|Year : 2019 | Volume
| Issue : 6 | Page : 244-249
Genotyping and molecular characterization of extended-spectrum beta-lactamases-producing uropathogenic Escherichia coli in and around Coimbatore district, Tamil Nadu, India
Mylsamy Muraleetharan1, Thirumoorthy Viswanathan2
1 Research and Development Centre, Bharathiar University, Coimbatore, Tamil Nadu, India
2 Department of Microbiology, LRG Government Arts College for Women, Tirupur, Tamil Nadu, India
|Date of Submission||29-Jun-2019|
|Date of Decision||17-Sep-2019|
|Date of Acceptance||01-Oct-2019|
|Date of Web Publication||23-Dec-2019|
Mr. Mylsamy Muraleetharan
Research and Development Centre, Bharathiar University, Coimbatore - 641 046, Tamil Nadu
Source of Support: None, Conflict of Interest: None
Background: Escherichia coli, an extraintestinal flora, develops virulence traits through a persistent encounter with host and constant exposure to antibiotics, which makes it line up as the most common urinary pathogen. Thus, this study aimed to screen the presence of virulence traits among multidrug-resistant urinary pathogenic E. coli among the urine samples collected from inpatients and outpatients of the multispecialty hospitals. Materials and Methods: Standard microbiological laboratory protocols were followed, and about 210 samples were processed and screened. Results: Among those samples, 114 were reported positive for the presence of uropathogenic E. coli(UPEC). Gender-wise distribution was found to be more among female patients (56%) than male patients (51%). During screening for hemolytic activity, 37% of test isolates were α hemolytic, 48% were β hemolytic, and 15% were γ hemolytic. Screening virulence among test isolates, sfa gene (49%), afa (42%), hly (68%), CNF (43%), and aer, accounts for 65%. Further, the multidrug resistance of the isolates was done by using ten antibiotics. All the isolates exhibited the multiple antibiotic resistance (MAR), and the highest percentage of resistance was found against ceftazidime (100%), and the least percentage of resistance was observed against imipenem (2%) followed by amikacin (8%). MAR index values of all the isolates ranged from 0.4 to 1. Conclusion: The presence of various virulence genes and the high degree of resistance among the isolates against the antibiotics used in this study confirm the prevalence of extended-spectrum β-lactamase producers among the UPEC.
Keywords: Escherichia coli, extended-spectrum beta-lactamases, multiple drug-resistant, urinary tract infection, uropathogen
|How to cite this article:|
Muraleetharan M, Viswanathan T. Genotyping and molecular characterization of extended-spectrum beta-lactamases-producing uropathogenic Escherichia coli in and around Coimbatore district, Tamil Nadu, India. Urol Sci 2019;30:244-9
|How to cite this URL:|
Muraleetharan M, Viswanathan T. Genotyping and molecular characterization of extended-spectrum beta-lactamases-producing uropathogenic Escherichia coli in and around Coimbatore district, Tamil Nadu, India. Urol Sci [serial online] 2019 [cited 2020 Aug 6];30:244-9. Available from: http://www.e-urol-sci.com/text.asp?2019/30/6/244/273880
| Introduction|| |
Urinary tract infection (UTI) is common among the human population worldwide. In 1987, it was accounted that 10% of the human societies have UTI at some time during their lives, as reported by Boscia and Kaye. However, this proportion was changed as nearly 50% of all women will have experienced at least one UTI in their lifetime. Not alone females, males are also prone to UTI, but the prevalence and incidence rate of UTI are high in females than males. Various organisms cause UTI, among which bacteria are the dominant causative organisms which stand for more than 95%. Escherichia More Detailscoli is the most predominant causative organism of UTI and is uniquely responsible for more than 80% of the infections.
E. coli is known to be a healthy flora in the intestine and the predominant causative agent of UTI as well as multidrug resistance (MDR). Uropathogenic E. coli (UPEC) has the potential to colonize and invade the host cell to establish the infection. UPEC exhibits various virulence factors and favors the adhesion of pathogens to the host to develop the infection in the host. S fimbria, one of the adhesion factors which causes the pathogen to adhere to the host, was encoded in the sfa gene. The fimbrial adhesion was also the vital virulence factor of UPEC, which is embedded in the afa gene reported in recurring cystitis as shown by Servin. After adhesion, the pathogen should establish infection in the host using secretory virulence factors such as hly (hemolysin) and CNF (cytotoxic necrotizing factor) genes., Siderophore encoding gene (aer) was also documented as an essential virulence factor of UPEC.
