Abstract: Objective: The aim of this study was to detect 10 virulence genes in 50 Escherichia coli strains isolated from patients with different infections. Methodology: Different phenotypic assays and multiplex PCR were used to detect virulence genes. Antimicrobial susceptibility testing was performed to 14 antimicrobials types by disk diffusion method. Results: The most common virulence gene was feoB (98%) and at lower prevalence were genes eaeA and stx1 (4%). Escherichia coli strains were highly resistant to most antimicrobials. Conclusion: Escherichia coli strains isolated from patients with bacteremia were highly virulence and more resistance to antimicrobials than those isolated from other sites.
INTRODUCTION
Escherichia coli is one of the most important opportunistic Gram negative bacteria. It resist many type of antimicrobials and cause different infections because it has many type of virulence genes encoding to many important virulence factors. It's known that Escherichia coli is a commensal bacterium, found as normal flora in intestines of human and animals1. Escherichia coli strains are classified into three different groups according to site of infection and pathogenicity; extraintestinal pathogenic E. coli (ExPEC) strains, commensal strains and intestinal pathogenic strains2. One of the most important group it is ExPEC, found as normal flora in the intestine but also can infect extraintestinal sites and causing many infections such as, Urinary Tract Infection (UTI), acute and chronic diarrhea and bacteremia3,4.
Escherichia coli is the most common cause of many infections in the worlds wide, such as; Urinary Tract Infection (UTI), heavy diarrhea and bacteremia5,6. Escherichia coli can cause these infections due to its ability to the acquisition of mobile genetic factors carrying many virulence genes7,8. Adhesions, iron uptake, toxins and capsules are the most common virulence factors responsible for attachment, adherence and invasion in the host and then lead to infection9.
Pathogenicity of E. coli is due to the presence of many virulence genes, located on chromosomes or plasmids or both that encodes important virulence factors, if present on the chromosome, these genes are typically found in specific regions called pathogenicity islands10.
Because of several strains of E. coli have plasmids that code for antimicrobial enzymes, which promote resistance to third generation cephalosporins, β-lactam antibiotics and other antimicrobials and are associated with the difficult of treatments and high morbidity and mortality rates, therefore, the aim of this study was to characterize 50 clinical isolates of E. coli isolated from urine, heavy diarrhea and bloodstream based on their resistance to antibiotics and the presence of ten virulence genes associated with their pathogenicity.
MATERIALS AND METHODS
Isolation and identification of Escherichia coli: A total of 390 clinical samples were collected from patients infected with urinary tract infection, heavy diarrhea and blood infection in Al-kufa hospital during the period of October, 2015 to April, 2016. All samples (urine and diarrhea) were collected in sterile cup and streaked immediately onto the surface of blood agar (Oxoid, UK), MacConkey agar (Oxoid, UK) and Chrome agar medium (Orientation company, France) by sterile loop and incubated overnight at 37°C. By a sterile syringe, 5 mL of blood were collected from adult patients and suspended with 45 mL of brain heart infusion broth (Oxoid, UK) and incubated at 37°C for 5-7 days and streaked onto the surface of blood (Oxoid, UK) and MacConkey agar (Oxoid, UK) by sterile loop and incubated overnight at 37°C11. All bacterial isolates were diagnosed by standard morphological and biochemical tests according to Faddin12. Finally, Vitek2® system (BioMerieux® -France) was used as a final identification step for all suspected E. coli isolates.
Detection of virulence factors by phenotypic methods
Capsule stain: This method was carried out according to Sorensen13 as follows: Fresh and pure of each bacterial colony was transferred on a slide, mixed with nigrosin stain and stained with methylen blue for 2 min and under light microscope the nigrosin stain provides a dark background to unstained capsule and metylene blue stain provides blue color to the cells.
Blood hemolysis production: Blood hemolysis production was detected with 5% sheep’s blood agar (Oxoid, UK). Strains producing a clear or semi clear zone of lysis after incubation for 24 h at 37°C were considered positives12.
Detection of siderophores production: In this method, nutrient agar (Oxoid, UK) supplemented with 200 mM of 2,2’-dipyridyl was used as iron-restricted agar medium according to Schwyn and Neilands14. All E. coli isolates streaked onto the surface of iron-restricted agar medium and incubated overnight at 37°C. Appearance of any bacterial growth considered as a positive results for ability of bacteria to siderophores production.
Biofilm formation testing: Fresh and pure colony of E. coli streaked on congo red agar plate and incubated at 37°C for 24-48 h. Formation of black or red colonies considered as a positive result for ability of bacterium to biofilm formation15.
