Detection of biofilm formation and correlation with antibiotic resistance in Acinetobacter baumannii

Acinetobacter baumannii is a major cause of hospital acquired infections worldwide and is associated with resistance to routinely used antibiotics. Many clinical isolates of Acinetobacter baumannii are found to be biofilm producers. Hence there is difficulty in treating patients with Multi Drug Resistant Acinetobacter baumannii (MDRAB). This present study aimed to study antibiotic resistance of A. baumannii isolates and to evaluate the biofilm formation of Acinetobacter baumannii by Tube Method (TM) and Microtiter Plate Method(MTPM).In this study, 73 A. baumannii isolates of various clinical specimens were evaluated. Confirmation was done through conventional methods. Testing for antimicrobial susceptibility was done by Kirby Bauer disc diffusion method. Biofilm formation was studied by TM and MTPM. Of the 73 isolates, 26(36%) were from urine, 19 (26%) from pus, 17 (23%) from sputum and 11 (15%) from other miscellaneous(body fluids excluding blood), out of which 81%(59/73) isolates were Multi Drug Resistant (MDR). 63% and 84% of isolates showed biofilm production in TM and MTPM, respectively. When comparing these two methods, MTPM assay was better than TM. Presence of a strong relationship between biofilm formation and MDRAB has been confirmed by the present study. Both methods used for detection of biofilm formation were found to be statistically significant. Sensitivity of MTPM was more than TM, which is supported by higher Positive Predictive Value of 87.5%. Therefore MTPM is a better method than TM and can be used as a screening method to detect biofilm production.

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Virulence Characteristics of Biofilm-Forming Acinetobacter baumannii in Clinical Isolates Using a Galleria mellonella Model

Microorganisms ◽

10.3390/microorganisms9112365 ◽

2021 ◽

Vol 9 (11) ◽

pp. 2365

Author(s):

Mahmoud A. F. Khalil ◽

Fatma A. Ahmed ◽

Ahmed F. Elkhateeb ◽

Eman E. Mahmoud ◽

Mona I. Ahmed ◽

...

Keyword(s):

Acinetobacter Baumannii ◽

Biofilm Formation ◽

Galleria Mellonella ◽

Microtiter Plate ◽

Diffusion Method ◽

Disk Diffusion Method ◽

Icu Patients ◽

Carbapenem Resistant ◽

Hospital Acquired

Acinetobacter baumannii is a Gram-negative coccobacillus responsible for severe hospital-acquired infections, particularly in intensive care units (ICUs). The current study was designed to characterize the virulence traits of biofilm-forming carbapenem-resistant A. baumannii causing pneumonia in ICU patients using a Galleria mellonella model. Two hundred and thirty patients with hospital-acquired or ventilator-associated pneumonia were included in our study. Among the total isolates, A. baumannii was the most frequently isolated etiological agent in ICU patients with pneumonia (54/165, 32.7%). All A. baumannii isolates were subjected to antimicrobial susceptibility testing by the Kirby–Bauer disk diffusion method, while the minimum inhibitory concentrations of imipenem and colistin were estimated using the broth microdilution technique. The biofilm formation activity of the isolates was tested using the microtiter plate technique. Biofilm quantification showed that 61.1% (33/54) of the isolates were strong biofilm producers, while 27.7% (15/54) and 11.1% (6/54) showed moderate or weak biofilm production. By studying the prevalence of carbapenemases-encoding genes among isolates, blaOXA-23-like was positive in 88.9% of the isolates (48/54). The BlaNDM gene was found in 27.7% of the isolates (15/54 isolates). BlaOXA-23-like and blaNDM genes coexisted in 25.9% (14/54 isolates). Bap and blaPER-1 genes, the biofilm-associated genes, coexisted in 5.6% (3/54) of the isolates. For in vivo assessment of A. baumannii pathogenicity, a Galleria mellonella survival assay was used. G. mellonella survival was statistically different between moderate and poor biofilm producers (p < 0.0001). The killing effect of the strong biofilm-producing group was significantly higher than that of the moderate and poor biofilm producers (p < 0.0001 for each comparison). These findings highlight the role of biofilm formation as a powerful virulence factor for carbapenem-resistant A. baumannii that causes pneumonia in the ICU.