Many β-lactam ring antibiotics were synthesized followed by the discovery of penicillin as reported by Sir Alexander Fleming in 1927, including derivatives of extended-spectrum β-lactams such as cephalosporins (cephems), monobactams, and carbapenems. First, antibiotic resistance was reported in the 1940s followed by MDR and extended-spectrum β-lactamase (ESBL) reported in the 1980s, which continues further. E. coli has the potential to produce β-lactamases, which hydrolyze cephalosporins and monobactams and mediate resistance against the extended-spectrum of antibiotics. ESBL producer's prevalence was reported worldwide, which differs from every locality and hospital based on the usage of antibiotics due to inappropriate antimicrobial therapy.,
Thus, characterizing the virulence genes improves our knowledge about understanding the pathogenicity of UPEC, and antibiotic resistance study helps the medical practitioners to be aware of treatment failures due to MDR and ESBL. Furthermore, it guides them to appropriate empirical antimicrobial therapy.
| Materials And Methods|| |
Isolation and identification of uropathogenic bacteria
Collection of samples
As E. coli is the most predominant causative agent for UTI, the study was focused on the isolation of UPEC. A total of 210 urine samples from multispecialty hospitals in and around Coimbatore were collected for the isolation of ESBL-producing UPEC. Luria Bertani (LB) broth was inoculated with 1 mL of the sample and incubated at 37°C overnight. After incubation for about 24 h, one loopful of the culture was streaked on eosin methylene blue (EMB) agar. Further, the isolates were identified by Gram staining and series of biochemical tests.
Maintenance of uropathogenic Escherichia coli
Nutrient agar slants were prepared in the test tubes, and a pure culture grown on the EMB agar was streaked on them. The tubes were incubated at 37°C for 24 h and refrigerated for preservation. The cultures inoculated onto the LB broth tubes were used for further study.
For all the isolates, the hemolytic activity was examined on blood agar plates. After 24 h of incubation at 37°C, hemolytic activity was identified as a zone of hemolysis around the colonies on blood agar plates containing 5% sheep red blood cells. UPEC isolates were cultured on blood agar plates (incubated at 37°C for 24 h) and observed for the α, β, or γ hemolytic activity.
Genotyping of virulence genes
Isolation of DNA
Bacterial culture grown in LB broth was distributed in 1 mL aliquots into microfuge tubes and centrifuged at 14,000 rpm for 5 min, and the obtained pellets were suspended in 200 μL of deionized water and subjected to boiling at 100°C for 15 min. After boiling, the microfuge tubes were placed immediately in an ice bath for 5 min and then centrifuged for 5 min at 14,000 rpm at room temperature. The supernatant containing DNA was transferred to another clean tube and stored at −20°C.
Polymerase chain reaction (PCR) was carried out for the screening of virulence genes in the test isolates. The genes responsible for fimbrial adhesion (sfa), afimbrial adhesion (afa), hemolysin (hly), cytotoxic necrotizing factor (CNF), and siderophore encoding gene (aer) were screened using specific primers. The primers used in this study are tabulated in [Table 1] and [Table 2]. Each reaction was carried out using 5 μL of PCR 2X master mix red dye with Tris-HCl (150 mM; pH 8.5), (NH4)2 (40 mM), SO4 (4.0 mM), MgCl2(1.5 mM), dNTPs (0.4 mM), Taq DNA polymerase (0.05 U/μL), inert red dye and a stabilizer (Ampliqon, Denmark), 2 μL of nuclease-free water, 1 μL of each forward and reverse primer (10 pM), and 1 μL of template DNA (50 ng). The PCR was carried out using a Mastercycler gradient (Eppendorf, Germany). The amplified product was separated by horizontal gel electrophoresis.