Adhesion test: This test was carried out according to Svanborg16, as follows: A fresh morning urine samples of healthy female were collected and centrifuged at 3000 rpm for 20 min. The pellet was washed three times with phosphate buffered saline. One milliliter of bacterial culture of each isolates (Compared with McFarland standard tube) was added to a same volume of epithelial cells suspension, mixed well and incubated at 37°C for 1 h with shaking.
| Table 1: | Sequence of primers used in multiplex polymerase chain reaction for 10 virulence genes of Escherichia coli |
| Table 2: | Thermo cycling conditions of multiplex polymerase chain reactions for 10 virulence genes of Escherichia coli |
The tubes were centrifuged and washed three times with phosphate buffer saline to discard unattached bacteria, the supernatant was decanted and the sediment cells were mounted on glass microscope slide and separated using the margin of other slide. The slides were dried by air and fixed with methanol for 10 min. The slides were stained with giemsa stain for 1.5 h to color uroepithelial cells. The excess stain removed by water and the slides were dried and examined by light microscope under oil immersion to calculate the number of bacteria adhered to cells. Adhesion of bacterial cells on 50 of epithelial cells is considered as a positive result for ability of bacterium to adhesion.
Multiplex PCR for detection of virulence genes
Extraction of DNA: This method was carried out according to Yang et al.17, as follows: Five pure and fresh colonies were suspended in 200 μL of dry weight and the bacterial cells were lysed by boiling at 100°C for 20 min (in water bath). The bacterial cells were placed in ice for 40 min and the other cellular components were removed by centrifugation at 9000 rpm for 10 min. Finally the supernatant was used as the DNA template17.
PCR detection of virulence-associated genes: A multiplex PCR was used to detect 10 genes encoding: Adhesins (fimH, papA and eaeA), siderophores production (feoB and iutA), capsule synthesis (kpsMTII), heamolysine (hlyF), toxin production (stx1 and stx2) and invasion (ibeA) (Table 1, 2).
Antimicrobial susceptibility testing: Antimicrobial susceptibility testing for the E. coli isolates was performed using a Kirby-Bauer method according to the Clinical Laboratory Standards Institute CSLI20. The following 14 antimicrobials were tested provided from (Bioanalyse, Turkey): amoxicillin+clavulanic acid (AMC) 30 μg, amoxicillin (AX) 25 μg, cefotaxime (CTX) 30 μg, ceftriaxone (CRO) 30 μg, ceftazidime (CAZ) 30 μg, imipenem (IMP) 10 μg, gentamicin (CN) 10 μg, tetracycline (TE) 30 μg, amikacin (AK) 30 μg, tobramycin (TM) 10 μg, doxycycline (DO) 30 μg, ciprofloxacin (CIP) 5 μg, chloramphenicol (C) 30 μg and nitrofurantoin (F) 300 μg. A fresh and pure colony from each E. coli isolate was grown overnight in Mueller Hinton broth (Oxoid, UK) at 37°C. Bacterial growth were adjusted to 0.5 on the MacFarland nephelometer scale (1.5×108 CFU mL–1) and plated on Mueller Hinton agar (Oxoid, UK) by the streaking method using a sterile swab. Antibiotics susceptibility and resistance was determined by growth zone diameter of isolate according to CLSI21. Escherichia coli ATCC 25922 strain was used as control.
Statistical analysis: Graphpad prism software (version 7.0) was used to statistically analyze the data.
RESULTS
Total isolates: Out of the 390 total specimens there were 190 specimens (48.717% stained with Gram negative) while there were 172 specimens (44.104% stained with Gram positive) and 28 specimens (7.179% without growth) (Table 3).
Also the results proved that, a total of 190 g negative bacterial isolates were collected from different infections (90 from urine, 75 from heavy diarrhea, 25 from blood) there were 50 E. coli isolate, 25 isolates (50%) from urine, 18 isolates (36%) from heavy diarrhea and 7 isolates (14%) from blood (Table 4).
