Abstract
Introduction
Antibiotic resistance and extensive use of antibiotics are amongst the major causes of failure in antibiotic treatment. The purpose of this study was to investigate antibiotic resistance patterns and to identify resistance genes of quinolones and colistin in Escherichia coli. There are a very few patents on E. coli isolated from colorectal cancer. So, this study demonstrates that some bacteria resistant to ciprofloxacin have not resistance genes.Moreover, new patterns for E. coli are presented for isolates of patients with colorectal cancer.
Materials and Methods
Of the three healthy people, inflammatory bowel diseases (IBD) patients and colorectal cancer patients, 40 E. coli strains isolated after confirmation by biochemical and molecular methods. The susceptibility of isolates to antibiotics was investigated using disk diffusion test. After deoxyribonucleic acid (DNA) extraction, polymerase chain reaction (PCR) was used to identify genes encoding resistance to ciprofloxacin (qnr A, qnr B) and colistin (mcr-1).
Results
The results showed that E. coli isolates from colorectal cancer patients had the highest resistance to piperacillin (67.5%), ceftazidime (47.5%), and cefepime (42.5%). Also, E. coli strains isolated from IBD patients showed resistance to antibiotic ceftazidime 13%. More than 95% of E. coli strains isolated from healthy people were susceptible to antibiotics. Based on the results, 18 (15%) E. coli strains showed resistance to ciprofloxacin. The qnr A gene was detected in 61.11% isolates; however, qnr B was detected in 9 (50%) isolates. Isolates resistant to colistin were not observed.
Conclusion
These findings indicate increased resistance of E. coli to ciprofloxacin in comparison with prior studies. Further research in this field will increase our knowledge and more effective exposure to the antibiotic resistance of the pathogenic microorganisms.
Keywords: Escherichia coli, ciprofloxacin, colistin, antibiotic resistance, colorectal cancer, inflammatory bowel diseases
1. Introduction
Enterobacteriaceae family are gram-negative bacilli shape bacteria that are typically considered to be the normal flora of the intestine. These bacteria are one of the common causes of nosocomial infections and human pathogens that can cause diseases such as cystitis, pyelonephritis, septicemia, pneumonia, peritonitis, meningitis, and associated infections with hospital equipment such as catheters [1, 2].
The most important species of this genus is Escherichia coli [2, 3]. The most common infections with E. coli include urinary tract infections, intra-abdominal infections, pneumonia, especially hospital pneumonia, neonatal meningitis, soft tissue infections, and ulcers, vascular infections, bacteremia and other infections. With the advent of resistance to fluoroquinolones and cephalosporins, the treatment of infections caused by this organism has become challenging [2, 3].
These resistances in the Enterobacteriaceae family, especially E. coli, are due to the production of ESBLs (Extended Spectrum B-lactamase) that have increased in most countries in recent years [2]. The organisms that ESBLs also produce can be resistant to other antibiotics, such as aminoglycosides, trimethoprim and sulfamethoxazole, and quinolones [3].
Quinolone resistance genes carrying on a plasmid called plasmid-mediated quinolone resistance (PMQR) are classified into five classes, which include qnr A, qnr B, qnr C, qnr D, qnr S. These genes encode proteins that prevent fluoroquinolones from binding to topoisomerase II [3].
