|Year : 2016 | Volume
| Issue : 4 | Page : 772-782
Distribution of class 1 integrons among multidrug-resistant Escherichia coli in Menoufia University Hospitals and commensal Escherichia coli isolates
Ghada R El-Hendawy, Nahla A Melake, Ahmed A Salama, Naira A Eissa, Wafaa A Zahran, Shymaa A Elaskary MSc
Department of Medical Microbiology and Immunology, Faculty of Medicine, Menoufia University, Menufia, Egypt
|Date of Submission||14-Oct-2014|
|Date of Acceptance||20-Nov-2014|
|Date of Web Publication||21-Mar-2017|
Shymaa A Elaskary
Menouf - Ahmed Orabi-St, 32511
Source of Support: None, Conflict of Interest: None
This study aimed to detect the prevalence of class 1 integrons among clinical as well as commensal Escherichia coli isolates. To investigate the distribution of class 1 integrons among E. coli isolates from different types of infection and E. coli isolates with different antibiotic susceptibility patterns, and to evaluate the contribution of class 1 integrons toward the dissemination of multidrug resistance (MDR) E. coli, particularly extended-spectrum b-lactamase (ESbL)-producing strains.
Class 1 integrons play a role in the emergence of multiresistant bacteria by facilitating the recruitment of gene cassettes encoding antibiotic resistance. Many of the most recently ESbL genes are frequently found within integron-like structures.
Materials and methods
The study was carried out by collecting specimens from 210 patients with nosocomial infection aimed to obtain 80 E. coli clinical isolates (CIs) from Menoufia University Hospitals and 20 E. coli commensals. MDR E. coli including ESbL producers were detected among the studied E. coli using standardized methods. Class 1 integrons (IntI1 gene) and ESbL (SHV, TEM, and CTX-M genes) were detected by PCR.
Urine, being the most frequent specimen, had E. coli CIs (56.25%), followed by sputum specimens (20%). About 64% of E. coli CIs and 30% of E. coli commensals had class 1 integron. All E. coli CIs and 70% of the E. coli commensals were MDR strains. Using the phenotypic confirmatory combined disk test, 31.1% of E. coli CIs and 5% of E. coli commensals were ESbL producers. In the PCR results, total SHV genes were 70.4% of E. coli CIs and 100% of E. coli commensals. Total TEM were 37% of E. coli CIs and total CTX-M were 29.6% of E. coli CIs. About 41% of the intI1 gene-positive E. coli CIs and 16.7% of the intI1 gene-positive E. coli commensals were ESbL producers.
The study highlights the prevalence of class 1 integrons in MDR E. coli CIs in our hospitals and E. coli commensal isolates. Fair agreement was found between the presence of class 1 integrons and ESbL production in E. coli CIs. A restrictive antibiotic subscription policy is needed to avoid increased selection pressure where integrons play a potentially significant role in the uptake and dissemination of resistance genes.
Keywords: class 1 integron, Escherichia coli clinical isolates, Escherichia coli commensals, ESβL genes
|How to cite this article:|
El-Hendawy GR, Melake NA, Salama AA, Eissa NA, Zahran WA, Elaskary SA. Distribution of class 1 integrons among multidrug-resistant Escherichia coli in Menoufia University Hospitals and commensal Escherichia coli isolates. Menoufia Med J 2016;29:772-82
|How to cite this URL:|
El-Hendawy GR, Melake NA, Salama AA, Eissa NA, Zahran WA, Elaskary SA. Distribution of class 1 integrons among multidrug-resistant Escherichia coli in Menoufia University Hospitals and commensal Escherichia coli isolates. Menoufia Med J [serial online] 2016 [cited 2017 Nov 21];29:772-82. Available from: http://www.mmj.eg.net/text.asp?2016/29/4/772/202521
| Introduction|| |
The integron system is a dynamic force in the evolution of multidrug resistance (MDR) and it helps bacteria to acquire novel combinations of resistance genes .
MDR among Enterobacteriaceae is a major concern in clinical settings and is a major public health issue . Recent data suggest that the resistance frequency of Escherichia More Details coli to antimicrobial has been increasing steadily .
Outbreaks because of extended-spectrum b-lactamase (ESbl)-producing E. coli have been reported from hospitals worldwide . Gene-encoding ESbLs are usually located on conjugative plasmids, although many of the most recently described ESbL genes have frequently been found within integron-like structures. The fact that ESbL genes could be acquired by strains harboring particular integrons may expand the possibilities of selection of these by a variety of different antimicrobials .