|Table 2: Polymerase chain reaction conditions for the amplification of various genes|
Click here to view
Antibiotic susceptibility testing
Antibiotic sensitivity assay was performed for all the test isolates. The third- and fourth-generation cephalosporin and carbapenems antibiotics such as cefotaxime, ceftazidime, cefapirin, amikacin, cefixime, vancomycin, nalidixic acid, cefazolin, gentamycin, and imipenem were used in this study. The antibiotic sensitivity test was carried out by the disc diffusion method using Muller–Hinton agar (MHA) (HiMedia, India). Pure cultures grown in nutrient broth overnight were swabbed over MHA using sterile cotton swabs. Antibiotic discs were placed on the agar surface with sufficient space and incubated at 37°C for 24 h after 30 min of prediffusion time. The inhibition zone was measured after the incubation and compared with the manufacturer's interpretative chart and classified as resistant, intermediate, and sensitive.
Multiple antibiotic resistance index
An isolate's multiple antibiotic resistance (MAR) index is the number of antibiotics to which the isolate has shown resistance over the total number of antibiotics to which the test organism was sensitive. The MAR index value >0.2 is considered to have originated from a high-risk contamination source where antibiotics are used very frequently, whereas the index value ≤0.2 indicates that the isolate has originated from sources where antibiotics are rarely or never used.
| Results|| |
Isolation and identification of uropathogenic Escherichia coli
The urine samples were collected from inpatients and outpatients of multispecialty hospitals around Coimbatore from both genders irrespective of the age group for the isolation of UPEC. Among 210 samples, 188 samples were from inpatients and 22 from outpatients. The isolates were subjected to Gram's staining and streaked on selective medium. A total of 114 isolates exhibited metallic sheen colonies on EMB agar plates, which confirm the presence of E. coli in the samples. Among 114 isolates, 36 were isolated from male and 78 isolates obtained from female patients.
Prevalence of uropathogenic Escherichia coli
The percentage of the prevalence of UPEC isolates was screened and was found to be high among outpatients (50%), whereas 55% of incidence was noticed among the inpatients [Table 3]. The gender-wise incidence among the patients was noticed. The highest percentage was seen among female patients (56%) compared to male patients (51%), and the results are tabulated [Table 4]. In age-wise prevalence, among the test isolates, 58% of female and 71% of male patients above the age of 35 and 42% of female and 29% of male patients above the age of 50 were found to be positive for UPEC.
|Table 4: Gender-wise distribution of uropathogenic bacteria in urinary tract infection samples|
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UPEC isolates obtained grown overnight on tryptic soy agar, and a single colony was streaked on 5% blood agar plates for determining the hemolytic activity. The tested isolates showed varying hemolysin (α, β, and γ) production. Among the isolates, 37% were α hemolytic, 48% were β hemolytic, and 15% were γ hemolytic.
Distribution of virulence gene among uropathogenic Escherichia coli
Virulence genes favor the fimbrial and afimbrial adhesion, and secretory factors are screened in all the test isolates. Among them, sfa gene encoding S fimbriae were found to be present in 49% of isolates, and afa gene encoding afimbrial adhesion was found in 42% of isolates. Hemolysin gene (hly) was present in 68% of isolates, and CNF gene was found to be present in 43%. Siderophore encoding gene (aer) was documented in 65% of test isolates.
Antibiogram of the isolates
All the 114 isolates obtained were screened for antibiotic sensitivity against ten commercially available antibiotics. The highest percentage (100%) of resistance was found against ceftazidime, and the least percentage of resistance was found against imipenem (2%). About 99% of isolates showed resistance against cefapirin, cefazolin, and cefixime, 97% against cefotaxime, 80% against vancomycin, 58% against nalidixic acid, 32% against gentamycin, and 8% against amikacin. This shows that these organisms acquired the resistance through transferable elements from close members of phylogeny, which were frequently exposed to antibiotic prophylaxis.
Multiple antibiotic resistance index
The MAR index values of all the isolates ranged from 0.4 to 1. MAR index of 0.9–1 and 0.8–0.9 was exhibited by 4% and 42% of the isolates, respectively. MAR index of 0.7–0.8 and 0.6–0.7 was exhibited by 35% and 10% of the isolates, respectively. About 6% of isolates exhibited a MAR index value of 0.5–0.6, and 3% exhibited index value between 0.4 and 0.5. None of the isolates exhibited MAR index <0.4. The results serve as evidence that all the isolates have exposure to the antibiotics used in this study, which confirms that the isolates were isolated from the area where antibiotics are intensively used.
| Discussion|| |
UTI is the most common clinical condition which can affect both genders at any age. The severity of infection depends on the presence of virulence factors and antibiotic resistance exhibited by the pathogen. Hence, the present study begins with the isolation of UPEC.
