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View of Prevalence of Class 1 and Class 2 Integrons in Extensively Drug-Resistant Escherichia Coliisolated from Iraqi Patients with Diabetic Foot Ulcers in Diyala Province

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Prevalence of Class 1 and Class 2 Integrons in Extensively Drug-Resistant Escherichia Coliisolated from Iraqi Patients with Diabetic Foot Ulcers in

Diyala Province

Lina Abdulameer S.Alsaadi*1,Iman Abbas Ali2,Saba Adnan Abbas3

1,2,3

Department of biology /College of science/Diyala university /Iraq [email protected]

Abstract

Diabetic foot infectionis a common and serious problem for all health systems in the world.

This study aimed tomolecularlydetect class (1)and class (2) integron genes responsible for antibiotic resistance by using PCR techniquein extensively drug-resistant Escherichia colibacteria isolated from clinical samples of (285) Iraqi patients with diabetic foot ulcers(DFUs) who attended different hospitals in Diyala province/Iraq during the period from July to the end of October /2020. The results showed that bacterial isolates appeared in (250) of these samples. The conventional microbiological methods and VITEK 2 automated system showed that 65 (26%) of the isolates were Escherichia coli isolates.For all the (65) pathogenic isolates of Escherichia coli, susceptibility testswere performed against 15antimicrobial agents.The results of antibiotic resistance showedthe following:Amoxicillin- Clavulanic acid 100%, cefotaxime 92.3%, piperacillin 84.%, ceftriaxone 78.4%, cefepime 76.6%,ceftazidime75.3%, ciprofloxacin 67.9%, levofloxacin 69.2%,while resistance rate to aminoglycosides was 92.3, 80% and 76.9% for gentamicin, amikacin and tobramycin respectively. Resistance rate foraztreonam was 52.3%, Imipenem 33.8% and Meropenem 43%.However, 20 (30.7%) isolatesof Escherichia coli were found to be extensively drug- resistant. Depending on the detection of integrase gene for the investigation of class 1 and class 2 integrons, PCR assay showed that 16(80%) of the extensively drug-resistant E. coli isolates were integrase gene positive, which confirms the extremely high dissemination of class 1 integron at the hospitals of Baquba city in Diyala province, while class2 integrase gene was not found. Analysis of class 1 integron variable regions showed the presence of (5) different fragment sizes of approximately 500, 600,700, 800 and 900 bp. On RFLP using restriction enzyme AluI, five different restriction patterns obtained Class1(500bp, 200-300 fragment), Class2 (±600bp, 90-200-300 fragment) Class3 (700bp, 200-500 fragment), Class4 (800bp, 100-200-500 fragment) and Class5(900bp, 100,200,600 fragment).

Keywords: Escherichia coli, Diabetic Foot ulcers, antibiotic resistance, PCR, Class 1 and Class 2 Integrons

Introduction

Diabetic foot ulcer (DFU) is a serious and common diabeticcomplication that significantly increases the cost of treatment.[1] the world health organization defined diabetic foot (DF) as a pathologic consequence including infections, ulcerations and/or destructions of deep tissuesrelated to neurologic abnormalities, different degrees of peripheral vascular disease and/or metabolic complication of diabetes in the lower limbs [2]. One of the bacteria in diabetic foot ulcer is Escherichia coli.Its long term complication represents a major health problem of high mortality and morbidity rates. According to bacterial culture and molecular approaches, DFU can be colonized with numerous aerobic and anaerobic polymicrobials[3,4].Among several types of bacteria isolated from diabetic foot ulcer patients, E .coliis considered one of the most important causes in the last years [5].

Escherichia coli, one of the most widely studied micro-organism worldwide, resides in the gastrointestinal tract of warm-blooded animals and reptiles and is an important pathogen in humans[6].In a world health organization report on antimicrobial resistance surveillance,