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Antibiofilm and antivirulence potential of silver nanoparticles against multidrug-resistant Acinetobacter baumannii

Scientific Reports ◽

10.1038/s41598-021-90208-4 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Helal F. Hetta ◽

Israa M. S. Al-Kadmy ◽

Saba Saadoon Khazaal ◽

Suhad Abbas ◽

Ahmed Suhail ◽

...

Keyword(s):

Silver Nanoparticles ◽

Antibacterial Activity ◽

Acinetobacter Baumannii ◽

Biofilm Formation ◽

Microtiter Plate ◽

Diffusion Method ◽

Resistance Pattern ◽

Infection Model ◽

The Impact

AbstractWe aimed to isolate Acinetobacter baumannii (A. baumannii) from wound infections, determine their resistance and virulence profile, and assess the impact of Silver nanoparticles (AgNPs) on the bacterial growth, virulence and biofilm-related gene expression. AgNPs were synthesized and characterized using TEM, XRD and FTIR spectroscopy. A. baumannii (n = 200) were isolated and identified. Resistance pattern was determined and virulence genes (afa/draBC, cnf1, cnf2, csgA, cvaC, fimH, fyuA, ibeA, iutA, kpsMT II, PAI, papC, PapG II, III, sfa/focDE and traT) were screened using PCR. Biofilm formation was evaluated using Microtiter plate method. Then, the antimicrobial activity of AgNPs was evaluated by the well-diffusion method, growth kinetics and MIC determination. Inhibition of biofilm formation and the ability to disperse biofilms in exposure to AgNPs were evaluated. The effect of AgNPs on the expression of virulence and biofilm-related genes (bap, OmpA, abaI, csuA/B, A1S_2091, A1S_1510, A1S_0690, A1S_0114) were estimated using QRT-PCR. In vitro infection model for analyzing the antibacterial activity of AgNPs was done using a co-culture infection model of A. baumannii with human fibroblast skin cell line HFF-1 or Vero cell lines. A. baumannii had high level of resistance to antibiotics. Most of the isolates harbored the fimH, afa/draBC, cnf1, csgA and cnf2, and the majority of A. baumannii produced strong biofilms. AgNPs inhibited the growth of A. baumannii efficiently with MIC ranging from 4 to 25 µg/ml. A. baumannii showed a reduced growth rate in the presence of AgNPs. The inhibitory activity and the anti-biofilm activity of AgNPs were more pronounced against the weak biofilm producers. Moreover, AgNPs decreased the expression of kpsMII , afa/draBC,bap, OmpA, and csuA/B genes. The in vitro infection model revealed a significant antibacterial activity of AgNPs against extracellular and intracellular A. baumannii. AgNPs highly interrupted bacterial multiplication and biofilm formation. AgNPs downregulated the transcription level of important virulence and biofilm-related genes. Our findings provide an additional step towards understanding the mechanisms by which sliver nanoparticles interfere with the microbial spread and persistence.

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Evaluation of the Role of Staphylococci in the Pathomechanism of Conjunctivitis

10.21203/rs.3.rs-191872/v1 ◽

2021 ◽

Author(s):

Ewa Jasińska ◽

Agnieszka Bogut ◽

Agnieszka Magryś ◽

Alina Olender

Keyword(s):