Isolation and prevalence of uropathogenic Escherichia coli
The urine samples were collected from inpatients and outpatients from both genders irrespective of the age group for the isolation of UPEC. In this study, a total of 114 (54%) isolates were identified as E. coli as reported by Singh et al. and Munkhdelger et al. in the samples by classical techniques. Tankhiwale et al. obtained 49.8% E. coli. Our results also coincide with those of Ali et al. who have isolated, altogether, 148 (59%) isolates from inpatients. Shrivastava et al. obtained 68.18% E. coli. A large spectrum of organisms has been reported by Jain et al. from patients of UTI with E. coli (65.95%). Hasan et al. have also reported a high incidence of E. coli (50.7%). This confirms that the occurrence of E. coli in UTI will be the account between the range of 45 and 65%. However to the contrary, Zahera et al. recorded 30% of the occurrence of UPEC. Kumari et al. isolated 76% E. coli from urine samples and Sabir et al. reported 80% of occurrence. From this, we can conclude that E. coli was the predominant causative agent of UTI.
Among 114 isolates tested, 36 (51%) were isolated from male and 78 (56%) were obtained from female patients in the present study. This is due to a short urethra and proximity to the anal opening. This corresponds with Jain et al. and Gupta and Maheshwari who reported female preponderance for this infection. This result will also be supported by the reports of Alqasim et al. who designed the study in which 76 (76%) belonged to female patients while 24 (24%) belonged to male patients. Fatima et al. isolated 76% E. coli, among which 72% were in females and 28% in males.
In the present study, the percentage of prevalence of UPEC was found to be high among inpatients (55%), whereas 50% of incidence was noticed among the outpatients. But Bharara et al. have also isolated ESBL from the outpatient department (76.7%), while only 23.3% were from the inpatient department. This result shows that the prevalence of UPEC was high among the inpatients. In age-wise prevalence, among the test isolates, 58% of female and 71% of male patients above the age of 40% and 42% of female and 29% of male patients below the age of 40% were found to be positive for UPEC. Similarly, Seyedjavadi et al. studied the prevalence of UPEC among the range of patients' age from 11 months to 75 years. We can conclude that the incidence of occurrence of UPEC will be high at sexually active stage and menopause. Tabasi et al. also stand as supporting evidence for this.
Virulence traits and distribution of virulence gene in uropathogenic Escherichia coli
Hemolysin is found to be an important virulent trait of the pathogen, which accounts for resisting serum activity and establishing itself inside the host. During screening hemolysin (α, β, and γ) production, the test isolates exhibited α hemolytic (37%), β hemolytic (48%), and γ hemolytic (15%) activity. But Zhao et al. obtained only β hemolytic and γ hemolytic activity in their study, and α hemolytic activity was not noticed. According to the results of Tabasi et al., hemolysin production was observed in 34% of the UPEC isolates.
Fimbriae-associated (sfa) and afimbriae-associated adherence (afa) are the important factors in the pathogenesis of UTI and cytotoxic necrotizing factor (CNF) that provide UPEC with the ability to cause tissue damage, facilitate bacterial dissemination, release host nutrients, and disable immune effector cells. Aerobactin (aer) accounts for iron uptake and also plays a major role in the pathogenicity of UPEC. In the present study, sfa gene was found to be present in 49% of isolates, and afa gene was found in 42% of isolates. Hemolysin gene (hly) was present in 38% of isolates, and CNF gene was found to be present in 43%. Siderophore encoding gene (aer) was documented in 65% of test isolates.