Recently, the study by Phongpaichitet al.  reported that class 1 integrons are widely prevalent among clinical isolates (CIs) of resistant E. coli, especially in ESbl producers, and are probably a reservoir for the development of MDR strains and nosocomial infections in hospitals.
The aim of this work was to detect the prevalence of class 1 integrons among clinical as well as commensal E. coli isolates, to investigate the distribution of class 1 integrons among E. coli isolates from different types of infection and E. coli isolates with different antibiotic susceptibility patterns, and to evaluate the contribution of class 1 integrons toward the dissemination of MDR in E. coli, particularly ESbL-producing strains.
| Materials and Methods|| |
The present study was carried out during the period from September 2012 to February 2014 and included 210 patients with various nosocomial clinical diseases who were admitted to Menoufia University Hospitals. In this study, we aimed to collect 80 E. coli CIs (group I) from hospitalized patients (one E. coli isolate from each patient) and 20 E. coli commensal isolates from the stool of healthy individuals (group II).
Group I included 34 men and 46 women with nosocomial infections admitted in different wards and ICUs. Group II included nine men and 11 women. They had not been exposed to antibiotics or a hospital environment in the 3 months before sample collection.
In group I, sample collection of urine, pus, wound swab, sputum, and blood was performed ,. All samples were inoculated on nutrient agar, blood agar, and MacConkey's agar and incubated aerobically at 37°C for 24 h. Culture and counting of urine samples on blood agar and/or CLED agar were performed using a sterile calibrated loop measuring 0.001 ml . Isolation and identification of organisms including E. coli were carried out using standard bacteriological methods .
Antimicrobial susceptibility testing for E. coli isolates was carried out using the disk diffusion method and interpreted according to the Clinical and Laboratory Standards Institute guidelines .
Screening for ESbLs production was performed by the disk diffusion test using ceftazidime (30 mg), cefotaxime (30 mg), ceftriaxone (30 mg), and aztreonam (30 mg). Suspected ESbL-producing E. coli was confirmed using the double disk synergy test and a confirmatory clavulinate combined disks test. In double disk synergy test, amoxicillin/clavulanic acid (20/10 mg) and cefotaxime (30 mg) disks were placed at a distance of 30 mm apart on an inoculated agar plate and incubated aerobically at 37°C for 18–24 h. E. coli that showed a clear extension of the cefotaxime inhibition zone toward the disk containing clavulanate were considered as synergy (ESbL producers). Antibiotic disks of ceftazidime (30 mg), ceftriaxone (30 mg), and aztreonam (30 mg) were also placed on the plate. When E. coli was suspected of harboring ESbLs, but was negative using the standard distance of 30 mm between disks, the test was repeated using a closer distance of 20 mm .
In the confirmatory clavulinate combined disk, ceftazidime (30 mg) and ceftazidime/clavulanic acid (30/10 mg) were used. E. coli was considered as an ESbL producer if there was at least 5 mm increase in the diameter of the ceftazidime/clavulanic disk than that of the ceftazidime disk alone. The same was done with cefotaxime (30 mg) and cefotaxime/clavulanic acid (30/10 mg) .
In group II, stool samples were collected  and cultivated on MacConkey agar and/or blood agar . Isolation, identification, and antibiotic susceptibility testing of isolated E. coli were performed according to the protocol followed for group I .
A molecular study was carried out using PCR. Detection of the IntI1 gene was carried out for all the studied E. coli CIs and commensal isolates. Thirty-two of E. coli CIs and 10 of E. coli commensals (including intI1 gene-positive and intI1 gene-negative isolates) were selected for the detection of ESbL genes.
DNA was extracted using the illustra bacteria genomicPrep Mini Spin Kit (GE Healthcare UK Limited Amersham Place, Little Chalfont, Buckinghamshire, UK). The primers used were designed and synthesized by Eurofins MWG Operon (Germany) ([Table 1]). PCR was carried out in a total volume of 45 ml with 30 ng DNA as a template. PCR conditions for the intI1 gene study were as follows: heating at 94°C for 12 min, followed by 30 cycles of 94°C for 1 min, 54°C for 30 s, and 72°C for 2 min, and a final elongation step of 72°C for 10 min ,. PCR conditions for the study of ESbL genes (TEM, SHV, and CTX-M) were as follows: heating at 94°C for 3 min, followed by 35 cycles of denaturation at 94°C for 45 s, annealing for 30 s (at 54°C for SHV, 51°C for TEM, and 50°C for CTX-M for primer optimization), and extension at 72°C for 1 min. A final extension was performed at 72°C for 3 min. The study was carried out using a Thermocycler apparatus (Biometra, Germany). Synthesized DNA fragments were detected on 1.5% agarose gels by ethidium bromide staining. A DNA ladder (100–3000 bp) was used to estimate allele sizes in base pairs for the gel .