Antibiotic Resistance ◽

Epithelial Cells ◽

Biofilm Formation ◽

Methicillin Resistance ◽

Microtiter Plate ◽

Host Cells ◽

Diffusion Method ◽

Microtiter Plate Assay ◽

Low Prevalence

Abstract Purpose: Determination of the association between ica genes and phenotypic biofilm formation in staphylococcal isolates involved in conjunctivitis, their antibiotic resistance as well as detection of selected virulence characteristics: adhesion to epithelial cells and in vitro cytotoxicity.Methods: The study included 26 Staphylococcus aureus (SA) and 26 Staphylococcus epidermidis (SE) isolates. The presence of icaAD genes and ica operon was determined by the PCR assay. Phenotypic biofilm formation was verified using the microtiter plate assay. Antibiotic resistance was performed using the disc diffusion method. Staphylococcal ability to attach to host cells was assessed by flow cytometry. Cytotoxicity on epithelial cells was evaluated by LDH assay.Results: The ica genes were detected in 26.9% of SE and in 42.3% of SA isolates. Only 15.3% of isolates (SE) were positive for both the icaAD and the ica operon. Phenotypically, 19.2% of SE isolates were strong biofilm producers, among which three were both icaAD- and ica operon-positive. 26.9% of SA isolates were strong biofilm producers. Methicillin resistance (MR) was detected in 34.6% of SE and 26.9% of SA isolates. 75% of MR isolates were multidrug resistant. SA isolates adhered to host cells more extensively than SE. SA isolates released higher level of LDH than SE.Conclusions: Adherence abilities were commonly observed in staphylococci associated with conjunctivitis. However, low prevalence of isolates positive for a complete and functional ica locus and low prevalence of strong biofilm producers was detected. SA adhered to a greater extent to eukaryotic cells than SE and were more cytotoxic.

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Correlation of biofilm formation and antibiotic resistance among clinical and soil isolates of Acinetobacter baumannii in Iraq

Acta Microbiologica et Immunologica Hungarica ◽

10.1556/030.66.2019.026 ◽

2019 ◽

pp. 1-10 ◽

Cited By ~ 1

Author(s):

Pakhshan A. Hassan ◽

Adel K. Khider

Keyword(s):

Acinetobacter Baumannii ◽

Congo Red ◽

Biofilm Formation ◽

Opportunistic Pathogen ◽

Microtiter Plate ◽

Test Tube ◽

Clinical Isolates ◽

Diffusion Method ◽

Biochemical Tests ◽

Disk Diffusion Method

Acinetobacter baumannii is an opportunistic pathogen that is reported as a major cause of nosocomial infections. The aim of this study was to investigate the biofilm formation by A. baumannii clinical and soil isolates, to display their susceptibility to 11 antibiotics and to study a possible relationship between formation of biofilm and multidrug resistance. During 8 months period, from June 2016 to January 2017, a total of 52 clinical and 22 soil isolates of A. baumannii were collected and identified through conventional phenotypic, chromo agar, biochemical tests, API 20E system, and confirmed genotypically by PCR for blaOXA-51-like gene. Antibiotic susceptibility of isolates was determined by standard disk diffusion method according to Clinical and Laboratory Standard Institute. The biofilm formation was studied using Congo red agar, test tube, and microtiter plate methods. The clinical isolates were 100% resistance to ciprofloxacin, ceftazidime, piperacillin, 96.15% to gentamicin, 96.15% to imipenem, 92.31% to meropenem, and 78.85% to amikacin. The soil A. baumannii isolates were 100% sensitive to imipenem, meropenem, and gentamicin, and 90.1% to ciprofloxacin. All A. baumannii isolates (clinical and soil) were susceptible to polymyxin B. The percentage of biofilm formation in Congo red agar, test tube, and microtiter plate assays was 10.81%, 63.51%, and 86.48%, respectively. More robust biofilm former population was mainly among non-MDR isolates. Isolates with a higher level of resistance tended to form weaker biofilms. The soil isolates exhibited less resistance to antibiotics than clinical isolates. However, the soil isolates produce stronger biofilms than clinical isolates.

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Correlation of biofilm production with antibiotic susceptibility pattern of Pseudomonas aeruginosa from various clinical specimens

Asian Journal of Medical Sciences ◽

10.3126/ajms.v13i1.39915 ◽

2022 ◽

Vol 13 (1) ◽

pp. 88-92

Author(s):

M Swapna ◽

G Sumathi ◽

M Anitha

Keyword(s):

