|Year : 2021 | Volume
| Issue : 3 | Page : 313-319
Antibiotic resistance, phylogenetic group, and genotyping investigation in Escherichia coli strains of gut flora in patients with colorectal cancer in Iranian population
Mahsa Mirzarazi1, Mojgan Bandehpour1, Ali Hashemi2, Mahmoud Vahidi3, Afsoon Taghavi4, Soroor Bashiri4, Bahram Kazemi5
1 Department of Medical Biotechnology, School of Advanced Technologies in Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran
2 Department of Microbiology, School of Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran
3 Department of Medical Laboratory Sciences, School of Allied Medical Sciences, Aja University of Medical Sciences, Tehran, Iran
4 Department of Pathology, School of Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran
5 Cellular and Molecular Biology Research Center, Shahid Beheshti University of Medical Sciences, Tehran, Iran
|Date of Submission||01-Jun-2021|
|Date of Acceptance||12-Jul-2021|
|Date of Web Publication||7-Sep-2021|
Dr. Mojgan Bandehpour
Cellular and Molecular Biology Research Center, Shahid Beheshti University of Medical Sciences, Tehran; Department of Medical Biotechnology, School of Advanced Technologies in Medicine, Shahid Beheshti University of Medical Sciences, Tehran
Source of Support: None, Conflict of Interest: None
Background: According to the World Health Organization, the prevalence of colorectal cancer (CRC) is the third-most common cancer in Iran. The population of Escherichia coli strains of the B2 phylogenetic group as one of the components of gut microbiota is increased in the gut of people with CRC and can also play a role in the process of cancerous intestinal cells or malignancy. The most common concern about treating infections and their association with cancer is multidrug resistance (MDR). Methods: In this study, stool samples of 20 patients with CRC were examined for sensitivity to 9 antibiotics and their genotypes were determined. Results: More than 90% of the isolates were MDR. Although among 20 isolates of E. coli gut flora in CRC patients, 11 (55%), 7 (35%), 0 (0%), and 2 (10%) strains belonged to phylogenetic groups B2, D, B1, and A, respectively. Conclusions: There was no significant relationship between the B2 phylogenetic group and MDR. Furthermore, the closeness of the species based on >90% similarity, seven clusters were observed that among five clusters, similarities were observed in terms of the phylogenetic group.
Keywords: Antibiotic resistance, colorectal cancer, Escherichia coli, genotyping, phylogenetic groups
|How to cite this article:|
Mirzarazi M, Bandehpour M, Hashemi A, Vahidi M, Taghavi A, Bashiri S, Kazemi B. Antibiotic resistance, phylogenetic group, and genotyping investigation in Escherichia coli strains of gut flora in patients with colorectal cancer in Iranian population. Biomed Biotechnol Res J 2021;5:313-9
|How to cite this URL:|
Mirzarazi M, Bandehpour M, Hashemi A, Vahidi M, Taghavi A, Bashiri S, Kazemi B. Antibiotic resistance, phylogenetic group, and genotyping investigation in Escherichia coli strains of gut flora in patients with colorectal cancer in Iranian population. Biomed Biotechnol Res J [serial online] 2021 [cited 2021 Dec 6];5:313-9. Available from: https://www.bmbtrj.org/text.asp?2021/5/3/313/325611
| Introduction|| |
Colorectal cancer (CRC) is the third-most common cancer in the world and Iran too. Unfortunately each year in the world, 1.36 million people are involved with this cancer., According to the World Health Organization, the prevalence of CRC is 9% and the mortality rate is 7.4% in Iran. It has been shown that a change in the diversity of the intestinal microbes, such as the Escherichia coli (E. coli) population or the digestive dysbiosis, can also play a role in the process of becoming cancerous intestinal cells or malignancy., According to other studies, the population of E. coli strains is increased in the gut of people with CRC in comparison with healthy people, which belonged to B2 phylogenetic group., On the other hand for all researchers in the world of medicine, it is clear that antibiotic resistance is a serious problem for health. Overdose of antibiotic consumption throughout one's life is one of the causes of this problem., Certainly, concerning antibiotic resistance, the most widespread concern is the multidrug resistance (MDR), which is a risk factor for human life at the time of bacterial infection., It seems that in the next 30 years, antibiotic-resistant bacteria will be the most common cause of death in humans compared to cancer. It is important to note, the big family of Enterobacteriaceae has a critical role in this problem. Since antibiotic resistance-inducer genes are transferable between the Enterobacteriaceae family and different E. coli strains., Therefore, it is reasonable to evaluate antibiotic resistance bacteria among intestinal flora such as E. coli. Our study aimed to investigate the MDR E. coli from people who have CRC and healthy ones, and determine phylogenetic groups, and genotype isolates.