|Table 1 Primers used for the detection of the IntI1 gene and ESβLs genes|
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| Results|| |
The study included two groups: group I included 80 patients with E. coli nosocomial infections (34 men and 46 women), mean age 43.60 ± 18.35 years, and group II included 20 healthy individuals (volunteers; nine men and 11 women), mean age 33.3 ± 22.47 years.
[Figure 1] shows the distribution of E. coli versus other organisms according to the types and number of specimens of group I (eight specimens had mixed bacterial growth and 33 specimens had no bacterial growth). There was a highly significant difference (P < 0.001) between different wards and specimens from which E. coli CIs were isolated. The ICU was the most represented ward for E. coli CIs (36.3%). Urine was the most prominent specimen that had E. coli CIs (56.25%; 40% were catheter and 16.25% were midstream urine samples).
|Figure 1: Distribution of Escherichia coli versus other organisms according to the types and number of specimens of group I (eight specimens had mixed bacterial growth and 33 specimens had no bacterial growth).|
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[Figure 2] shows a significant difference (P< 0.05) between the studied E. coli CIs and commensals in the distribution of class 1 integron, where 63.8% of E. coli CIs had the intI1 gene, whereas 30% of E. coli commensals had the intI1 gene.
|Figure 2: Distribution of the intl1 gene among the Escherichia coli CIs and E. coli commensals studied. CIs, clinical isolates.|
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[Table 2] shows a highly significant difference (P < 0.001) between E. coli CIs and E. coli commensals in their resistance to different antibiotics. All E. coli CIs were resistant to three or more antibiotics classes (i.e. MDR). There was a highly significant difference (P < 0.001) between CIs and commensal isolates with the intI1 gene in relation to resistance to most antibiotics. [Table 3] shows the relation between the presence of the intl1 gene and resistance phenotypes of E. coli CIs and E. coli commensals. The more the resistance against different antibiotics, the higher the likelihood of the presence of the intI1 gene. Sixteen E. coli CIs (12 had the intI1 gene and four did not have the intI1 gene) and one E. coli commensal (had intI1 gene) were resistant to all 21 antibiotics tested in this study (pan-resistance) including ESbLs, quinolones, imipenem, amoxicillin/clavulanic acid, and cefepime. However, 14 E. coli CIs (had intI1 gene) were resistant to all the above-mentioned drugs, except cefepime.
|Table 2 Antibiotic resistance patterns of the E. coli isolates studied and their relation to the presence of the intl1 gene|
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|Table 3 Relation between resistance phenotypes of the E. coli isolates studied and the presence of the intI1 gene|
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[Figure 3] shows the results of different phenotypic methods for ESbL detection among the E. coli isolates studied. There was a highly significant difference (P < 0.001) between the three phenotypic methods used for ESbL detection among the E. coli CIs studied.
|Figure 3: Phenotypic methods for ESβL detection among the studied Escherichia coli isolates, (a) E. coli CIs and (b) E. coli commensals. CIs, clinical isolates; ESβL, extended-spectrum β-lactamase.|
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[Table 4] shows the results of detection of ESbL genes among 42 E. coli isolates (32 E. coli CIs and 10 E. coli commensals) studied by PCR. ESbL genes were positive among 28 out of 42 (66.7%) the E. coli isolates studied(24/32; 75% for E. coli CIs and 1/10; 10% for E. coli commensals). In terms of PCR results, the sensitivity of the cephalosporin/clavulanate combination disks test was 89.3% and its specificity was 92.8%, with 90.4% accuracy for the detection of ESbL-producing isolates. [Table 5] shows the distribution of ESbL genes among the E. coli isolates studied. It shows that the SHV gene was present among 70.4% of E. coli CIs and 100% of E. coli commensals. TEM and CTX-M genes were present among 37 and 29.6% of E. coli CIs, respectively, and they were not present among E. coli commensals [Figure 4] and [Figure 5]. There were significant differences in ESbL gene types between E. coli CIs and commensals (P< 0.05).