Munkhdelger et al. obtained the least occurrence of hlyA (8.1%),sfa (8.8%), and afa (15.5%), but aer gene was present in 56.1% of test isolates obtained from the patients. Zhao et al. also got the least percentage of occurrence of afa (4%), sfa (49%), hly D (11%), and CNF 1 (9%). This shows the evidence for the emergence of commensal as a pathogen, which later develops the machinery required to establish its virulence. On the other hand, the results of Morales-Espinosa et al. show the highest level of occurrence of virulence genes sfa (100%), CNF (95.6%), aer (91.3%), and hly (73.9%). This gives the evidence that these isolates may be associated with recurrent infection. Raeispour and Ranjbar also reported the prevalence of aer (90%) and hly (60%) as reported by Tabasi et al. In their study, they recorded the presence of afa (34.9%), sfa (18.2%), hly (68.2%), CNF (63.6%), and aer (90.9%). However, Momtaz et al. obtained a higher proportion of CNF 1 (62%) and hly (62%) when compared to the iron gene (52%), but Santo et al. got aer as the most prevalent iron gene receptor (73.1%) compared to CNF1 (36.8%) and hlyA gene (30.8%). From all these reports, we come to inference that there is variation in notch in the prevalence of virulence genes according to the level of exposure of pathogen to the host.
Antibiogram of the isolates
UPEC is well known for exhibitors of MDR and ESBL. Among ten antibiotics, the test isolates showed the highest percentage (100%) of resistance against ceftazidime. About 99% of isolates showed resistance against cefapirin, cefazolin, and cefixime, 97% against cefotaxime, 80% against vancomycin, 58% against nalidixic acid, 32% against gentamycin, and 8% against amikacin, and the least percentage of resistance was found against imipenem (2%).
Like the present study, Momtaz et al. performed a disk diffusion method to study antibiotic resistance against various antibiotics. The maximum resistance was against penicillin (100%), and the minimal resistance was against nitrofurantoin (5.69%). Kumari et al. reported that the antibiotic sensitivity pattern of E. coli showed that 73% were sensitive to imipenem, amikacin, and cefoperazone-sulbactam, while 26% were sensitive to norfloxacin and 25% to cefepime. Here, the resistance against imipenem and amikacin was higher than the present study. Same disk diffusion method was followed by Munkhdelger et al. who indicated that the test isolates were ESBL-producing MDR strains that are sensitive toward imipenem.
Ali et al. got the maximum percentage of resistance against co-trimoxazole (82%) and cephalosporin (80%), followed by other generations of cephalosporin. However, the percentage of resistance against nitrofurantoin, tetracycline, carbapenem, and β-lactam inhibitors was <10%. Our results nearly correspond with the results of Raeispour and Ranjbar. The isolates used in their studies showed 60% resistance to nalidixic acid (63%), amikacin (8%), and imipenem (0%). Similarly, Fatima et al. got resistance index as imipenem (6%), amikacin (4%), gentamicin (40%), cefixime (66%), and cefotaxime (60%). Many other reporters also studied the degree of resistance of UPEC. For example, Seyedjavadi et al. reported the susceptibility rates of isolates and concluded high susceptibility toward amikacin and imipenem.
Similarly, the result of the current study is found to be supported by the study results of Harwalkar et al., Tabasi et al., and Alqasim et al. Nevertheless, Gupta and Maheshwari reported that few isolates found the drugs like imipenem, which is used as last resort in the healthcare settings, resist. The isolates obtained in the present study also exhibited MAR index of >0.4, which confirms that the isolates were constantly exposed to antibiotics. The antibiotic resistance pattern of the present study and former reports gives us clear evidence that imipenem and amikacin can be suggested as a drug of choice for treating ESBL-producing UPEC.
| Conclusion|| |
The present study demonstrates the importance of screening the virulence factor and antibiotic resistance of significant pathogens to have a better understanding of the causative agent since virulence genes and antibiotic resistance play a major role in the disease progression and indefatigable treatment. Nowadays, commensal and UPEC share the same phylogenetic background and antibiotic resistance due to the adaptation of extraintestinal lifestyle and cause infection. Thus, molecular screening of virulence traits and antibiotic resistance gives us knowledge about the disease progression and problems in treating them. UPEC screened in the present study was positive for various virulence and promising resistance. This shows the prevalence of medically imperative UPEC in the surveyed area. Degree of antibiotic resistance of the test isolates showed a range of exposure of the isolates to antibiotics and difficulties in treating them. This shows the importance of using antibiotics in empirical antimicrobial therapy.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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[Table 1], [Table 2], [Table 3], [Table 4]