| Methods|| |
Sample collection and isolation of bacteria
All sampling principles are observed and stool samples were taken from patients and healthy individuals with full verbal satisfaction and in some cases hospitalized patients were written([Code of Ethics: IR SBMU.RETECH.REC.1398.028]. To identify bacterial agents, from 20 people (9 females and 11 males) who were referred to health centers of University of Medical Sciences and the pathologist diagnosed CRC during colonoscopy, stool samples were collected before any treatment. Samples were cultured on a MacConkey agar and Eosin Methylene Blue agar (Merck, Germany) plates by the standard loop and incubated for 24 h at 37°C. Stand to reason that, to use statistical analysis and comparison of isolates of E. coli from people who have CRC with E. coli isolated from healthy people, stool sampling was necessary from them. Thus, stools of 50 (25 females and 25 males) people who were sure of their health and did not show the symptoms and any relevance of the disease were collected, similar to the patient's feces, they were cultured and isolated. It should be noted that all healthy and patient people received a questionnaire asking questions about annual the amount of antibiotic intake.
In our study, all isolated bacterial DNAs were extracted with the phenol–chloroform protocol. Closely with the use of spectrophotometry and measuring the ratio of 260 nm to 280 nm, extracted DNA absorbance was considered and all samples that their absorbance ratio was 1.8–2 were considered as a pure DNA.
Molecular confirmation for Escherichia coli strains
By polymerase chain reaction (PCR) method and sequencing of the uspA (Universal stress protein-coding) gene, this is possible that conclusively all isolated samples were E. coli strains., The nuleotide sequence of primers and PCR condition were shown in [Table 1].
Phylogenetic groups of the isolated Escherichia coli strains
In this study, phylogenetic groups of all E. coli isolated from the bacterial flora were investigated with PCR performing for chuA (outer membrane hemin receptor ChuA), YjaA (Stress response protein) stress response protein, and TspE4C2 (tail-specific protease) genes [Table 1]., Demonstrably isolates that have chuA were divided into B2 or D phylogenetic groups and that which have not this gene were divided into B1 or A phylogenetic groups [Figure 1].
|Figure 1: Stages of polymerase chain reaction to determine phylogenetic groups of isolated Escherichia coli strains to B2 (brawn), D (red), B1 (yellow), and A (green)|
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Antibiotic susceptibility test
Antimicrobial susceptibility test was performed using the disc diffusion method as described by the Clinical and Laboratory Standards Institute (CLSI) for 9 antibiotics that are most commonly used and had a report of most resistance to them for pathogenic E. coli strains., Briefly, colonies were taken from overnight growth on 5% sheep blood agar (16–20 h, at 35°C) and suspended in Mueller–Hinton broth (Merck, Germany). The turbidity of the suspensions was adjusted to an equivalent 0.5 McFarland. The suspensions were used to inoculate on Mueller–Hinton agar (Merck, Germany) plates. Sulfamethoxazole (SXT), tetracycline (TE), ciprofloxacin (CP), amoxicillin (AMX), nalidixic acid, cotrimoxazole, ampicillin (AM), ceftazidime (CAZ), and kanamycin A (Padtan Teb, Iran) disks were placed on Mueller–Hinton agar. The cultures were incubated at 35°C for 16–20 h. and then, the inhibition zone was measured and compared with data of inhibition zones of test cultures by CLSI. Data were interpreted by the percent of susceptible, intermediate, or resistant isolates as defined by CLSI breakpoint interpretative criteria. E. coli American Type Culture Collection 25922 was used as a quality control strain for disk diffusion.