|Table 4 Sensitivity and specificity of the cephalosporin/clavulanate combination disks test in relation to the PCR study for the detection of ESβL-producing isolates|
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|Figure 4: Gel electrophoresis of PCR amplification products of SHV, TEM, and CTX-M ESβL genes. Lane 1; DNA ladder (100–3000 bp), lanes 2 and 3; negative for all genes, lanes 4 and 5; positive SHV gene (293 bp), lanes 6 and 7; positive TEM gene (403 bp) and lanes 8 and 9; positive CTX-M gene (569 bp).|
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|Figure 5: Gel electrophoresis of PCR amplification product of the intI1 gene. Lane 1; DNA ladder (100–3000 bp), lanes 2 and 6; intI1 negative genes, and lanes 3, 4, 5, 7, 8, 9 and 10; intI1 positive genes (1.9 kbp).|
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[Figure 6] shows the distribution of ESbL-producing E. coli isolates detected by PCR according to the wards and specimen type. [Table 6] shows the risk factors of group I patients in the study of class 1 integron. There was a highly statistically significant difference (P < 0.001) between individuals with the intI1 gene and those who did not have the gene according to the age group. There was a statistically significant difference (P< 0.05) in the presence of the intI1 gene between patients admitted to the ICU and those not admitted. Eighty percent of the patients admitted to the ICU had the intI1 gene. Eighty percent of the patients who were in the hospital for more than 4 weeks had the intI1 gene. The class 1 integron increased in patients exposed to invasive procedures, and those who were administered antibiotics, corticosteroids, and/or cytotoxic drugs. There was no significant difference (P > 0.05) between the reasons for hospital admission and associated comorbidities and the presence of the intI1 gene.
|Figure 6: Distribution of ESβL-producing Escherichia coli isolates detected by PCR (a) in wards and (b) in terms of specimen type. ESβL, extended-spectrum β-lactamase.|
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|Table 6 Risk factors of the patients in group I in the study of class 1 integron|
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[Table 7] shows the relation between the presence of the intI1 gene and ESbL production among the E. coli isolates studied. About 41% of the intI1 gene-positive E. coli CIs and 16.7% of the intI1 gene-positive E. coli commensals were ESbL producers, with no statistically significant difference (P > 0.05). In terms of κ agreement, there was fair agreement (κ = 0.21) between the presence of the intI1 gene and the phenotypic presence of ESbL among E. coli CIs and slight agreement (κ = 0.14) between the presence of the intI1 gene and the phenotypic presence of ESbL among E. coli commensals. In terms of the type of ESbL genes, the predominant gene was the SHV type (71.4% among E. coli CIs and the only gene detected in E. coli commensals). There was no statistically significant difference (P > 0.05) between the presence of the intI1 gene and the ESbL gene type.
|Table 7 Relation between the presence of the intI1 gene in the studied E. coli isolates and the ESβL study|
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| Discussion|| |
In the present study, 45% of the urine specimens showed E. coli infections. Similar results were reported by Salem et al.  in Egypt as E. coli was found in 58.75% of all urinary isolates from hospitalized patients. Our results showed that E. coli was the most frequent microorganism isolated from urine. The same result was reported bySalem et al. , Mohamed Al-Agamy et al. , Evans et al.  andKang et al. .
In our results, E. coli CIs (36.3%) were found the most in ICUs, followed by the urosurgery ward (16.3%) and the general surgery ward (13.8%). Urine was the major source (56.25%) of E. coli CIs, followed by sputum specimens (20%). This was in agreement with the results of Salem et al. , who reported that E. coli is one of the main causes of nosocomial infections, especially in patients admitted to ICUs, and the main cause of urinary tract pathogens in the developed countries.
In our study, there was a significant difference between the E. coli CIs and commensals studied in the class 1 integron distribution as the intI1 gene was detected among 63.8% of E. coli CIs and among 30% of E. coli commensals. This was in agreement with the results of Cambray et al. , who reported that integrons are more prevalent in Enterobacteriaceaeisolated from hospital-acquired infections than from commensal fecal flora in the community. They reported that the intI1 gene found in nosocomial pathogens can spread to and circulate in commensal fecal flora in the community. The IntI1 gene has been reported as the most common and widespread, especially in clinical settings, and is one of the main contributors to antibiotic resistance dissemination. Our results were also in agreement with those reported by Chang et al.  in Taiwan, (52%), Muhammad et al.  in Pakistan, (43.56%), Salem et al.  in Egypt, (54%), and Machado et al. .