Pseudomonas Aeruginosa ◽

Antibiotic Resistance ◽

Biofilm Formation ◽

Antibiotic Sensitivity ◽

Microtiter Plate ◽

Resistance Pattern ◽

Sensitivity Testing ◽

Clinical Specimens ◽

Biofilm Production ◽

Microbiological Methods

Background: Pseudomonas aeruginosa is one of the most prevalent nosocomial pathogens that cause a life-threatening infection. One of the important characteristics of P. aeruginosa is biofilm formation which leads to antibiotic resistance. Aims and Objectives: The aim of the study was to study the antibiotic resistance pattern of P. aeruginosa isolates and correlation with their biofilm-production. Materials and Methods: A total of 87 P. aeruginosa isolates from different clinical specimens were processed and confirmed by conventional microbiological methods as per standard methodology. Antibiotic sensitivity testing was done for all isolates. Biofilm producing isolates were identified by the microtiter plate method (MTPM). Results: Of 87 P. aeruginosa isolates, majority were from pus 33 (38%), followed by urine 26 (30%), sputum 19 (22%), body fluids 7 (8%), and blood 2 (2%). Biofilm producing isolates showed more resistance in comparison to non-biofilm producers. The observed difference between biofilm formation for multidrug resistant and susceptible isolates was found to be statistically significant. Conclusion: MTPM method was an effective test for detection of biofilm formation and was also able to verify biofilm production by P. aeruginosa. This indicated a higher propensity among the clinical isolates of P. aeruginosa to form biofilm and revealed a positive correlation between biofilm formation and antibiotic resistance. This indicates the need for testing of even susceptible isolates for virulence factors such as biofilm production.

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Molecular Analyses of Biofilm-Producing Clinical Acinetobacter baumannii Isolates from a South Indian Tertiary Care Hospital

Medical Principles and Practice ◽

10.1159/000508461 ◽

2020 ◽

Vol 29 (6) ◽

pp. 580-587

Author(s):

Balaram Khamari ◽

Manmath Lama ◽

Chanakya Pachi Pulusu ◽

Amarendra Pratap Biswal ◽

Sai Manoz Lingamallu ◽

...

Keyword(s):

Antibiotic Resistance ◽

Acinetobacter Baumannii ◽

Virulence Genes ◽

Random Amplified Polymorphic Dna ◽

Tertiary Care ◽

Care Hospital ◽

Drug Resistant ◽

Class 1 Integron ◽

Biofilm Production ◽

Class 1

Objectives: The aim of the study was to determine the presence of antimicrobial-resistance (AMR) genes, virulence genes, and mobile genetic elements (MGEs) in 14 biofilm-producing clinical isolates of Acinetobacter baumannii. Materials and Methods: PCR amplification was performed to analyse the prevalence of genes associated with antibiotic resistance (extended-spectrum β-lactamases [ESBLs] and metallo-β-lactamases [MBLs]), virulence factors, MGEs (class 1 integron, Tn1213, and A. baumannii antibiotic resistance [AbaR]), and comM among the study isolates. Random amplified polymorphic DNA (RAPD) PCR was then deployed to understand their phylogenetic relationship. All the isolates were investigated for biofilm production. Results: Two isolates were antibiotic-sensitive (AS), 3 were multi-drug-resistant (MDR), and the remaining 9 were extensively drug-resistant (XDR). The majority of the isolates were found to be positive for biofilm production and were sensitive against tetracycline and colistin only. Ab14 and Ab11 were found to be resistant to minocycline and colistin, respectively. blaTEM, blaOXA, blaNDM, blaVIM, blaSIM, and blaPER-1; class 1 integron; composite transposon Tn1213; AbaR island, and virulence factor genes were detected among the isolates. These pathogens were found to have originated from multiple clonal lineages. Conclusion: Biofilm-producing A. baumannii with multiple virulence and AMR genes pose serious clinical challenges. The presence of MGEs further compounds the situation as these isolates serve as potential reservoirs of AMR and virulence genes. Together with their capacity for natural competence, A. baumannii, if left unchecked, will lead to the spread of resistance determinants to previously sensitive bacteria and may aid in the emergence of untreatable pan-drug-resistant phenotypes.

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Defining Gene-Phenotype Relationships in Acinetobacter baumannii through One-Step Chromosomal Gene Inactivation

mBio ◽

10.1128/mbio.01313-14 ◽

2014 ◽

Vol 5 (4) ◽

Cited By ~ 67

Author(s):

Ashley T. Tucker ◽

Emily M. Nowicki ◽

Joseph M. Boll ◽

Gregory A. Knauf ◽

Nora C. Burdis ◽

...