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E.coli is listed as one of the 9 bacteria of global concern responsible for common infections in hospitals and communities [7].The rapid dissemination of multidrug-resistant bacteria has become a concern worldwide and complicates the treatment of infections. This phenomenon is a consequence of the bacterialability for acquiring exogenous genes by mobile elements (e.g. conjugative plasmid, transposons & integrons), and has been recognized as a major cause of widespread multidrug resistance pheno- and genotypes[8].Although integrons are not mobile by themselves, they can be transferred horizontally. Resistance integrons are elementscontaining the components of a system for sitespecific recombination, distinguishing, capturing and carrying resistance genes in mobile cassettes. For these reasons, integrons are the major contributors to the spread and maintenance of MDR. [9,10]. Integrons are classified into several classes based on the sequence of their intI genes. Class 1–3 integrons are widely associated with resistance determinants in human clinical isolates. Class 1 integron is primarily associated with capture gene cassettes from a huge pool of resistance genes conferring antibiotic resistance[10]. Moreover, class 1 integron is the most frequently element found in nosocomial and community environments[11].Because the integron system is able to create novel combination of resistance gene, it could be a dynamic force in the MDR bacteria evolution. Moreover, the entire integron element is usuallyfound within other mobile genetic elements like the plasmid & transposon. The integron element containing its gene cassettes is able disseminate horizontally through a bacterial population [12].This study aimed to assess the occurrence of class1& class2 integrase-specific int1 and int2 genes integrons and to investigatethe diversity of their variable regions carried by commensalE. coli isolates collected from Iraqi Patients with Diabetic Foot ulcers(DFUs) in Diyala Provence.

Materials and methods Sample collection

In our study,(285) swab specimens were collected from diabetic foot infection ulcers from Baquba hospital/Central health LabsfromJuly to the end of October /2020.The specimens collected were (21 males and 15 females) with their ages ranging between (35-80)years. A single colony was selected from each primary positive culture on blood agar, MacConkey agar and mannitol salt agar, and identified depending on its morphological and cultural characteristics. The biochemical tests were used to confirm the diagnosis of E.coli using the Vitek-2 system [13].

Antimicrobial SusceptibilityTest

For the detection of the sensitivity of 65E.coli isolates, 15 antimicrobial discswere used in accordance withthe method of Bauer and Kurby[14].Muller Hinton agar plates were prepared. In (5) ml of normal saline, the isolated colonies were suspended and mixed by vortex, and the turbidity was compared with the standard McFarland solution. On Muller Hinton agar,aliquot of 2ϻl bacterial suspensionswere placed anddistributed by cotton swabs in three different directions via rotating the plates 60o for each direction. The agar plates were placed upside down at room temperature for few minutes. Antibiotic discs were put on the agar plates and incubated at 37oC overnight. The zone inhibition ruler was used to measure the inhibition zones in (mm) and the results were compared with the National committee for clinical laboratory standards[15].

Any bacterialstrain that resisted the minimum of at least 3 differentclasses of antibiotics was considereda multi-drug resistant(MDR), while any bacterial strain that remained susceptible toonly one or twoclasses of antibiotics wasconsidered anextensive- drugresistant(XDR),whereas any bacterial isolate which resistedall sub classes of antibiotics was considered a pandrugresistant (PDR)[16].

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DNA Extraction

From all bacterial isolates, the genomic DNA was extractedby using the extraction kit of genomic DNA. The purification was based on the manufacturing company guidelines (Promega USA). The DNA concentration was misheard by using Quantus Florometer (Promega, USA). All twenty extensively-drug resistant (XDR) isolates were examinedusingthe standard PCR conventional by applying specific primers for class 1 integrase gene, class 2 integrase gene and variable regions of class 1 integron CS genes as shown in table (1).

Table (1): The primers used fortarget gene detection

Product Size (bp) Annealing

Temp . (C) Seq.

Gene Target

280 60

5-CCT CCC GCA CGA TGA TC -3 5’TCC ACG CAT CGT CAG GC -3’

int1-F int1-R

Class 1

integrase gene

466 5’- GCAAATGAAGTGCAACGC -3’ 48

5’- ACACGCTTGCTAACGATG -3’

Int2-F Int2-R Class 2

integrase gene

Variable 5’-GGCATCCAAGCAGCAAG-3’ 55

5’AAGCAGACTTGACCTGA-3’

In5`CS-F In3`CS -R Variable region

of class 1 integron

The PCR conditions started with a thermocycler program according to data shown in table (2). Amplified PCR products were detected by agarose gel electrophoresis.

Table (2): The PCR thermocycler program for Escherichia coli target genes Cycle

m:s

°C Steps

1 30 30 30 1 1 05:00

00:30 00:30 01:00 07:00 10:00 95

95

60,48or 55 72

72 10 Initial denaturation

Denaturations Annealings Extensions Final extensions Holds

* Elongation in 72ºC/ 1 minute and final extension 72°C/ 7 min for all genes The restriction fragment length polymorphism (RFLP)

To confirm that the different bacterial isolates were carrying identicalintegrons, RFLP typing was used to compare amplicons with similar size by using the restriction endonucleasesAluI.