Virulence factors: Different phenotypic procedures were used to investigate virulence factors, siderophores production, biofilm formation and adhesion were observed in 50 isolates (100%). The capsule productions and hemolysis were observed in 18 isolates (36%) and 8 isolates (18%), respectively (Fig. 1-3, Table 5).
| Table 3: | Total specimens isolated from patients in the current study (N = 390) |
| Table 4: | Prevalence of Escherichia coli strains according to source of infections (N = 190) |
| Fig. 1: | Escherichia coli on congo red agar plate, showing red-black colonies as a positive result for biofilm formation |
| Fig. 2: | Aggregation of Escherichia coli around epithelial cells as a positive result for ability to adhesion |
| Fig. 3: | Escherichia coli strain on blood agar plate surface after 24 h showing hemolysis phenomenon |
| Table 5: | Prevalence of virulence factors among Escherichia coli by phenotypic characterization according to source of infections (n = 50) |
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| Table 6: | Prevalence of virulence genes among 50 Escherichia coli strains according to sample source |
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| Table 7: | Profile of virulence genes among 50 Escherichia coli strains according to sample source |
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Also, out of total 50 isolates, there were 45 isolates (90%) were positive for fimh gene, 18 isolates (36%) were positive for papA gene, 49 isolates (98%) were positive for feoB gene, 26 isolates (53%) were positive for kpsMTI (Fig. 4), 5 isolates (10%) were positive for ibeA gene, 25 isolates (50%) were positive for iutA gene and 8 isolates (16%) were positive for hlyF gene (Fig. 5), 2 isolates (4%) were positive for stx1 and eaeA gene and 4 isolates (8%) were positive for stx2 gene (Fig. 6). The prevalence of virulence factors and virulence genes were given in Table 6 and 7.
Antimicrobial susceptibility test: Antimicrobial susceptibility test was conducted for 50 strains of E. coli. This study indicated that the antimicrobial resistance rates of the 50 isolates to amoxicillin, amoxicillin+clavulanic acid, cefotaxime, ceftriaxone and ceftazidime were all high 90-100%. The lowest resistance rate was observed for imipenem (6%). Resistance test to 14 antibiotics of E. coli strains were given in Table 8 and 9.
| Fig. 4(a-c): | Multiplex PCR amplified products from extracted DNA of 50 Escherichia coli isolates amplified with diagnostic genes; fimH, papA, kpsMTII and feoB show positive results at 508, 720, 272 and 470 bp, respectively, L: DNA molecular size marker, (a) Strain No. 1-17, (b) Strain No. 18-34 and (c) Strain No. 35-50 |
| Fig. 5(a-c): | Multiplex PCR amplified products from extracted DNA of 50 Escherichia coli strains amplified with diagnostic genes; iutA, ibeA and hlyF show positive results at 587, 814 and 444 bp, respectively, L: DNA molecular size marker, (a) Strain No. 1-17, (b) Strain No. 18-34 and (c) Strain No. 35-50 |
| Fig. 6: | Multiplex PCR amplified products from extracted DNA of Escherichia coli amplified with diagnostic genes; eaeA, stx2 and stx1 show positive results at 384, 225 and 180 bp, respectively, L: DNA molecular size marker |
| Table 8: | Antimicrobial sensitivity test to 14 antimicrobials for 50 Escherichia coli strains isolated from patients with different infections |
| Table 9: | Profile of 50 clinical isolates of Escherichia coli according to antimicrobials resistance and prevalence of virulence genes |
AMC: Amoxicillin+clavulanic acid, AX: Amoxicillin, CTX: Cefotaxime, CRO: Ceftriaxone, CAZ: Ceftazidime, IMP: Imipenem, CN: Gentamicin, AK: Amikacin, TM: Tobramycin, TE: Tetracycline, DO: Doxycycline, CIP: Ciprofloxacin, C: Chloramphenicol, F: Nitrofurantoin | |
DISCUSSION
Escherichia coli is one of the most important Gram negative bacteria causing urinary tract infections, acute diarrhea and bloodstream infections worldwide. Escherichia coli have many important virulence factors are associated with adhesion, colonization and survival in different human tissues9,22. These virulence factors are fimbriae, proteins, toxins, siderophores and capsular polysaccharides23.
In this study, the results indicated that the fimH adhesion gene was present in 45 of all isolates (90%), 23 isolates (92%) from urine, 15 isolates (83.33%) from heavy diarrhea and 7 isolates (100%) from blood. The papA was the second prevalent adhesion gene (36%) of all isolates; 8 isolates (32%) from urine, 6 isolates (33.33%) from heavy diarrhea and 4 isolates (57.14%) from blood. The third prevalent gene in all our isolates was eaeA (4%), it was prevalent in two isolates from heavy diarrheal samples only. Some previous studies indicated that fimH, papA and eaeA were most important virulence genes in E. coil strains isolated from urine and fecal samples9,24-26.
The most important step to development of UTI is adherence to urinary epithelia, allowing the bacteria to stay and persist in the urinary tract against flushing by urine flow and activation of host signaling pathways27,28.