Keyword(s):

Oxidative Stress ◽

Antibiotic Resistance ◽

Acinetobacter Baumannii ◽

Genome Editing ◽

Biofilm Formation ◽

Content Type ◽

Stress Protection ◽

Biocide Resistance ◽

Hospital Acquired ◽

Oxidative Stress Protection

ABSTRACTRates of infection with hospital-acquiredAcinetobacter baumanniihave exploded over the past decade due to our inability to limit persistence and effectively treat disease.A. baumanniiquickly acquires antibiotic resistance, and its genome encodes mechanisms to tolerate biocides and desiccation, which enhance its persistence in hospital settings. With depleted antibiotic options, new methods to treatA. baumanniiinfections are desperately needed. A comprehensive understanding detailingA. baumanniicellular factors that contribute to its resiliency at genetic and mechanistic levels is vital to the development of new treatment options. Tools to rapidly dissect theA. baumanniigenome will facilitate this goal by quickly advancing our understanding ofA. baumanniigene-phenotype relationships. We describe here a recombination-mediated genetic engineering (recombineering) system for targeted genome editing ofA. baumannii. We have demonstrated that this system can perform directed mutagenesis on wide-ranging genes and operons and is functional in various strains ofA. baumannii, indicating its broad application. We utilized this system to investigate key gene-phenotype relationships inA. baumanniibiology important to infection and persistence in hospitals, including oxidative stress protection, biocide resistance mechanisms, and biofilm formation. In addition, we have demonstrated that both the formation and movement of type IV pili play an important role inA. baumanniibiofilm.IMPORTANCEAcinetobacter baumanniiis the causative agent of hospital-acquired infections, including pneumonia and serious blood and wound infections.A. baumanniiis an emerging pathogen and has become a threat to public health because it quickly develops antibiotic resistance, making treatment difficult or impossible. While the threat ofA. baumanniiis well recognized, our understanding of even its most basic biology lags behind. Analysis ofA. baumanniicellular functions to identify potential targets for drug development has stalled due in part to laborious genetic techniques. Here we have pioneered a novel recombineering system that facilitates efficient genome editing inA. baumanniiby single PCR products. This technology allows for rapid genome editing to quickly ascertain gene-phenotype relationships. To demonstrate the power of recombineering in dissectingA. baumanniibiology, we use this system to establish key gene-phenotype relationships important to infection and persistence in hospitals, including oxidative stress protection, biocide resistance, and biofilm formation.

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Bacterial Profile of Urinary Tract Infections: Evaluation of Biofilm Formation and Antibiotic Resistance Pattern of Uropathogenic Escherichia coli

Journal of Pure and Applied Microbiology ◽

10.22207/jpam.14.4.33 ◽

2020 ◽

Vol 14 (4) ◽

pp. 2577-2584

Author(s):

Tariq Ahmad Shah ◽

P. Preethishree ◽

Ashwini ◽

Vidya Pai

Keyword(s):

Escherichia Coli ◽

Antibiotic Resistance ◽

Urinary Tract ◽

Biofilm Formation ◽

Urine Samples ◽

Diffusion Method ◽

E Coli ◽

Biofilm Production ◽

Tract Infections

Urinary tract infection (UTI) is one of the most common complaints in the outpatient clinic and a major health problem owing to the emergence of antibiotic resistance and biofilm formation. The objective of this study was to isolate and identify the causative bacterial agent of UTI and detect in vitro biofilm formation by Escherichia coli and investigate its correlation with antibiotic resistance. Urine samples from 519 patients with suspected UTIs were collected and processed by conventional microbiological procedures. Antimicrobial susceptibility testing for E. coli isolates was performed on Mueller Hinton agar (MHA) plates using the Kirby-Bauer disk diffusion method. Biofilm production was evaluated using the tissue culture plate method. Of 519 urine samples, 115 (22.1%) showed significant bacteriuria. The most common isolate was E. coli (n=57, 49.6%), followed by Klebsiella spp. (n=23, 20%). All E. coli isolates were evaluated for their ability to form biofilms in vitro. Of 57 isolates, 50 (87.7%) were biofilm producers and 7 (12.3%) were non-biofilm producers. Antibiogram of E. coli isolates revealed the highest resistance to ampicillin (96.5%) and nitrofurantoin (91.2%), followed by amoxyclav (82.5%), ceftazidime (73.7%), cefepime (71.9%), and tetracycline (71.9%). A significant association (p<0.05) was observed between biofilm formation and resistance to amoxyclav, ceftazidime, cefepime, imipenem, and nitrofurantoin. A significant correlation was noted between biofilm production and antibiotic resistance. Hence, screening of all isolates of uropathogenic E. coli for biofilm production and studying their antibiogram would allow appropriate choice of antibiotic therapy.