This was in contrast to the Phongpaichit et al.  study, in which the intI1 gene was detected in 59.5% of E. coli CIs, which was comparable with 63% of E. coli commensals. Also, they reported that integrons had been identified as a primary source of resistance genes and are considered to be the reservoir of antimicrobial resistance genes within microbial populations and they could be a responsible for the development of MDR and nosocomial infections in hospitals.
The prevalence of the IntI1 gene was higher in the Pongpech et al.  study, in which the intI1 gene was detected in 99% of E. coli CIs and 87% of fecal specimen E. coli isolates. Other comparable prevalences of the intI1 gene in gram-negative CIs have been reported in Western and Central Europe, 43%, more than 50% in the Netherlands, 59% in France, and 75% among aminoglycosides-resistant isolates from the USA .
In the present study, there was a highly significant difference between E. coli CIs and E. coli commensals in their susceptibility to different antibiotics. All E. coli CIs and 70% of E. coli commensals were MDR strains. This was in agreement with the study of Lee et al. , who reported that the rate of resistance to antimicrobial agents was higher among nasocommial E. coli (98.5%) than those of E. coli commensals.
A difference in the resistance to antibiotics in our study was observed between the CIs and commensals; about 100% of E. coli CIs were resistant to ampicillin, amikacin, tobramycin, gentamycin, and trimethoprim/sulfamethoxazole versus 50–55% of E. coli commensals. About 90–95% of E. coli CIs versus 20–35% of E. coli commensals were resistant to piperacillin, amoxicillin/clavulinate, streptomycin, chloramphenicol, and nitrofurantoin. About 75–85% of E. coli CIs versus 10–30% of E. coli commensals were resistant to norfloxacin, ciprofloxacin, cefmanadole, cefipime, and tetracycline. However, 40–50% of E. coli CIs versus 5–10% of E. coli commensals were resistant to azteronam, imipenem, ceftriaxone, and ceftazidime. The most effective drugs against the E. coli CIs studied were cefepime and imipenem.
In the study of Salem et al.  in Egypt, the most effective drugs against clinically isolated E. coli were imipenem and levofloxacin. Their percentages are similar to our results for ampicillin, tetracycline, and chloramphenicol, but differ in trimethoprim/sulfamethoxazole (69.7%), amoxicillin/clavulinate (49.5%), ciprofloxacin (35.6%), and norfloxacin (34%).
Our study showed a higher antibiotics resistance pattern of E. coli CIs than that in the study of Phongpaichit et al.  in Thailand for ciprofloxacin, norfloxacin, amikacin, gentamycin, ceftriaxone, cefotaxime, and imipinem, whereas a resistance pattern of E. coli commensals similar to that of our results was observed by the study of Lee et al.  in Korea, who reported that the least resistance was against third-generation cephalosporins and the most effective drugs were imipenem and azteronam.
In the present study, there was a highly significant difference between E. coli CIs and commensal isolates with the intI1 gene in relation to resistance to most antibiotics (e.g. ampicillin, piperacine, amoxicillin/clavulinic acid, amikacin, gentamycin, tobramycin, streptomycin, chloramphenicol, and nitrofurantoin) and less significant difference to ciprofloxcacin, norfloxacin, cefamandole, cefoxitin, tetracycline, and trimethoprim/sulfamethoxazole. No significant difference was detected between strains with the intI1 gene in relation to resistance to cefotaxime, ceftriaxone ceftazidime, cefepime, azteronam, and imipenem. This was in agreement with Muhammad et al. Hall et al. , and Guerin et al. ; they documented that there is a strong association between the presence of integrons and antimicrobial resistance established by several studies on E. coli in various regions of Asia, Europe, and USA.
Fluit and Smith  andLee et al.  documented that integrons are associated with a variety of resistance gene cassettes, especially to trimethoprim/sulphamethoxazole and aminoglycosides, and the presence of the intI1 gene is associated significantly with MDR E. coli isolates. This is not surprising because trimethoprim/sulphamethoxazole has been a therapeutic combination that has been used frequently. However, Sepp et al.  found that the resistance of integron-carrying and non-carrying strains is more dependent on influencing factors (hospitalization and administration of antibiotics) than the presence or absence of integrons.