The feoB and iutA genes which are two of the bacterial iron acquisition systems. In this study, the feoB gene was present in 49 of all isolates (98%), 18 and 7 isolates (100%) from heavy diarrhea and blood. respectively and 24 isolates (96%) from urine. The iutA gene was present in 25 of all isolates (50%); 7 isolates (100%) from blood, 12 isolates (48%) from urine and 6 isolates (33.33%) from heavy diarrhea. These findings concerning the frequency of the iutA gene in E. coli isolates are in accordance with previous reports of 30% Moreno et al.29 for faecal isolates. Because of there was low concentrations of free iron (in human host) for multiplication of pathogenic bacteria. Therefore, many bacteria such as E. coli have several means for iron uptake from the host through the expression of iron-acquisition systems30.
Siderophore production is important for each pathogen especially in pathogenic bacteria for persisting and survival in the blood stream and other human tissues such as epithelial tissues. When iron availability is become limited in the host, siderophore system play an important role as a virulence factor that contributes to bacterial growth in fluids and host tissues31,32. The prevalence of the iutA gene found in our E. coli isolates correlates with results published by Moulin-Schouleur et al.19, it is noted that the iutA gene, which encodes the aerobactin receptor was present in a high rate of strains isolated from patients with bloodstream infection.
The iutA gene is commonly associated with ExPEC and those isolated from blood infection in human33,34. The relationship between bacterial iron acquisition systems and many types of infections in human had been suspected in previous studies35,36. Zhao et al.37 and Yun et al.9 reported that feoB and fimH were the most prevalent virulence genes in E. coli strains isolated from human with UTI. The feoB and iutA genes might have a role in iron acquisition in bloodstream infection and UTI. The feoB gene seems to be work differently and has an additional mechanism in bloodstream infection strains in addition to in UTI.
In this study, genes related to hemolysin and toxins production (hlyF, stx1 and stx2) were researched. The hlyF gene is frequently detected in all blood isolates (7 isolates 100%) and detected in only one isolate from urine (4%). While the stx1 and stx2 genes were present in diarrheal samples only, 2 isolates (11.11%) and 3 isolates (16.66%), respectively. Mohsin et al.38 showed that 11 (78.5%), 11 (78.5%) and 6 (42.8%) were positive for stx1, stx2 and eaeA genes, respectively.
The utilization of many toxins secreted by some strains of E. coli is not fully understood. Among these toxins, putative hemolysin encoded by hlyF gene is an extracellular cytolytic protein toxin that is produced by more than 30% of total strains of E. coli. This toxin has been associated with both clinical severity in UTI patients and bloodstream infection39. Haemolytic activity in E. coli has been attributed to many types of haemolysin genes. A new class of haemolysin represents a novel bacterial gene designated hlyF gene40.
These results suggest that this gene (hlyF) is strongly associated with E. coli isolates from bloodstream infection. Each enterohemorrhagic Escherichia coli (EHEC) strains cause dangerous infections in humans and have at least one Shiga like toxin (stx1 or stx2) gene. Shiga like toxins are also is the major virulence factors of Shiga toxin-producing Escherichia coli (STEC) strains, among these toxins, stx1 and stx2 encoded by stx1 and stx2 genes which have an important role in human pathogens linked to non-blood and bloody diarrhea, hemorrhagic colitis and hemolytic uremic syndrome41.
The stx1 genotype is one of the most important factors of clinical outcome of shiga toxin producing Escherichia coli (STEC) infection and that pathogenicity for humans was higher in the stx2 genotype strains42.
The eaeA gene which has been shown to be necessary for attaching and effacing activity, encodes a protein which is called intimin. Numerous investigators have underlined the strong association between the carriage of eaeA gene and the capacity of STEC strains to cause severe human illnesses, such as blood stream infection and haemolytic uraemic syndrome. But the production of this virulence factor is not a very essential for pathogenesis, because of many cases of bloodstream infection in human have been caused by STEC were negative for eaeA gene26,43.
This current study revealed that the ibeA gene was present in 10% of total isolates, 2 isolates (8%) from urine and 3 isolates (16.66%) from stool. The percentage of E. coli isolates with the ibeA gene was not similar to that found for strains isolated from faeces by Watt et al.44 (4%) and Kaczmarek et al.45 (4.5%). These differences in percentage might be that according to the differences between types of virulent strains associated with their different phylogenetic backgrounds.