RFLP of the variable region class 1 integronCS gene was performed using the restriction enzyme AluI (promega U.S.A) in accordance with the manufacturer’s guidelines. In this technique, 2U of the AluI enzyme was used to digest 10 µl of the CS gene PCR products and incubated for 4 hours at 37oC in a waterbath.Then the 2%agarose of gel electrophoresis was used to separate the restricted fragments.

Resultsand discussion

From the (150) diabetic patients with foot ulcers,(185) bacterial isolates were taken. The ages of the patients ranged between (35-80) years, and the highest number of diabetic foot ulcers (DFUs) patients was found within the age group (60-65) years.The results showed thatStaphylococcus spp was the most frequent isolate 76 (30%) followed by Escherichia coli

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(26%) thenEnterococcus spp. (21%). All isolates were identified through morphological, cultural and some biochemical tests by using Vitek-2-GN system. The bacterial antibiotic susceptibilitytesting was also performed. The resistance of the (65) E. coli isolates was studied against (15) different antibiotics. The table (3) shows different sensitivity and resistance patterns to the (15) antibiotics.

Table (3): Antibiogram susceptibility of E. coli isolatestoward antibiotics Percentage%

Resistance Number of isolates n=65 Antibiotics

)84.6%) )92.3%) (100%) )92.3%) (78.4%) (75.3%) (76.9%) (76.9%) (69.2%) )92.3%) (76.9%) (80%) (52.3%) (33.8%) (43%) 55

60 65 60 51 49 50 50 45 60 52 50 34 22 28 Pipracilin

Ticarcilin

Amoxicillin/Glavulanic acid

Cefotaxime Ceftriaxone Ceftazidime Cefepime Ciprofloxacin Levofloxacin Gentamycin Amikacin Tobramicin Azetreonam Impenem Meropenem

The higher resistance was shown tothe β-lactams, aminoglycosides and flouroquinolones. A moderate resistance was shown to monobactams, as (52.3%) of the isolates were resistant to azetreonam, whereascarbapenemes showed the lowest resistance (43%) and (33.8%) for meropenem and imipenem respectively. The bacterial isolates exhibitedthe highest resistance (100%) to Amoxicillin/Glavulanic acid followed by Cefotaxime & Gentamicin (92.3%), Pipracilin (84.6%), Amikacin (80%), Ceftriaxone (78.4%), Cefepime and Ciprofloxacin (76.9%). Resistance to other antibiotics was (75%) to Ceftazidime, (69%) to Levofluxacin.

Table (3) showed various antibiotic resistance patterns among the studied isolates. In the current study, 18(27.6%) of isolateswere classified as multidrug sensitive (MDS),27(41.5%) of isolates were multidrug resistant (MDR) because they showed resistance to three antibiotic classes at least. Among these isolates, 20(30.7%)were resistant to 5 or 6 classes of antibiotics meeting criteria for extensively-drug resistant (XDR) organisms.

Astudy by Naqid [17] inKurdistan Region of Iraq revealed that E. coliisolates were highly sensitive to eretapenem andimpenem (96.4%) and (97.6%) respectively, but showed (87.8%) resistance to ampicillin. Results in the present study agreed with Al–Zubaidi [18] who stated that the isolates ofE. coli inDiyala hospitals demonstrated resistance to various antibiotic types, such as floroquinolones and carbapenemsand 50%, 25% of isolates were resistant to Levofloxacin and Imipenem, respectively.

Detection and analysis of integrons

Class 1 integron gene intI1 was found in 16 (80%) of theextensively drug-resistant E. coli isolates (Figure 1,2). No strain was shown to harbor class 2 integrase geneintI-2, which confirms the extremely high dissemination of class 1 integron at the hospitals of Baquba in Diyala province. This result agrees with Singh [19] whose PCR results showed that 75% of Escherichia coli isolates carried class 1 integron but it differs from the Iranian study

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presented by Halaji et al[20]who revealed 47% integron positive Escherichia coli isolates.Among the multidrug resistant Gram-(-ve) bacteria, integrons play a key role because of their higher ability to transfer antibiotic resistance genes [21]. In our study, a highly prevalence of integrons was observed; however, intl-1 integrons were found to be much more than intl-2 integrons.

Figure (1):Agarose gel electrophoresis of the amplified PCR productsto detectIntgron class1(IntI1) genes (280bp) run on 1.5%agarose (at 70 volts for 90 minutes), stainingby ethidium bromide, Lane 1-10Escherichia coliisolate ;M: Marker DNA ladders (100bp);

Lane2,3,5,7,8,9,10 positive to class1 Intgron IntI1 gene.