Colistin belongs to polymyxins (cationic polypeptides) with a wide range of effects on Gram-negative bacteria, especially the Enterobacteriaceae family [3]. Recently, in clinical practice, two types of polymyxins (polymyxin B and polymyxin E or Colistin) were used. These polymyxins are similar in biological activity and differ only in their structure in an amino acid. The mechanism of resistance to polymyxin is modifications to lipid A, which is to reduce the affinity to colistin [3]. Colorectal cancer (CRC) is the third leading cause of cancer related deaths and causes mortality of 600,000 people per year in the world [4]. Various factors such as inheritance, environmental factors and inflammatory syndromes associated with infectious agents are effective in creating CRC [5]. Among these factors, infectious agents are associated with cancer by about 20% and play an important role in the development of cancer [6, 7]. In the human gastrointestinal tract, there are about 1014-1013 microorganisms that are symbiotic, called microbiota, and are essential for the maintenance of homeostasis [8]. The disorder of this microbial balance has been observed in CRC patients [9]. Resistance to antibiotics among pathogen bacteria is a topic that has been considered today as a worldwide problem. Regarding increasing growth of resistance in hospital infections and treatment centers, the determination of antibiotic resistance patterns in common pathogens can be of great importance in experimental and specific treatments against a particular pathogen. Therefore, due to the importance of this issue, this study was designed. The aim of this study was to investigate antibiotic resistance patterns and to identify genes of quinolones (qnr A and qnr B) and colistin (mcr-1) in E. coli isolated from mucosal samples of patients with colorectal cancer (CRC) and inflammation of the intestine and healthy people.
2. Materials and methods
2.1. Study Design
In this experimental study, sampling was done at Digestive Disease Research Institute, Shahid Beheshti Hospital in Hamedan, Iran. Their study was performed from March to May 2018. The conditions for entering the study population in this research were based on a questionnaire completed by each patient. Items of the questionnaire included: sex, age, history of the disease, gastrointestinal symptoms, native and resident of the region. The patients and control groups included 54 men and 66 women, with a mean age of 56 years (age range 16-81 years) and mean disease duration of 5-13 years provided a signed agreement for this study, and the protocol was approved by the local ethics committee of the Hamadan University of Medical Science (IR.UMSHA.REC.1395.298).
2.2. Clinical Characteristics of Patients
Intestinal biopsies were obtained of the terminal ileum and the colon of 40 patients with CRC and 40 patients with inflammatory bowel diseases (IBD). Moreover, biopsies of the ileum and colon of 40 individuals who had no significant pathological findings following endoscopic examination for changes in stool habits, abdominal pain, upper gastrointestinal bleeding or cancer were considered as controls [10]. During a colonoscopy, two biopsy samples were taken from each person for routine pathological assessment and bacteriological study
2.3. Culture and Isolation of E. coli
Biopsy samples (50 to 100 mg) taken by the gastroenterologist from the intestinal tissue of the patients were immediately placed in a tube containing 100 ml of sterile phosphate salt buffer (PBS) and transferred to the microbiological laboratory in the vicinity of the ice. First, the biopsy specimens were three times rinsed with PBS, the purpose of this was to eliminate fecal specimens. After the wash, the biopsy specimens were chopped and turned into a homogeneous state. The aim was to release the E. coli bacteria in the intestinal mucosal tissue. The sample chopped with a sterile loop on Blood agar and MacConkey agar was incubated for 24 hours at 37°C. The E. coli strains isolated from the biopsy specimens using conventional microbiological (SIM, Methyl-Red/Voges Pros-kauer (MR-VP) Broth, Simmons citrate agar) tests strains were identified. E. coli strains from controls were retrieved only from descending colon biopsy specimens.
2.4. Sensitivity to Antibiotics
In this study, sensitivity to 8 antibiotics was determined using disk agar diffusion method,which employed the clinical and laboratory standards institute (CLSI) criteria [11].
2.5. DNA Extraction
The amount of 300 μl of the suspension bacteria in a 1.5 ml microtube was dispensed and centrifuged for 2 minutes for precipitation, containing bacterial culture media at 1400 rpm. To wash the resulting precipitate, 300 μl sterilized distilled water was added and then centrifuged at 1400 rpm for 5 minutes after two steps. Next, 100 μl of 5% NaOH solution was added and placed at 95°C for 30 minutes and then with a round of 1400 rpm for 5 minutes centrifugation.
For final purification of DNA, 100 μl of tris solution was added to the precipitate and centrifuged at 1400 rpm for 5 minutes. The supernatant liquid, which contained pure DNA, was collected in a 0.5 μl microtube. The concentration and absorbance of the purified DNA at 260/280 nm wavelengths were determined by a Nanodrop Spectrophotometer (Thermo Fisher Scientific).