Partridge et al.  reported that antibiotic pressure has probably played an important role in the mobile integrons (MIs) selection and dissemination in bacteria. More than 130 gene cassette (GCs) conferring resistance to antibiotics and more than 60 GCs of unknown functions have been described in MIs. This is in contrast to the study of Hocquet et al. , who reported that no published studies have shown the direct in-vivo selection of resistance through the acquisition of an integron. However, Guerin et al. , in recent in-vitro studies, reported that antibiotics can induce integrase transcription, both in chromosomal integrons and in MIs, through the SOS response. The activation of the SOS response in bacteria results in integrase overexpression, which leads to an increase in GCs recombination events . This was in agreement with Goerke et al.  who had established that some widely used antibiotics, such as fluoroquinolones, trimethoprim, and b-lactams, can trigger SOS induction and can thus promote the dissemination of antibiotic-resistance genes. The study carried out by Heir et al.  showed the ability of integrons to capture and collect novel genes encoding resistance to classes of antibiotics whose resistance has not been associated previously with integrons. This expands the growing list of class 1 integron gene cassettes that confer resistance to an increasing number of antibiotics within many classes.
Our results showed that, the greater the resistance against different antibiotics, the greater the likelihood of the presence of the intI1 gene. Sixteen E. coli CIs (12 had the intI1 gene and four did not have the intI1 gene) and one E. coli commensal (had intI1 gene) were resistant to all 21 antibiotics tested in this study (pan-resistance) including ESbLs, quinolones, imipenem, amoxicillin/clavulanic acid, and cefepime, whereas 14 E. coli CIs (which had the intI1 gene) were resistant to all the above-mentioned drugs, except cefepime. This was not in agreement with Chang et al.  who found that 39 E. coli isolates were resistant to chloramphenicol and 22 were resistant to gentamycin and only one isolate contained an integron-carrying resistant gene cassette. Also, they found that a few isolates (2–7%) were resistant to ceftazidime and azteronam drugs and no integron-associated resistant determinants were found.
In the PCR results, the sensitivity of the cephalosporin/clavulanate combination disks test was 89.3% and its specificity was 92.8%, with 90.4% accuracy in the detection of ESbL-producing isolates. A total of 78.1% (25/32) of E. coli CIs were found to be ESbL producers by the cephalosporin/clavulanate combination disks test, of which 75% were ESbL gene positive and 3.13% of the isolates were ESbL gene negative by the molecular method. However, 9.38% of the isolates were non-ESbL-producers by the cephalosporin/clavulanate combination disks test and by the molecular method, they were ESbL gene positive for E. coli CIs. No difference was observed between phenotypic and genotypic methods in E coli commensals. This was in agreement with the results of Färber et al.  and Bali et al. , who reported similar results for ESbL E. coli detection by phenotypic and molecular methods.
The prevalence of ESbL-producing E. coli isolates from different countries was documented by Phongpaichit et al. : 12.6% in Thailand, 5–8% in Korea, Japan, Malaysia, and Singapore, 12–24% in Taiwan, Philippines, Indonesia, Hong Kong, and China, and 31% in Bangkok. The Pan European Antimicrobial Resistance using Local Surveillance (PEARLS) study reported the highest rates of ESbL-producers in Egypt (38.5%) and Greece (27.4%) and the lowest rates in the Netherlands and Germany (3.6%) .
In our study, class 1 integrons were detected in 50% of the isolates with only one ESbL genotype, but in 7.4 and 14.8% of the isolates with two or three different ESBL genotypes, respectively. The predominant ESbL genewasthe SHV gene. It was present among 70.4% of E. coli CIs and 100% of E. coli commensals. TEM and CTX-M genes were present among 37 and 29.6% of E. coli CIs, respectively, and they were not present among E. coli commensals. There were significant differences in ESbL gene types between E. coli CIs and commensals. Our results were in agreement with those of Färber et al. , and not in agreement with those of Mohamed Al-Agamy et al.  andBali et al. , who reported thatthe TEM gene wasthe most prevalent ESbL gene among CIs of Enterobacteriaceaeworldwide. In the study carried out by Chen et al. , 90% of the ESbL-producing E. coli isolates harbored CTX-M genes, whereas only 59 and 32% had TEM and SHV genes, respectively. The study by Machado et al.  showed that conjugative transfer of an integron with the genetic elements carrying ESbL genes was observed more frequently for CTX-M (95%) than for SHV (33%) and TEM (23%).