Figure(2):Agarose gel electrophoresis of amplified PCR productsto detect Intgron class1(IntI1) genes (280bp) run on 1.5%agarose ( at 70 volts for 90 minutes), staining

with ethidium bromide, lane 11-20 Escherichia coliisolates;M: Marker DNA ladder (100bp); Lanes 11,12,13,14,16,17,18,19,20 positive for class1 Intgron( IntI1) gene.

The analysis of class 1-integron variable regionshowed existence of five(5) variable fragment sizes of about 500, 600,700, 800 and 900 bp( Table 4)(Figure 3).

Table (4): Class1 Intgron variable regionsCS gene typing No. of isolates &

% PCR product (pb) class1 Intgron variable

regions typing

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2( 12.5%) 3 (18.7%) 8(50%) 2( 12.5%) 1(6.25%) 500

600 700 800 900 Type I

Type II Type III Type IV Type V

In our study, PCR amplification of CS gene of the sixteen extensively drug-resistant E.

coliisolates (class 1 integron positive) were digested with AluI enzyme and obtained five distinct RFLP patterns with different number of fragments that varied from 1 to 4 with sizes of fragments varied from (90 to 600 bp).On RFLP using restriction enzyme AluI, five different restriction patterns obtained Class1(500bp, 200-300 fragment), Class2 (±600bp, 90- 200-300 fragment) Class3 (700bp, 200-500 fragment), Class4 (800bp, 100-200-500 fragment) and Class5 (900bp, 100,200,600) (Figure 3).

The class 1 integron variable regions (CS) gene and PCR–RFLP of the CSgenotyping is an essentialtechnique to investigate infectious diseaseoutbreaksaiming to explainthe temporal and local increase in infection incidence causedbysome bacterial species [22]. The outbreak strain typingmakes theoutbreak control plans easier, definies thedegree of spreading of bacterial types and clone numberscontributed toinfection transmissions, observingthe reservoir of epidemic cloneand controling the assessment of the control plan,like monitoring vaccinations process effectiveness[23].

FIGURE 3 :The ethidium bromide stained gel showing the typical banding patternexamined with the uniplex PCR assay, PCR amplification of the class1 Intgron variable regions using primersIn5'CS and In3'CSgene from Escherichia coli, with the amplicon size 500-900bp. DNA amplification products were separated by

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electrophoresis in an (2%) agarose gel. The electrophoresis was done at 70 volts for 1.5 hours. The symbol “M” refers to ladder marker, Lane ( 10,14): class1 Intgron variable regionsgene type I (500bp), lane(8,9,11) type II (600bp), lane (4,5,6,7,12,13,16)type III(700) , lane (1,15)type IV(800bp),lane 3 type V (900bp), RFLP patterns obtained of class1 Intgron variable regions PCR products digested byAluI endonuclease. Lane (10,14)type1500bp (fragments : 200, 300 bp), lane(8,9,11) typeII 600 pb (fragments:

90,200,300bp),lane(4,5,6,7,12,13,16)typeIII700bp (fragments : 200, 300,400 bp), lane (1,15)typeIV 800bp (fragments :100, 200, 500 bp), lane 3 type V (900bp) (fragments:

100,200,600).

Conclusions

Thecurrent study demonstratedsuperiority of extensively drug-resistantstrainin the isolates ofE. colifrom patients with DFUs. Knowing the isolate's antibiotic susceptibility patterns canhelp todetermine the empirical treatment of diabetic ulcer. Therefore, indiscriminate antibiotic use and chances of resultant antibiotic resistance emergencemay also be decreased.It can be concluded from this study that integrons were prevalent and playedbasic roles in multidrug resistant E. coli, which cangive some essential surveillance information representing the antibiotic selective pressures in this specific area. It is important to concentrate on investigating the infection source in order to reduce the emergence of the multi-resistant strains.

References

1- Lipsky BA.(2004). A report from the international consensus on diagnosing and treating the infected diabetic foot. Diabetes Metab Res Rev. 20(Suppl 1):68–77.

2- World Health Organization. Classification of diabetes mellitus,(2019).

3- Center for Disease Control and Prevention.(2011). National Diabetes Fact Sheet: National Estimates and General Information on Diabetes and Prediabetes in the United States.

Atlanta, GA, USA: Department of Health and Human Services, Center for Disease Control and Prevention.

4- Huang Y, Cao Y, Zou M, Luo X, Jiang Y, Xue Y, et al.(2016). Acomparison of tissue versus swab culturing of infected diabetic foot wounds. Int J Endocrinol. 2016:8198714.