Molecular genotyping by rep-polymerase chain reaction (box polymerase chain reaction) for Escherichia coli strains
In this study, repetitive element sequence-based PCR (rep-PCR) was performed for all of the samples with the single primer BoxA1R., The nucleotide sequence of primers and temperature conditions were prepared, as shown in [Table 1]. The PCR products were loaded on the 1.5% agarose gel. To determine the proximity of species, rep-PCR profiles were analyzed by GelCompar II software, version 4.0 (Applied Maths, Belgium). In this software, type analysis was performed based on unweighted pair group method with arithmetic mean at 90% similarity level. Similarity level based on dice, optimization 1%, and position tolerance 1%.,
In this study, for data analysis, the independent samples t-test, Pearson Chi-square test, or Fisher's exact test were performed by SPSS software version 16.0. (IBM SPSS software version 16.0).
The study was approved by Scientific Committee of Shahid Beheshti University of Medical Sciences, Tehran, Iran .Under Ethics codes of IR SBMU.RETECH.REC.1398.028.
| Results|| |
The uspA gene amplification
By the uspA gene fragment amplification and sequencing, it was confirmed that all of the 70 isolates of bacteria from the control and patient groups were E. coli.
Isolated Escherichia coli phylogenetic groups
As seen in [Figure 2] and [Figure 3], using standard PCR methods for three genes that were explained above, it was identified among 20 isolates of gut flora E. coli strains in patients with CRC, 11 (55%), 7 (35%), 0 (0%), and 2 (10%) strains belonged to phylogenetic groups B2, D, B1, and A, respectively. Concerning the 50 commensal E. coli isolates of healthy groups, 13 (26%), 12 (24%), 5 (10%) and 20 (40%) bacteria were allocated into B2, D, B1, and A groups, respectively. It is worth noting that a comparison of phylogenetic groups of E. coli isolates and division into two groups was significant (P = 0.02). Hence, we can say that the prevalence of E. coli with phylogenetic group B2 is higher in people with CRC certainly. It should be noted that in both of the control and patients groups, respectively, 3 (6%) and 6 (30%) of E. coli strains belonged to the B2 phylogenetic group, isolated from people who had first- and second-degree family members with types of cancer precedent. This difference was statistically significant P < 0.05.
|Figure 2: Investigation of phylogenetic groups of Escherichia coli strains. (a) B2 and D phylogenetic groups, 1) Marker, 2) Negative control (Escherichia coli that has not chuA gene), 3 and 4) B2 phylogenetic group (chuA+, yjaA+), 3 and 5) D phylogenetic group (chuA+, yjaA−). *chuA = 279bp, yjaA = 211bp. (b) B1 and A phylogenetic groups, 1) Marker, 2) Positive control for chuA gene (Escherichia coli EcoR62), 3 and 4) B1 phylogenetic group (TspE4c2+, chuA−), 3 and 5) A phylogenetic group (TspE4c2−, chuA−)|
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|Figure 3: Division of phylogenetic groups of Escherichia coli strains. (1) 100bp DNA ladder. (2) Positive control (Escherichia coli EcoR62) that has chuA, yjaA, and TspE4C2 genes. (3) B2 phylogenetic group (chuA+, yjaA+). (4) D phylogenetic group (chuA+, yjaA−). (5) B1 phylogenetic group (TspE4c2+, chuA−). (6) A phylogenetic group (TspE4c2−, chuA−)|
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Categorization of the patient and healthy groups based on susceptibility to antibiotics
We have created four categories by people answering to questionnaire about antibiotic intake per year: (1) people who had no consumption ever, (2) people who had one or two times consumption, (3) people who had two or three times consumption, and (4) people who had more than three times consumption.
