Tube method and Congo red agar versus tissue culture plate method for detection of biofilm production by uropathogens isolated from midstream urine: Which one could be better?

Background: Microorganisms that infect humans differ in pathogenesis, virulence factors and antimicrobial resistance profiles. In natural settings, bacterial cells are most often found in close association with surfaces and interfaces, in the form of multicellular aggregates commonly called biofilms. Given their ubiquity and importance in the microbial world, it is hardly surprising that biofilms have attracted the attention of the scientific community. Biofilm formation on medical implant devices such as catheters is also a major problem that is closely tied to the adhesion and resistance-related abilities of the biofilm.Methodology: The ability of 216 bacterial isolates from mid-stream urine (100), catheter-stream urine (52) and catheter tips (64) to form biofilms was investigated using the tissue culture plate method, the tube and Congo red agar methods as well as their antibiotic resistance profiles using the agar disc diffusion method.Results: These revealed that Klebsiella spp. was the predominant bacterial genera accounting for 45.8% of the total isolates. A total of 50 isolates were biofilm-formers with 22% identified by the tissue culture plate method and 78% identified by the Congo red agar method. Klebsiella spp. had the highest ability to form biofilm while antibiotic resistance profiles showed all the biofilmformers to be multiply antibiotic resistant with least resistance to ofloxacin.Conclusion: It can therefore be concluded that some bacterial isolates associated with urinary tract infections have a propensity to form biofilm, thereby becoming multiply antibiotic resistant, and ofloxacin remains the antibiotic of choice in the treatment of such infections.

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Phenotypic and genotypic characterization of biofilm producing clinical coagulase negative staphylococci from Nepal and their antibiotic susceptibility pattern

Annals of Clinical Microbiology and Antimicrobials ◽

10.1186/s12941-021-00447-6 ◽

2021 ◽

Vol 20 (1) ◽

Author(s):

Sarita Manandhar ◽

Anjana Singh ◽

Ajit Varma ◽

Shanti Pandey ◽

Neeraj Shrivastava

Keyword(s):

Tissue Culture ◽

Congo Red ◽

Antibiotic Susceptibility ◽

Clinical Samples ◽

Susceptibility Pattern ◽

Coagulase Negative Staphylococci ◽

Antibiotic Susceptibility Pattern ◽

Biofilm Production ◽

Culture Plate ◽

Tissue Culture Plate

Abstract Background Coagulase-negative staphylococci (CNS) survive as commensals of skin, anterior nares and external canals of human and were regarded as non-infectious pathogens. However, they are emerging as a major cause of nosocomial infectious due to their ability to form biofilms and high resistance to several classes of antibiotics. This study examines the biofilm forming abilities of 214 clinical CNS isolates using phenotypic and genotypic methods, and determines their antibiotic susceptibility patterns. Methods A total of 214 clinical isolates collected from different clinical samples were identified as CNS and their antibiotic susceptibility determined by CLSI guidelines. The biofilm forming ability of all isolates was determined by three phenotypic methods; Congo red agar (CRA) method, tube adherence method (TM) and tissue culture plate (TCP) method and by genotypic method for the detection of icaAD genes. Results Among all the isolates, S. epidermidis (57.5%) was found the most frequently, followed by S. saprophyticus (18.7%), S. haemolyticus (11.2%), S. hominis (7%), and S. capitis (5.6%). Antibiotic susceptibility pattern demonstrated 91.6% isolates were resistant to penicillin and 66.8% to cefoxitin while 91.1% isolates were susceptible to chloramphenicol. Constitutive and inducible clindamycin resistant phenotype as measured by D-test was seen among 28% and 14.5% of isolates respectively. Tissue culture plate method detected biofilm production in 42.1% isolate followed by 31.8% through tube method while 20.1% isolates were found to produce slime in Congo red agar method. The genotypic assay revealed presence of icaA and icaD genes in 19.2% isolates. Conclusion The study shows a high prevalence of biofilm formation and inducible clindamycin resistance in CNS isolates, indicating the importance of in-vitro biofilm production test and D-test in routine laboratory diagnostics. Implementation of efficient diagnostic techniques for detection of biofilm production in clinical samples can help manage staphylococcal infections and minimize risks of treatment failures in hospitals.

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In-Vitro Evaluation of Biofilm and Hemolysis activity of Candida albicans Isolated from Oral Cavity

International Journal of Applied Sciences and Biotechnology ◽

10.3126/ijasbt.v8i4.32971 ◽

2020 ◽

Vol 8 (4) ◽

pp. 394-399

Author(s):

Bijay Kumar Shrestha ◽

Jenish Shakya

Keyword(s):

