in the susceptible group 8 (40%) isolates harboured the virulence genes with variable distribution. It was found that hlyA, cnf-1 and papC genes were detected in the following percentages respectively 25%, 30% and 20%. There was no statistically significant difference between the resistant and susceptible groups regarding the 3 virulence genes.
UPEC is the most frequent cause of community and hospital-acquired urinary tract infections (Calhau et al., 2015). These infections can vary from a simple cystitis to a serious parenchymatous attack. The degree of severity depends on the virulence of the responsible isolates (Srinivasan et al., 2003; Soto et al., 2011).
Several virulence factors such as fimbriae, toxins, and siderophores contribute to the colonization and pathogenicity of UPEC (Qin et al., 2013). Measuring a phenotype in vitro does not always correlate with in vivo expression and very often underestimates the presence of a virulence factor in vivo (Kaper et al., 2004). The distribution of virulence properties can vary depending upon host characters, type of infection and predisposing factors which determine the host parasite interaction in vivo which can culminate in an active infection (Vijayalakshmi et al., 2015). In addition there is always the possibility of mutation at the level of the corresponding gene, leading to the absence of its detection. Therefore, a positive PCR shows the presence of the virulence gene, but a negative PCR does not point to the absence of the corresponding operon (Tarchouna et al., 2013).
This study was designed to assess some important phenotypic virulence factors of E. coli: haemolytic activity, cytotoxic activity and haemagglutination and their relations to the following genes: hlyA, cnf-1 and papC.
We found that 30 % of E. coli isolates showed [beta]- haemolysis on sheep blood agar. Similar results were obtained by other studies done by Fatima et al. (2012), EL-Mosallamy et al. (2015), Tabasi et al. (2015) and Vijayalakshmi et al. (2015) who reported that 30%, 24%, 34% and 25% of the isolates were [beta]-haemolytic, respectively.
Higher rates of [beta]-haemolytic E. coli were detected by Mandal et al. (2001), Raksha et al. (2003), Rebecca and Elizabeth (2005), Santo et al. (2006), Regua-Mangia et al. (2010) and Sai et al. (2013) who reported that 45.5%, 41.36%, 40.7%, 96%, 47 % and 40% of E. coli isolates were [beta]- haemolytic respectively. Several reasons explain the differences in the percentage of haemolysin production by UPEC like: source of blood, type of haemolysin produced, source of bacteria and method to screen the production ability (Al-Chalabi et al., 2010).
Regarding cytotoxic effects of E. coli in this study, it was found that no change was detected in the HEp-2 cell line during the first hour of its incubation with E. coli suspension. However, 2 hours post infection; 92% of the tested E. coli isolates showed morphological changes and almost complete detachment in HEp-2 cells was detected 3 hours post infection. Our results were in accordance with Guignot et al. (2000) and Ghadir et al. (2010).
Agglutination of human erythrocytes by E. coli strain is an indirect evidence of the presence of fimbriae on that strain. MRHA of human RBCs is the phenotypic expression of P fimbriae on E. coli, while MSHA is the phenotypic expression of type 1 fimbriae and both of them mediate adherence and they are important in bladder colonization (Vijayalakshmi et al., 2015).
In the present study, it was found that 18% of the isolates showed haemagglutination. Similar results were obtained by Vidhya et al. (2016) who found that 20% of the isolates were positive for haemagglutination. Higher rates were reported by Fatima et al. (2012), Desai et al. (2013), Sai et al. (2013) and Vijayalakshmi et al. (2015) who found that 48%, 66%, 59% and 68% of the isolates were haemagglutinating, respectively.
In this study, higher prevalence for MRHA (12%) than MSHA (6%) was observed. Our results were in agreement with Fatima et al. (2012) and Desai et al. (2013). On the other hand, higher prevalence for MSHA than MRHA was recorded by Sai et al. (2013) and Vijayalakshmi et al. (2015).
The genes coding for alpha-haemolysin (hlyA), cytotoxic necrotizing factor (cnf-1) and P fimbrial adhesin (papC) were detected by PCR in the current study. It was observed that 13 isolates (26%) of E. coli isolates were positive for hlyA gene which was in agreement with Bingen-Bidois et al. (2002), Tiba et al. (2008), Bashir et al. (2012) and Fatima et al. (2012). Lower rates were obtained by other studies done by Arisoy et al. (2006), Farshad and Emamghorashi (2009) and Tarchouna et al. (2013). Higher rates were obtained by Santo et al. (2006) and Karimian et al. (2012) who reported that 96% and 50.4% of the isolates were positive for the gene, respectively.