In the present study, ICUs were the epicenter for ESbL gene-positive E. coli isolates, where 61.5% of E. coli CIs that carried the SHV gene alone were isolated from ICUs. However, the TEM gene alone was found in 33.3% of E. coli CIs in the ICU and 33.3% E. coli CIs in the pediatric department. This indicated that ICUs had a high prevalence of ESbL-producing E. coli CIs. This result was in agreement with that of Paterson and Bonomo , who found that ICUs were often the epicenter of ESbL production in hospitals, and in one large outbreak, more than 40% of all the ESbL-producing organisms in the hospital were from patients in ICUs; ESbLs evolve spontaneously outside ICUs. Nursing homes and chronic care facilities may be a focus of infections with ESbL-producing organisms .
Catheter urine specimens represented by 69.2% of ESbL gene-positive E. coli CIs that had SHV gene alone and represented by 33.3% of that had TEM gene alone. This was in agreement with the result of Moor et al. , who found that urine was the most common source (97%) of ESbL-producing E. coli. Also, 33.3% of E. coli CIs that had the TEM gene alone was found in the wound swabs. Sputum specimens were present in 50% of E. coli CIs that had the CTX-M gene alone and in 75% of E. coli CIs that had SHV, TEM, and CTX-M genes. Our results showed that catheter urine and sputum specimens were the major sources of ESbL-producing E. coli.
Admission to ICUs was the main risk factor among our patients with IntI1 gene-positive E. coli (80%). This was in agreement with the results of Grape et al. , who found that higher numbers of IntI1 gene- positive isolates were found in patients in the ICU compared with non-ICU patients and outpatients (82 vs. 10 and 8%, respectively).
Other risk factors detected in our study affecting IntI1 gene- positive E. coli results were prolonged stay in the hospitals, exposure to invasive procedures, administration of antibiotics, corticosteroids, and/or cytotoxic drugs, and associated comorbidities. This was in agreement with the study of Machado et al. , who attributed this to alinkage with particular strains and/or transferable genetic elementsin the hospital. However, Sepp et al.  found that the resistance of IntI1 gene-positive and gene-negative strains was more dependent on influencing factors (hospitalization and antibiotic administration) than the presence or absence of integrons.
In our study, there was no significant difference between ESbL-producers and non-ESbL-producers E. coli CIs and E. coli commensals in the presence of the intI1 gene. A phenotypic ESbL study showed that 41.2% of intI1 gene-positive E. coli CIs and 16.7% of intI1 gene-positive E. coli commensals were ESbL producers. This was in agreement with the study of Tacao et al. , in which the prevalence of intI1 gene-positive E. coli was 56.41% among ESbL-producer strains and 27.67% among non-ESBL-producer strains. This is also comparable with the study of Phongpaichit et al. , in which class 1 integrons were found more frequently among ESbL-producing (56/75; 74.7%) than among non-ESbL-producing (19/75; 57.3%) E. coli strains. The results of Chen et al.  are also in agreement with our results.
For the ESbL gene type, no statistically significant difference was found in relation to the presence of the intI1 gene with the predominant SHV gene type (71.4% among E. coli CIs and the only gene detected in E. coli commensals). This was in agreement with Chen et al. , who reported that the prevalence of class 1 integrons among SHV, TEM, and CTX-M gene carriers was 84.4, 72.9, and 68.9, respectively.
| Conclusion|| |
Class 1 integrons are widely prevalent among resistant E. coli CIs, especially urinary isolates, suggesting an important role as a reservoir in producing drug resistance. They were associated with admission to ICU, long hospital stay, exposure to invasive procedures, and a history of drug intake.
A restrictive antibiotic subscription policy is needed to avoid an increased selection pressure where integrons play a potentially significant role in the uptake and dissemination of resistance genes.
The prevailing trend of the co-occurrence of class 1 integrons and antimicrobial multiresistance is an additional threat for the spread of the antimicrobial multiresistance, which may further complicate future strategies for empirical therapy.
Class 1 integrons circulate in commensal fecal flora in the community and associate with MDR in commensal E. coli isolates. Therefore, careful monitoring is necessary for the prevention of a wide dissemination of integrons and an increase in community-acquired infections by MDR pathogens.
Although class 1 integrons were associated more strongly with resistance against older antibiotic classes, a fair agreement was found between class 1 integrons and ESbL production.
| Acknowledgements|| |
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], [Table 4], [Table 5], [Table 6], [Table 7]