Tissue Culture ◽

Candida Albicans ◽

Human Blood ◽

Hemolytic Activity ◽

Congo Red ◽

Plate Method ◽

Oral Rinse ◽

Culture Plate ◽

Tissue Culture Plate

Candida albicans is a member of the healthy human microflora, colonizing several niches in the body and can cause opportunistic infection under host debilitated and immunocompromised condition. The present study aimed to investigate the in-vitro hemolytic activity of C. albicans isolated from oral cavity and screen biofilm through three different methods. During the study, 200 oral rinse samples from general human population were analyzed in microbiology laboratory of Central Campus of Technology, Tribhuvan University, Hattisar, Dharan. Nepal. Candida albicans were isolated and identified by conventional microbiological procedures. The hemolytic activity was evaluated through two different Sabouraud dextrose broth media (SDB) containing 7% defibrinated human blood, one supplemented with 3% glucose (SDBwG) and the other without glucose (SDBwoG). The biofilm formation was screened through congo red agar, tube method and tissue culture plate method. In this present study, 42 (21%) isolates of Candida albicans were isolated from 200 oral rinse samples. Isolated Candida albicans exhibited mean hemolysis activity of 28.66% on human blood SDB without glucose and 43.55% on human blood SDB with 3% glucose. Tissue culture plate method was considered sensitive, specific and accurate method for quantitative screening of biofilm in comparison to tube method and congo red agar method. This research concluded that Candida albicans exhibited greater hemolytic activity in human blood with glucose (SDBwG) than in human blood without glucose (SDBwoG). This finding explains that an increased blood glucose concentration may contribute to increased hemolysis activity of Candida albicans that could play pathogenic role for inducing infection like oral candidiasis in debilitated host like diabetic patients. Tissue culture plate method can accurately screen biofilms than tube and congo red agar method. Int. J. Appl. Sci. Biotechnol. Vol 8(4): 394-399

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Inducible Clindamycin Resistance and Biofilm Production among Staphylococci Isolated from Tertiary Care Hospitals in Nepal

Infectious Disease Reports ◽

10.3390/idr13040095 ◽

2021 ◽

Vol 13 (4) ◽

pp. 1043-1052

Author(s):

Sarita Manandhar ◽

Raju Shrestha ◽

Ratna Shova Tuladhar ◽

Sunil Lekhak

Keyword(s):

Tissue Culture ◽

Biofilm Formation ◽

Tertiary Care ◽

Plate Method ◽

Methicillin Resistant ◽

Inducible Clindamycin Resistance ◽

Biofilm Production ◽

Resistant Strains ◽

Tissue Culture Plate ◽

Tertiary Care Hospitals

Resistance to antibiotics, biofilm formation and the presence of virulence factors play important roles in increased mortality associated with infection by staphylococci. The macrolide lincosamide streptogramin B (MLSB) family of antibiotics is commonly used to treat infections by methicillin-resistant isolates. Clinical failure of clindamycin therapy has been reported due to multiple mechanisms that confer resistance to MLSB. This study aims to find the incidence of different phenotypes of MLSB resistance and biofilm production among staphylococci. A total of 375 staphylococci were isolated from different clinical samples, received from two tertiary care hospitals in Nepal. Methicillin resistance was detected by cefoxitin disc diffusion method and inducible clindamycin resistance by D test, according to CLSI guidelines. Biofilm formation was detected by the tissue culture plate method and PCR was used to detect ica genes. Of the total staphylococci isolates, 161 (42.9%) were Staphylococcus aureus, with 131 (81.4%) methicillin-resistant strains, and 214 (57.1%) isolates were coagulase-negative staphylococci, with 143 (66.8%) methicillin-resistant strains. The overall prevalence of constitutive MLSB (cMLSB) and inducible MLSB (iMLSB) phenotypes was 77 (20.5%) and 87 (23.2%), respectively. Both iMLSB and cMLSB phenotypes predominated in methicillin-resistant isolates. The tissue culture plate method detected biofilm formation in 174 (46.4%) isolates and ica genes in 86 (22.9%) isolates. Among biofilm producing isolates, cMLSB and iMLSB phenotypes were 35 (20.1%) and 27 (15.5%), respectively. The cMLSB and iMLSB were 11 (12.8%) and 19 (22.1%), respectively, in isolates possessing ica genes. Clindamycin resistance in the form of cMLSB and iMLSB, especially among MRSA, emphasizes the need for routine D tests to be performed in the lab.

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Effect of Glucose Induction on Biofilm Density in Clinical Isolate Acinetobacter baumannii Patients in Intensive Care Unit of Dr. Soetomo Hospital, Surabaya

Folia Medica Indonesiana ◽

10.20473/fmi.v56i2.21230 ◽

2020 ◽

Vol 56 (2) ◽

pp. 118

Author(s):

Wira W Lindarto ◽

Eddy Bagus Wasito ◽

Kartuti Debora

Keyword(s):

Tissue Culture ◽

Acinetobacter Baumannii ◽

Clinical Isolate ◽

Growth Media ◽

Plate Method ◽

Culture Plate ◽

Tissue Culture Plate ◽

Glucose Induction ◽

Sugar Levels ◽

Effect Of Glucose

This study aimed to analyze the effect of glucose induction on the clinical isolate biofilm density of Acinetobacter baumannii. Thirteen clinical isolates of A. baumannii non biofilm forming were collected from non-DM patients who were treated at the ICU of Dr. Soetomo Hospital, Surabaya, was treated with the addition of 0.08% glucose, 0.15% glucose, 0.2% glucose, and 0.4% glucose in TSB growth media, followed by biofilm density examination with Tissue Culture Plate Method (TCPM) using 96 wells flatbottomed polyesterene tissue culture plate and read by autoreader ELISA with a wavelength of 630 nm (OD630). Biofilm density obtained was analyzed using ANOVA statistical analysis. The results of OD630 showed that the biofilm density increased significantly at the addition of 0.2% and 0.4% glucose. There was a significant increase in biofilm density at the addition of 0.2% and 0.4% glucose so that the management of blood sugar levels in ICU patients was needed before and when medical devices were installed.