As regard the cnf-1 gene, it was found that 15 isolates (30%) of E. coli isolates in this study were positive for the gene. Similar results were obtained by Adwan et al. (2015) and Calhau et al. (2015). Lower results were obtained by Arisoy et al. (2006), and Tarchouna et al. (2013). On the other hand, higher rates were obtained by Karimian et al. (2012) who reported that 50.4% of the isolates were positive for the gene.
In the present study, it was found that 13 isolates (26%) were positive for papC gene. This finding was in accordance with Farshad and Emamghorashi (2009), Qin et al. (2013) and Calhau et al. (2015). Higher papC gene carriage rates of 36%, 32%, 32.7% and 34.6% were reported by Usein et al. (2001), Santo et al. (2006), Tiba et al. (2008), Firoozeh et al. (2014), respectively. Lower papC gene carriage rates of 23% and 24% were obtained by Arisoy et al. (2006) and Bashir et al. (2012), respectively. The diversity in frequency of pap gene among different studies can be attributed to the fact that UPEC strains can utilize a variety of adhesins to bind to the urinary epithelial cells, and start the infection. Hence, the strains lacking the pap operon may use other adhesins encoding operons such as afa, and sfa for binding (Neamati et al., 2015).
In addition, the differences in prevalence of UPEC virulence genes showed that the virulence properties of UPEC strains is closely depending on geographic region, weather climate of each regions, diets, the levels of public health, hospital's health and even methods of sampling (Karimian et al., 2012).
In the present study, the simultaneous presence of papC, cnf-1 and hlyA was observed in only one UPEC isolate. Bashir et al. (2012) found that the three genes were found collectively in 4 isolates (out of a total of 59 isolates). The cumulative presence of papC, cnf-1 and hlyA is the evidence of the presence of pathogenicity island IIJ96, which is highly prevalent among UTI isolates (Piatti et al., 2008).
Regarding the relation between the phenotypic virulence factors and the virulence genes, it was found that hlyA gene was detected in a higher percentage in [beta]-haemolytic E. coli 53% (8/13) than non- haemolytic E. coli 14% (5/13) and this difference was statistically significant (Pvalue = 0.011). The presence of the gene in some non-haemolytic strains may be explained by the possibility of modification of the gene leading to inactive haemolysin (Landraud et al., 2000). Also, since alpha haemolysin is a pore-forming toxin, at low concentration its binding does not always lead to lysis (Boehm et al., 1990; Ostolaza and Goni, 1995). On the other hand, 7 [beta]-haemolytic E. coli isolates in this study were negative for hlyA gene which means that haemolysis on sheep blood agar might be due to haemolysins other than a-haemolysin (Fatima et al., 2012).
In our study, we found that all isolates positive for hlyA gene exhibited cytotoxic activity on HEp-2 cell line; 11 of which harboured cnf-1 gene in addition. Out of the 13 isolates positive for papC gene, only 2 isolates were positive for MRHA. This can be explained by Wullt (2002) who found that some papC positive isolates, especially those isolated form asymptomatic infections, do not express P fimbriae. In addition, the expression of P fimbriae is subjected to phase variation so in vitro growth conditions differing in glucose concentration, temperature and pH may switch the fimbriae on or off. We found that the remaining 4 isolates positive for MRHA were negative for papC gene. According to Sabitha et al. (2014), MRHA can be mediated by P fimbriae and also by X, F1C and Dr fimbriae.
Regarding sensitivity of E. coli to fluoroquinolones, it was observed that 30 isolates (60%) were resistant to all tested fluoroquinolones. This result was in agreement with Lupez-Banda et al. (2014), Calhau et al. (2015) and Gururaju et al. (2015).
In the current study, although there was a higher percentage of both virulence factors and virulence genes in the fluoroquinolones resistant group than in the fluoroquinolones sensitive group, this difference was not statistically significant. This was in accordance with Oliveira et al. (2011). According to previous studies, Johnson et al. (2003), Horcajada et al. (2005), Moreno et al. (2006), Takahashi et al. (2009), and KawamuraSato et al. (2010) found that quinolones and fluoroquinolones resistant UPEC strains display overall reduced virulence while, susceptible E. coli strains were more virulent. Piatti et al. (2008) found a lower incidence of papC, hlyA, and cnf-1 among fluoroquinolones resistant group than among susceptible group, which seems to be due to a loss of the corresponding PAI, probably as a result of the mutation that causes resistance.