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Biofilm Formation in Acinetobacter Baumannii: Genotype-Phenotype Correlation

Molecules ◽

10.3390/molecules24101849 ◽

2019 ◽

Vol 24 (10) ◽

pp. 1849 ◽

Cited By ~ 13

Author(s):

Cheng-Hong Yang ◽

Pai-Wei Su ◽

Sin-Hua Moi ◽

Li-Yeh Chuang

Keyword(s):

Antibiotic Resistance ◽

Acinetobacter Baumannii ◽

Biofilm Formation ◽

Diffusion Method ◽

Multiple Drug ◽

Opportunistic Pathogens ◽

Antibiotic Resistant ◽

Multiple Drug Resistant ◽

Polymerase Chain ◽

And Control

Strains of Acinetobacter baumannii are commensal and opportunistic pathogens that have emerged as problematic hospital pathogens due to its biofilm formation ability and multiple antibiotic resistances. The biofilm-associated pathogens usually exhibit dramatically decreased susceptibility to antibiotics. This study was aimed to investigate the correlation of biofilm-forming ability, antibiotic resistance and biofilm-related genes of 154 A. baumannii isolates which were collected from a teaching hospital in Taiwan. Biofilm-forming ability of the isolates was evaluated by crystal violet staining and observed by scanning electron microscopy. Antibiotic susceptibility was determined by disc diffusion method and minimum inhibitory concentration; the biofilm-related genes were screened by polymerase chain reaction. Results showed that among the 154 tested isolates, 15.6% of the clinical isolates were weak biofilm producers, while 32.5% and 45.4% of them possessed moderate and strong biofilm formation ability, respectively. The experimental results revealed that the multiple drug resistant isolates usually provided a higher biofilm formation. The prevalence of biofilm related genes including bap, blaPER-1, csuE and ompA among the isolated strains was 79.2%, 38.3%, 91.6%, and 68.8%, respectively. The results indicated that the antibiotic resistance, the formation of biofilm and the related genes were significantly correlated. The results of this study can effectively help to understand the antibiotic resistant mechanism and provides the valuable information to the screening, identification, diagnosis, treatment and control of clinical antibiotic-resistant pathogens.

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The Frequency of Fluoroquinolone Resistant Enterotoxigenic Escherichia coli from Stools of Diarrheic Children in Ebonyi State, Nigeria

10.21203/rs.2.10616/v1 ◽

2019 ◽

Author(s):

Ebuka David ◽

MA Yameen ◽

IO Igwenyi ◽

IR Iroha ◽

HC Nzelibe ◽

...

Keyword(s):

Escherichia Coli ◽

Biofilm Formation ◽

Enterotoxigenic Escherichia Coli ◽

Fluoroquinolone Resistance ◽

Diffusion Method ◽

Drug Resistant ◽

Disk Diffusion Method ◽

E Coli ◽

Biofilm Production ◽

Rapid Emergence

Abstract Enterotoxigenic Escherichia coli (ETEC) is an important cause of acute childhood diarrhea. The evaluation of ETEC in children is important for therapeutic and economic purposes. Hence, this study aimed to evaluate the frequency of ETEC among diarrheic children, their multidrug and fluoroquinolone resistant pattern. A total of twenty diarrheagenic E. coli (DEC) isolates were gotten from hundred diarrheal samples using biochemical and molecular methods. Multiplex PCR was used to detect the presence of four different pathological types of DEC. Disk diffusion method was used to determine the antibiotic susceptibility of the organisms. Biofilm formation was detected by thiazoylblue tetrazolium bromide dye in a 96-well plate. Results showed that ETEC represented 30% of the DEC, of which 80% were multidrug and fluoroquinolone resistant. The biofilm production abilities of all the ETEC were found to exist within weak, moderate and strong biofilm producers. We observed a high ETEC frequency and rapid emergence of multidrug/fluoroquinolone resistance, suggesting that it is one of the most important causes of frequent drug resistant diarrhea in children in this region.

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