2.6. PCR Reaction to Determine the Resistance Genes
We used three qnr A, qnr B and Mcr-1 genes to detect resistance genes with fluqinolones and colistin. PCR was used to amplify these genes Table 1.
3. Results
A total of 120 strains of E. coli isolated, and 40 isolates from colorectal cancer, were diagnosed, along with 40 isolates from patients with intestinal inflammation. In addition, 40 strains of E. coli from healthy were also collected.
The resulting antibiograms showed that E. coli strains isolated from patients with colorectal cancer had the highest resistance to piperacillin (67.5%), ceftazidime (47.5%), and cefepime (42.5%). Also, E. coli strains isolated from patients with intestinal inflammation showed the highest resistance to ceftazidime (13%). Samples isolated from healthy individuals showed most antibiotics 95% sensitivi-ty. Based on the results of the disk diffusion test, 18 isolates (15%) showed resistance to ciprofloxa-cin. However, colistin-resistant strains were not detected in E. coli (Fig. 1).
3.1. Distribution of Genes Encoding Re-sistance to Ciprofloxacin and Colistin in E. Coli Strains
According to the results of disc diffusion test, 18 isolates of 15% of group 1: E. coli isolated from patients with colorectal cancer (10 isolates), group 2: E. coli isolated from patients with inflammatory disease (3 isolates), group 3: E. coli isolated healthy (1 isolate) showed resistance to ciprofloxacin. And in the strains of E. coli, colistin-resistant strains were not found. PCR test was performed to detect qnr A and qnr B genes (Fig. 2 and Table 2).
4. Discussion
In the current study, antibiotic resistance patterns (with an emphasis on fluoroquinolones) of E. coli isolated from patients with colorectal cancer and IBD and healthy subjects as control were assessed. Out of 120 strains, 18 strains of 15% E. coli were resistant to ciprofloxacin. Ten strains of 35% of E. coli isolated from patients with colorectal cancer showed resistance to ciprofloxacin. Also, antibiotic susceptibility test results showed that among the 120 strains of E. coli in this study, the high susceptibility to antibodies to colistin, amikacin and imipenem was 100%, 90% and 90%, respectively. Resistance to these antibiotics in strains isolated from patients with colorectal cancer was observed as follows: resistance to pipracillin was 67.5%, to ceftazidime being 47.5%, and to cefepime being 42.5%. The results of this study, similar other studies in different areas of Iran and Europe, showed a high level of resistance to fluoroquinolones. Aibinu et al., isolated 103 strains of E. coli from cancer patients, their results showed resistance to ciprofloxacin being 21.4% that was consistent with our study [14]. In another study, Kheirabadi et al., reported resistance to ciprofloxacin being 22.7% [15]. Also, Jamshidi et al., investigated E. coli isolated from people with urinary tract infections, which reported resistance rate to ciprofloxacin 40% [16].
Since fluoroquinolones are commonly used antibiotics in the treatment of urinary tract infections in Iran, the increasing resistance to these antibiotics has raised concerns about the choice of treatment for these infections. In various studies, the rate of resistance has been reported to be between 3% and 10%, but there are many differences between the results of studies that have been responsible for the causes of E. coli infection. Resistance to fluoroquinolones, especially ciprofloxacin, up to 20% has also been reported to confirm the results of resistance to this antibiotic in our study area [17]. According to some studies, resistance to fluoroquinolones has increased from 3% to 23% over 8 years [18, 19]. Ruiz et al., suggested the treatment of urinary tract infections with fluoroquinolones. In addition, resistance to fluoroquinolones 10% to 20% have been reported which is similar to the results of our study [20]. Given the high resistance to ciprofloxacin in this study and other studies conducted in Iran, this
indicates that the resistance to antibiotics of fluoroquinolones, especially ciprofloxacin, is increasing in Iran [21]. In a study conducted in the United States in 2006, resistance to E. coli isolates was also reported for quinolones to be 21% and 12% for fluoroquinolones, which is less relevant to the current study [22]. In an investigation accomplished in Pakistan in 2011, E. coli resistance rate to ciprofloxacin was 36.45%, which is similar to the results of the present study [23]. Kheir Abadi et al., found the resistance of E. coli isolates to ciprofloxacin 22.7%, which is similar to the results of our study [15]. Sanchez et al., evaluated the antimicrobial resistance of E. coli between 2002 and 2010, ad found that the resistance to ciprofloxacin increased from 3% to 17.1%, and resistance to co-trimoxazole increased from 17.9% to 24.2%; the resistance to these antibiotics in the current study also increased much higher in comparison with previous studies [24]. Mohajeri et al., reported that E. coli were resistant to cefotaxime, ceftazidime and ciprofloxacin in 27%, 22.5%, and 26%, respectively [25]. Farshad et al., reported an antibiotic resistance pattern in E. coli isolated from the urinary tract infection patient. E. coli isolates were resistant to co-trimoxazole, amikacin and ciprofloxacin, in 76%, 3% and 3.8%, respectively, and resistance to imipenem was not observed [26].