According to this grouping, in the healthy group, 17 (34%), 27 (55%), 5 (10%), and 1 (2%) of members were belonging to 1, 2, 3, and 4 categories, respectively.
Furthermore, with this arrangement in the CRC group, 6 (34%), 11 (55%), 0 (0%), and 3 (15%) of individuals were categorized. As inserted in [Table 2], among the isolates of the healthy group, the first place for the most sensitivity and resistance in order belonged to CP and AM. While in the other group, utmost sensitivity and resistance were for CAZ and SXT, respectively. As well as, in the two groups that were before mentioned, statistical differences in the abundance of resistance to all antibiotics, except TE and ceftazidime, were significant. According to the diagram that is drawn based on [Figure 4], the frequency of nondrug resistance isolates from healthy individuals and patients was 4 (8%) and 1 (5%), respectively. Among these isolates, sensitivity to total antibiotics was only in strains that isolated from healthy individuals. Also depending on whether the strains are resistant to how many antibiotics or not, for both of the groups, the strains were divided into nine categories and the large differences between the two groups were significant in most categories.
|Table 2: The pattern of susceptibility and resistance of Escherichia coli to antibiotics in this study|
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|Figure 4: Column diagram of the percentage of multidrug resistance among Escherichia coli strains. The commensal strains in healthy people. The commensal strains in people with colorectal cancer|
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The rep-PCR products were loaded on the 1.5% agarose gel for 1.5–2 h with voltage 95 [Figure 5].
|Figure 5: BoxA1R gene products by Rep-polymerase chain reaction on 1.5% agarose gel electrophoresis. (1) Marker, (2-15) polymerase chain reaction products patterns of the samples|
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By Gelcompar II software, Dendro Gram was drawn for both groups of CRC and control together, respectively [Figure 6]. Therefore it was made possible that the closeness of the isolates in each group individually investigated. Moreover, in all Dendro Grams, the isolates were named not only based on being in the normal and CRC groups but also based on phylogenetic groups. So that in the next stages of this study, we can assay the closeness of species accordingly. According to [Figure 6], an attempt was made to checking the dendrogram based on >90% similarity. Accordingly, seven clusters were observed and marked with a red rectangle. According to [Table 3], out of seven clusters, three cases are related to the isolates of the control group and four cases are related to the isolates of the CRC group. Therefore, it was observed that although the number of isolates in the control group was more than the other group, the number of clusters that are genetically related to each other was higher in the CRC group.
|Figure 6: Rep-polymerase chain reaction genotyping Dendro Gram for all of the isolates. It shows seven clusters or groups of the closeness of species with >90% similarity|
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|Table 3: Relationship between each factor of the phylogenetic groups and the multidrug resistance in the clusters|
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| Discussion|| |
E. coli strains are categorized into four phylogenetic groups as B2, D, B1, and A that have the most virulence gene., Because of what was mentioned before, consideration of E. coli strains in the gut of CRC patients was important in terms of the antibiotic resistance and phylogenetic group, especially that generally people with cancer are more disposed to E. coli infections such as urinary tract infection.,, In this study, more than half of the E. coli strains of CRC patients were categorized in the B2 phylogenetic group (55%). Although compared to a similar study in the United States of America, it has been reported 73.7%. In another study in France over a decade even in healthy people, E. coli of gut flora which belonged to the B2 phylogenetic group has gone up. These data were notable but in our study fortunately in comparison with our other research in 2015, there was no noticeable change in the percentage of this phylogenetic group for the strains of bacterial flora in healthy individuals. As mentioned in the results of the antibiogram test, in the control group, the most resistance to antibiotics was correlated to AM (88%) and AMX (76%). However, this resistance was observed among similar strains in the patient group. These antibiotics are classified in the penicillin family antibiotics and are commonly prescribed or even used arbitrarily by most people., Therefore, increasing the antibiotic resistance in this family can be predicted even among commensal E. coli strains. However, in this study, the percentage of AM resistance of intestinal flora strains of healthy individuals was further than the other studies., SXT is another common antibiotic that was used., In this research, among E. coli strains that isolated from the intestinal flora of people with CRC, the most antibiotic resistance was observed for this antibiotic (95%). It is important to note that resistance to SXT in the CRC group was more than the pathogenic strains in another study that was 58%. This resemblance could raise the question that, as most commensal E. coli strains in people with CRC, similar to most pathogenic strains of the bacterium belongs to the B2 phylogenetic group, whether the pattern of antibiotics resistance of these strains is similar to pathogenic strains?