5- Zubair M, Malik A, Ahmad J. (2010). Clinico-bacteriology and risk factors for the diabetic foot infection with multidrug-resistent microorganisms in North India. Biology and Medicine, 2 (4), 22-34.

6- Tenaillon O, Skurnik D, Picard B, Denamur E.(2010). The population genetics of commensal Escherichia coli. Nat Rev Microbiol;8:207–217.

7- World Health Organization. (2014). Antimicrobial resistance: global report on surveillance.

www.who.int/drugresistance/documents/ surveillancereport/en/ [accessed 9 November 2016].

8- Berendonk TU, Manaia CM, Merlin C, Fatta-Kassinos D, Cytryn E et .al.(2015) Tackling antibiotic resistance: the environmental framework. Nat Rev Microbiol;13:310–317.

9- Gillings M.R. (2014). Integrons: past, present, and future.Microbiol. Mol. Biol. Rev. 78:257–

277.

10- Deng, Y., X. Bao, L. Ji, L. Chen, J. Liu, J. Miao, D. Chen, H. Bian, Y. Li, and G. Yu. 2015.

Resistance integrons: class 1, 2 and 3 integrons. Ann. Clin. Microbiol. Antimicrob. 14:45.

11- Wei, Q., X. Jiang, M. Li, G. Li, Q. Hu, H. Lu, G. Chen, Y. Zhou, and Y. Lu. (2013).

Diversity of gene cassette promoter variants of class 1 integrons in uropathogenic Escherichia coli. Curr. Microbiol. 67:543–549.

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12- Solberg O.D., R.M. Ajiboye, and L.W. Riley.( 2016). Origin of class 1 and 2 integrons and gene cassettes in a population-based sample of uropathogenic Escherichia coli. J. Clin.

Microbiol. 44:1347–1351.

13- Collee J. In: Collee J, Fraser G, Marimon AG, Simmons BP, editors.(2007). Mackie

&Mccartney practical medical microbiology. Elsevier.

14- Bauer, A.W., Kirby, W.M.M., Sherris, J.C. and Turck, M.(1996). Antibiotic susceptibility testing by a standardized single disk method. Am. J. Clin. Pathol., 45(4): 493-496.

15- Patel JB, Weinstein M, Eliopoulos G, Jenkins S, Lewis J, Limbago B, Mathers AJ, Mazzulli T.(2017).M100 Performance standards for antimicrobial susceptibility testing.

United State: Clinical and Laboratory Standards Institute(CLSI), p.240.

16- Magiorakos A.P.(2011). Multidrug Resistant (MDR), Extensively Drug Resistant (XDR) and Pandrug-1 Resistant (PDR) Bacteria in Healthcare Settings. Expert Proposal for a Standardized International Terminology.

17- Naqid I A, Balatay A A, Hussein N R, Saeed K A, Ahmed H A, et al. Antibiotic Susceptibility Pattern of Escherichia coli Isolated from Various Clinical Samples in Duhok City, Kurdistan Region of Iraq, Int J Infect. 2020 ; 7(3):e103740.

18- AL–ZUBAIDI, S. J. J. (2021). Molecular typing using RAPD–PCR of Multidrug resistance Escherichia coli isolated from patients in Diyala province/Iraq. International Journal of Pharmaceutical Research, 13(2).‏

19- Singh NS, Singhal N, Kumar M and Virdi JS (2021). High Prevalence of Drug Resistance and Class 1 Integrons in Escherichia coli Isolated From River Yamuna, India: A Serious Public Health Risk. Front. Microbiol. 12:621564.

20- Halaji, M., Feizi, A., Mirzaei, A., Sedigh Ebrahim-Saraie, H., Fayyazi, A., Ashraf, A., &

Havaei, S. A. (2020). The Global Prevalence of Class 1 Integron and Associated Antibiotic Resistance in Escherichia coli from Patients with Urinary Tract Infections, a Systematic Review and Meta-Analysis. Microbial Drug Resistance, 26(10), 1208-1218.‏

21- Fluit, A.C., Schmitz, F.J., (2004). Resistance integrons and super-integrons. Clin. Microbiol.

Infect. 10, 272–288.

22- Rehman, M.U. (2017). Characteristics of integrons and associated gene cassettes in antibiotic-resistant Escherichia coli isolated from free-ranging food animals in China J.

Food Sci. 82, 1902–1907.

23- Yuan, Q., et al., (2018). Antibiotic resistance genes and intl1 prevalence in a swine wastewater treatment plant and correlation with metal resistance, bacterial community and wastewater parameters. Ecotoxicol. Environ. Safe. 161, 251–259.

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