Resistance to various antibiotics varies based on the therapeutic patterns that occur in different areas. By comparing the results of antibiograms and evaluating the percentage of resistance to antibiotics in diverse regions, we found that in this study, resistance to pepracillin was more than in similar studies, while in the case of ciprofloxacin, amikacin and cotrimoxazole, variable results were observed and resistance to antibiotics such as cefotaxime and cefipime was more than that observed in the present study.
When comparing various antibiotic percentages with similar tests, it was noted that regional variation in different parts of the world or even one country provides different therapeutic responses to antimicrobial drugs.
The results conclude that sensitivity to antibiotics is due to factors such as irrational antibiotics prescription, enzymatic mutations, as well as transfer of resistance through plasmids has decreased [27, 28]. The results of statistical analysis of the chi-square test showed that the resistance level of isolated E. coli in cancer patients was higher than in healthy subjects with IBD (P <0.05).
Based on the findings of antibiograms, 15% of E. coli isolates showed resistance to ciprofloxacin. And in the strains of E. coli, colistin-resistant strains were not found. The PCR test was performed to identify the genes qnr A and qnr B.
The results showed that the prevalence of qnr A and qnr B genes in isolates resistant to ciprofloxacin was 11 (61%) and 9 (50%) respectively.
Jamshidi et al., reported resistance to ciprofloxacin (46%), although the rate of resistance to ciprofloxacin was higher than their study, but the presence of qnr A gene was lower than our study [16].
Zhou et al., showed that the prevalence of qnr A, qnr B genes in E. coli strains was reported to be 0.4%, and 1.2%, respectively, which showed a lower outbreak than the present study [29]. Robicsek et al., in between 1999 and 2004 reported the presence of an abundance of qnr A genes in isolates of E. coli (4%) [12].
In the study by Akya et al., the antibiotic resistance to ciprofloxacin 56 (84.8%) was observed to be high. The prevalence of qnr A and qnr B genes was 28.7% and 40.9%, respectively [30]. Although the main cause of resistance to quinolones is mutation in quinolone target genes, in recent years, plasmid-mediated quinolone resistance genes (PMQRs) have contributed to the high horizontal transmissivity in the development of quinolone-resistant isolates [31].
In the current study, qnr B was the most abundant gene, as reported in other studies [30]. In the investigation of Rajaei et al., the prevalence of qnr A and qnr B genes was 10.66% and 8.33%, respectively [32]. Soleimani et al., found the prevalence of qnr A gene in the isolates resistant to ciprofloxacin to be 14.3%, being less prevalent than our study. Studies have shown that there is a direct correlation between the amount of consumption, quinolones and the rate of resistance to ciprofloxacin [33].
In studies conducted around the world, the prevalence of ciprofloxacin-resistant strains was different. Corkill et al., reported an incidence of qnr A gene in 32% of the isolates studied [34]. In another study Oktem et al. found that out of the 34 isolates of E. coli resistant to ciprofloxacin, 5 isolates contained 6.3% of the qnr A gene [35].