The ibeA is one of the most important virulence gene encoded for invasion protein has been identified as an important virulence factor involved in the invasion of brain micro-vascular endothelial cell that enables penetration across the blood brain barrier45,46. Soto et al.47 suggested that ibeA gene may has an important role in the early steps of E. coli sepsis and meningitis in neonatal because of he reported that the ibeA gene is significantly more prevalent in E. coli strains causing early infection than in those involved in late infection.
Capsular polysaccharides is another important virulence factor in bacteria, which protects this pathogen from phagocytosis, bactericidal effects of serum factors and antimicrobials effect.
Escherichia coli have many capsular virulence genes responsible for more than 80 chemically and serologically distinct capsules, called K antigens48,49. The majority of ExPEC strains of E. coli associated with invasive disease express group 2 capsules50. The kpsMTII gene is one of the most important virulence genes that encodes proteins required for translocation of E. coli group II capsular polysaccharide across the inner membrane51. In these results the kpsMTII gene was prevalence in 26 of total isolates (52%), 11 isolates (44%) in urine, 11 isolates (61.11%) in heavy diarrhea and 26 isolates (52%) were detected in blood samples.
Bacterial capsule is well known as a virulence determinants that contribute to resistance against different human host defenses. Capsular polysaccharides (type K) is associated with ExPEC and uropathogenic E. coli (UPEC) infection, often to different degrees depending on the serotype of the strain52. These results suggest that the kpsMTII gene is particularly associated with pathogenicity of E. coli strains.
In recent years, antibiotic resistance has increased and became a major problem in many countries especially in developing countries23,53-55. Antimicrobial resistant E. coli strains cause more severe diseases for longer periods, therefore, the treatment of diseases caused by this bacterium often require many classes of antimicrobial therapy.
In this study, most E. coli strains isolated from bloodstream were resistant to most antimicrobials particularly β-lactam antibiotics and third generation cephalosporins. It might be that long term exposure to these antimicrobials by patients infected with bacteremia lead to horizontal transfer of plasmid resist antimicrobial genes between different strains of bacteria.
The results of this study demonstrated that there was positive relationship between the ability of E. coli strains to antimicrobial resistance and the prevalence of virulence genes and source of isolates. Almost all E. coli strains isolated from bloodstream were resistant to most antimicrobials, in the same time they have most of virulence genes. The AX, AMC, CTX, CRO, CAZ, IMP, CN, TM, TE, DO, CIP, C and F resistance has been associated with a higher prevalence of feoB, iutA, fimH, kpsMTII, hlyF and papA genes.
There was strong relationship between resistance of antimicrobials and prevalence of some virulence factors in bacteria. Virulence factors such as capsular polysaccharides, outer membrane lipoproteins and biofilms play an important role in protecting bacteria from antimicrobials exposure when compared with those do not have these virulence factors56. Bacteria with biofilm forming are generally more resistant to many antimicrobials57. Capsular polysaccharides, outer membrane lipoproteins and biofilms act as biodegradable effect on β-antimicrobials. The β-lactamase enzymes are secreted and maintain their activity inside of biofilm matrix and decompose β-lactam antibiotics before reach to the bacterial cells57. Antimicrobials penetration inside of capsular polysaccharides, outer membrane and biofilms could be blocked by other factors, such as the presence of surfaces with negatively-charged, particularly for large polar molecules such as aminoglycosides with positively charged. Also, trace amount of metabolic rates and limited oxygen are may be important factors contributing to increasing resistance to aminoglycosides, β-lactamase, fluoroquinolones and cephalosporins58,59.
Many investigations have been reported that E. coli strains have multidrug resistance and virulence factors10,60,61. Also, Ibrahim et al.62 proved that the clinical E. coli isolates had the highest resistance to β-lactam antibiotics.
Multiple resistances of E. coli strains to β-lactams antibiotics including sulphonamides, fluoroquinolones, expanded spectrum aminoglycosides, cephalosporins and tetracycline have been reported previously by Bradford et al.63. While, De Verdier et al.64 reported that 61% of all E. coli strains were resistant to more than one antimicrobial. These differences in virulence genes and antibiotic resistance properties in the clinical samples could be related to types of bacterial strains, differences in geographical area, type of samples tested and antibiotic prescription preference among clinicians.
CONCLUSION
In conclusion, these results revealed that E. coli isolates from bloodstream showed higher level of antibiotic resistance and have many virulence factors than those isolates from urine and heavy diarrhea. The feoB, iutA, fimH, kpsMTII, hlyF and papA genes were implicated as important virulence genes for development of bloodstream infection in human.