Resistance to TE, in both of the healthy and patient groups, was observed, but, despite some studies, there was no significant relationship between the percentage of resistance to TE (10% in fecal strains, 20% in pathogenic strains) in both the groups. What is certain, this study alike other relevant studies, reported an increasing trend in resistance to common antibiotics, even among commensal strains, which is certainly worrying. More than 90% of the isolates in both of the control and CRC groups were MDR, that is, the abundance was similar to the abundance of the pathogenic strains of this bacterium in another research. However, according to another study about the antibiotic-resistant normal flora strains, the estimated MDR bacteria were lower. Under the contents of our results, there was no significant relationship between antibiotic intake and MDR in both the groups. Also strangely unlike a similar study on the commensal strains of E. coli, there was no significant association between the abundance of phylogenetic group B2 and MDR; in fact, of the 13 isolates from the control group and 11 isolates from the patient group which belonged to the B2 phylogenetic group, 12 and 10 isolates were MDR, respectively. Howbeit, it was possible, as in some studies,, to investigate the association of this phylogenetic group with the frequency of resistance to each antibiotic. But for us, the issue of MDR was more important because resistance to multiple antibiotics is problematic and worrying., Anyhow it should be mentioned, investigation about the antibiotic resistance and phylogenetic groups of this bacterium are important. Regarding the genotyping section, it should be mentioned, rep-PCR is one of the most common techniques for investigating the genetic closeness of bacteria including E. coli.,, In most studies on this bacterium, dendrograms were investigated based on different similarity percentages, videlicet in a study, dendrogram was analyzed based on >75% similarity. In another one, despite the analysis based on >95% similarity, no association was seen between genetic closeness and the factors considered in that study. It is interesting that similar results in another study with analysis based on >80% similarity were seen too. Based on the mentioned in the results section, seven clusters were observed. The relationship between the phylogenetic groups and MDR with species similarity was considered too. In five clusters (3 cases in the CRC group and 2 cases in the control group), the isolates had a similar phylogenetic group. In addition, MDR was similar in only two clusters (one cluster in the CRC group and one cluster in the control group).
| Conclusion|| |
More than half of the E. coli commensal population in CRC patients were categorized in the B2 phylogenetic group (55%). In the control group, the most resistance to antibiotics was correlated to AM and AMX. However, this resistance was observed among similar strains in the patient group.
Declaration of patient consent
All sampling principles were observed and stool samples were taken from patients and healthy individuals with full verbal satisfaction and in some cases, hospitalized patients were written.
The authors would like to thank the staff of Cellular and Molecular Research Center at Shahid Beheshti University of Medical Sciences.
Financial support and sponsorship
This work was supported by the Student Research Committee, Shahid Beheshti University of Medical Sciences [NO -1398/10467]. We also appreciate the staff of the Cellular and Molecular Biology Research Center and Departments of Medical Biotechnology and also Microbiology at the Shahid Beheshti University of Medical Sciences.
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6]
[Table 1], [Table 2], [Table 3]