Wang et al., in a total of 78 isolates of ciprofloxacin-resistant E. coli in Brazil between 2002 and 2003 reported only one qnr A positive [36]. Since the two recent studies have a difference in time for about a decade with our study, the results of these studies confirm the increasing trend of qnr genes. Pakzad et al., reported a total of 14 isolates of E. coli in the 24 resistant strains of qnr A gene (37.5%) [37].
Mohamadbigi et al., similar to our study, found resistance to ciprofloxacin to be 72.77% and also the prevalence of qnr A and qnr B genes was found to be 37.33% and 67.92%, respectively [38].
Given that between quinolones, ciprofloxacin has the most effective action against gram-negative bacteria and gram-positive bacteria [39]. Ciprofloxacin inhibits bacterial DNA from DNA repair and translates by inhibiting DNA gyrase in gram-negative bacteria and topoisomerase IV in gram-positive bacteria [40, 41].
Yosofi et al., investigated the relationship between qnr genes in inducing resistance to ciprofloxacin in E. coli. The results of their study showed that 139 ciprofloxacin-resistant patients were present in 309 samples. Out of these, 103 samples 74.1% were observed to carry the qnr B gene and 8.5% carriedthe qnr A gene [42]. Bouchakour et al., studied 39 strains of E. coli; the frequency of qnr A and qnr B genes was found to be 2.65% and 10.25, respectively [31]. The difference between the results of the study and the present study in terms of the observed resistance level can be due to more precise monitoring programs in that country and the inaccessibility of these important drugs.
Conclusion
Considering the results of this study and other studies and due to increasing resistance to various types of antimicrobial agents such as ciprofloxacin and imipenem, which are the therapeutic indices for E. coli infections, further studies in this field as well as reduction in the use of antibiotics and irrational antibiotics prescription by physicians are necessary. The results of this study showed that qnr genes play an important role in resistance to quinolones in the virulence of E. coli. The high prevalence of these genes in strains as well as the presence of plasmids, which facilitate their rapid transfer and spread, is clinically important. Therefore, there is a need to monitor and investigate the genetic pattern of resistance to fluoroquinolones, the existence of screening programs and serious preventive measures. The specific invention has not been made in this study; only in this study, new patterns for E. coli are presented in samples of patients with colorectal cancer.
Current and Future Developments
There is need to monitor and investigate the genetic patterns of resistance to fluoroquinolones and colistin, the existence of screening programs and serious preventive measures. It is necessary to control the prophylactic use of antibiotics, especially those that are critical for human health, to reduce the abundance of resistance organisms. At the same time, systematic surveillance of antimicrobial resistance considering a One Health approach should be implemented to guide the intervention strategies directed to control antimicrobial resistance.
Fig. (1).
Resistance and sensitive of E. coli strains isolated (A); Patients with colorectal cancer (B); Patients with inflammatory bowel diseases (C); Healthy subjects. (A higher resolution / colour version of this figure is available in the electronic copy of the article).
Abbreviations: CIP; Ciprofloxacin. CO; Colistin. AMK; Amikacin. SXT; Trimethoprim/sulfamethoxazole. IPM; Imipenem. PIP; Piperacillin. CAZ; Ceftazidime. FEP; Cefepime.
Fig. (2).
Agarose gel electrophoresis of qnr A, qnr B gene in ciprofloxacin resistant strains PCR products. (A higher resolution / colour version of this figure is available in the electronic copy of the article).
Lane 1; (PCR product of qnr A and qnr B gene) and Lane 2; (positive control of qnr A and qnr B gene), Lane 3; (negative control), M; size of DNA marker (50 bp).
Table 1.
The primers used in this study.
Table 2.