Virulence determinants may help us to get insights into pathogenesis of UPEC isolates and management of UTIs. Phenotypic methods are simple but not very specific, while multiplex PCR is rapid, easy and more specific in detecting virulence genes. The tested E. coli isolates exhibited a high resistance to fluoroquinolones with no significant difference was found between fluoroquinolones resistant and sensitive E. coli isolates regarding the virulence genes as well as virulence factors.
(Received: 02 August 2018; accepted: 25 September 2018)
This study was funded partly by Faculty of Medicine, Cairo University and partly by the authors.
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Noha Mahmoud Gohar *, Hanaa Fathy Aly and Magda Ibrahim Ayoub
Department of Medical Microbiology and Immunology, Faculty of Medicine, Cairo University, Cairo, Egypt.
* To whom all correspondence should be addressed. Tel.: 01001332681; E-mail: firstname.lastname@example.org
Caption: Fig. 1. HEp-2 cell line, control cell line
Caption: Fig. 2. The cytotoxic effect of the tested E. coli isolate on HEp-2 cells with mild detachment 2 h post infection
Caption: Fig. 3. The cytotoxic effect of the tested E. coli isolate on HEp-2 cells with almost complete detachment 3 h post infection
Caption: Fig. 4. Multiplex PCR for virulence genes in E. coli isolates. Lane (1): cnf-1 (band at 498 bp), Lane (2): papC (band at 328 bp), Lane (M): 100 bp DNA ladder
Caption: Fig. 5. Multiplex PCR for virulence genes in E. coli isolates. Lane (1): hlyA and cnf-1, Lane (2): hlyA and papC, Lane (3): cnf-1 and papC, Lane (4): hlyA, cnf-1 and papC, Lane (M): 100 bp DNA ladder
Caption: Fig. 6. Fluoroquinolones susceptibility of isolated E. coli to different fluoroquinolones using disk diffusion method (A): An E. coli isolate showing sensitivity to all used fuoroquinolones (B): An E. coli isolate showing resistance to all used fuoroquinolones
Table 1. Primers' Sequences Primer Sequence Reference hly A F: 5'AACAAGGATAAGCACTGTTCTGGCT3' Yamamoto et al. (1995) R: 5'ACCATATAAGCGGTCATTCCCGTCA3' cnf-1 F: 5'AAGATGGAGTTTCCTATGCAGGAG3' Arisoy et al. (2006) R: 5'CATTCAGAGTCCTGCCCTCATTAT3' papC F: 5'GACGGCTGTACTGCAGGGTGTGGCG3' Daigle et al. (1994) R: 5'ATATCCTTTCTGCAGGGATGCAATA3' Table 2. Expected PCR product size Genes Amplicon size Reference (bp) hly A 1177 Yamamoto et al. (1995) cnf-1 498 Arisoy et al. (2006) papC 328 Daigle et al. (1994) Table 3. Interpretation of sensitivity testing of E. coli with fluoroquinolones disks Strain Antimicrobial Disk Zone diameter agent content R E. coli Ciprofloxacin 5 Mg [less than or equal to] 15 Levofloxacin 5 Mg [less than or equal to] 13 Norfloxacin 10 Mg [less than or equal to] 12 Ofloxacin 5 Mg [less than or equal to] 12 Strain Antimicrobial agent I S E. coli Ciprofloxacin 16-20 [greater than or equal to] 21 Levofloxacin 14-16 [greater than or equal to] 17 Norfloxacin 13-16 [greater than or equal to] 17 Ofloxacin 13-15 [greater than or equal to] 16 Table 4. Distribution of Virulence genes among E. coli isolates No. of Virulence Virulence No. of genes genes isolates 3 cnf-1, hlyA, papC 1 2 cnf-1, hlyA 10 2 cnf-1, papC 2 2 hlyA, papC 2 1 cnf-1 2 1 papC 8 Total 25
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|Author:||Gohar, Noha Mahmoud; Aly, Hanaa Fathy; Ayoub, Magda Ibrahim|
|Publication:||Journal of Pure and Applied Microbiology|
|Date:||Sep 1, 2018|
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