Distribution of resistance genes to ciprofloxacin and colistin antibiotics in E. coli strains.
| Strains Resistant to Ciprofloxacin | Genes Studied | Antibiotic Patterns | |||
|---|---|---|---|---|---|
| qnr A | qnr B | mcr-1 | |||
|
E. coli isolated from patients with colorectal cancer |
S1 | + | -- | - | CIP (R), AMK(S), CO(S), SXT(R), CAZ(S), FEP(S), IPM(S), PIP(R) |
| S2 | + | + | - | CIP (R), AMK(R), CO(S), SXT(S), CAZ(S), FEP(R), IPM(S), PIP(R) | |
| S3 | -- | + | - | CIP (R), AMK(S), CO(S), SXT(R), CAZ(S), FEP(S), IPM(S), PIP(R) | |
| S4 | + | -- | - | CIP (R), AMK(R), CO(S), SXT(S), CAZ(R), FEP(R), IPM(S), PIP(R) | |
| S5 | -- | + | - | CIP (R), AMK(S), CO(S), SXT(S), CAZ(S), FEP(R), IPM(S), PIP(R) | |
| S6 | + | + | - | CIP (R), AMK(R), CO(S), SXT(R), CAZ(S), FEP(S), IPM(S), PIP(R) | |
| S7 | -- | + | - | CIP (R), AMK(R), CO(S), SXT(S), CAZ(R), FEP(R), IPM(S), PIP(R) | |
| S8 | + | -- | - | CIP (R), AMK(S), CO(S), SXT(S), CAZ(R), FEP(S), IPM(S), PIP(R) | |
| S9 | + | + | - | CIP (R), AMK(R), CO(S), SXT(R), CAZ(S), FEP(R), IPM(S), PIP(R) | |
| S10 | + | + | - | CIP (R), AMK(S), CO(S), SXT(S), CAZ(R), FEP(S), IPM(S), PIP(R) | |
|
E. coli isolated from patients with IBD |
S11 | + | + | - | CIP (R), AMK(S), CO(S), SXT(R), CAZ(S), FEP(R), IPM(S), PIP(R) |
| S12 | + | -- | - | CIP (R), AMK(R), CO(S), SXT(S), CAZ(R), FEP(S), IPM(S), PIP(S) | |
| S13 | + | -- | - | CIP (R), AMK(S), CO(S), SXT(S), CAZ(S), FEP(S), IPM(S), PIP(R) | |
|
E. coli isolated healthy subjects |
S14 | + | + | - | CIP (R), AMK(R), CO(S), SXT(S), CAZ(S), FEP(S), IPM(S), PIP(S) |
Abbreviations: CIP; Ciprofloxacin. CO; Colistin. AMK; Amikacin. SXT; Trimethoprim/sulfamethoxazole. IPM; Imipenem. PIP; Piperacillin. CAZ; Ceftazidime. FEP; Cefepime S: Sensitive R: Resistance.
Acknowledgements
We would like to thank all members of the microbiology Laboratory of Hamadan University of Medical Science.
list of Abbreviations
- E. coli
Escherichia coli
- CRC
Colorectal Cancer
- GI
Gastroenteritis
- MEM
Meropenem
- IMI
Imipenem
- CIP
Ciprofloxacin
- CO
Colistin
- PCR
Polymerase Chain Reaction
Funding Statement
This research was funded by a grant (no. 9508114597) from the Student Research Center Hamadan University of Medical Sciences, Hamadan, Iran.
Ethics Approval and Consent to Participate
The present study was ethically approved by the Hamadan University of Medical Sciences, Institutional Review Board (9508114597).
Human and Animal Rights
No animals were used for studies that are the basis of this research. All human procedures followed were in accordance with the Helsinki Declaration of 1975.
Consent for Publication
Informed consent was obtained from the participants of this study.
Availability of Data and Materials
The authors confirm that the data supporting the findings of this research are available within the article.
Funding
This research was funded by a grant (no. 9508114597) from the Student Research Center Hamadan University of Medical Sciences, Hamadan, Iran.
Conflict of Interest
The authors declare no conflict of interest, financial or otherwise.
Limitations
One of the most important limitations of this study was the number of E. coli isolated from patients with colorectal cancer.
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Data Availability Statement
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