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Evaluasi Metode Skrining Biofilm Enterococcus faecalis Isolat Klinik Kateter Urin

Jurnal Kedokteran Meditek ◽

10.36452/jkdoktmeditek.v24i68.1701 ◽

2019 ◽

Author(s):

Wani Devita Gunardi ◽

Mohamad Yanuar Prasetyo Nugroho ◽

Elisabeth D. Harahap

Keyword(s):

Tissue Culture ◽

Enterococcus Faecalis ◽

Congo Red ◽

National Healthcare ◽

National Healthcare Safety Network ◽

Culture Plate ◽

Tissue Culture Plate

Laporan National Healthcare Safety Network dari tahun 2006 - 2008, menunjukan penyebab paling umum kedua infeksi saluran kemih (ISK) terkait kateter adalah genus Enterococcus setelah Eschericia coli. Pada infeksi saluran kemih terkait kateter urin, faktor yang berperan penting dalam patogenesis infeksi ini, yaitu: pembentukan biofilm pada kateter urin. Peranan Enterococcus faecalis sebagai penyebab infeksi saluran kemih berkaitan dengan kemampuannya dalam membentuk biofilm. Ada beberapa variasi metode untuk mendeteksi pembentukan biofilm seperti Tissue Culture Plate (TCP), Tube method (TM), dan Congo Red Agar (CRA). Tujuan penelitian ini untuk mendapatkan metode deteksi pembentukan biofilm dari E. faecalis yang tepat, cepat, dan mudah dilakukan. Total tigabelas isolat bakteri E. faecalis yang didapat dari hasil isolasi kultur kateter urin dilakukan uji deteksi pembentukan biofilm dengan metode TCP sebagai baku emas dan CRA sebagai pemeriksaan pembanding. Hasilnya, didapatkan 61,5% dan 69,2% bakteri E. faecalis mampu menghasilkan biofilm menggunakan metode TCP dan CRA. Hasil uji diagnostik metode CRA dibandingkan dengan metode TCP untuk deteksi pembentukan biofilm, didapatkan sensitivitas dan spesifisitas dari CRA sebesar 75% dan 40%. CRA merupakan metode yang cepat dan mudah untuk dilakukan, namun memiliki spesifitas yang kurang baik. Hal ini dapat disebabkan pembacaan hasil yang bersifat subjektif dan menimbulkan kesalahan paralaks. Kata kunci : Enterococcus faecalis, Kateter Urin, Biofilm.

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Biofilm-Formation in Clonally Unrelated Multidrug-Resistant Acinetobacter baumannii Isolates

Pathogens ◽

10.3390/pathogens9080630 ◽

2020 ◽

Vol 9 (8) ◽

pp. 630 ◽

Cited By ~ 1

Author(s):

Aisha M. Alamri ◽

Afnan A. Alsultan ◽

Mohammad A. Ansari ◽

Amani M. Alnimr

Keyword(s):

Tissue Culture ◽

Antibiotic Resistance ◽

Acinetobacter Baumannii ◽

Congo Red ◽

Biofilm Formation ◽

Multidrug Resistant ◽

Control Measures ◽

Infection Control Measures ◽

Culture Plate ◽

Tissue Culture Plate

This study analyzed the genotype, antibiotic resistance, and biofilm formation of Acinetobacter baumannii strains and assessed the correlation between biofilm formation, antibiotic resistance, and biofilm-related risk factors. A total of 207 non-replicate multi-drug-resistant A. baumannii strains were prospectively isolated. Phenotypic identification and antimicrobial susceptibility testing were carried out. Isolate biofilm formation ability was evaluated using the tissue culture plate (TCP), Congo red agar, and tube methods. Clonal relatedness between the strains was assessed by enterobacterial repetitive intergenic consensus-PCR genotyping. Of the 207 isolates, 52.5% originated from an intensive care unit setting, and pan resistance was observed against ceftazidime and cefepime, with elevated resistance (99–94%) to piperacillin/tazobactam, imipenem, levofloxacin, and ciprofloxacin. alongside high susceptibility to tigecycline (97.8%). The Tissue culture plate, Tube method, and Congo red agar methods revealed that 53.6%, 20.8%, and 2.7% of the strains were strong biofilm producers, respectively, while a significant correlation was observed between biofilm formation and device-originating respiratory isolates (p = 0.0009) and between biofilm formation in colonized vs. true infection isolates (p = 0.0001). No correlation was detected between antibiotic resistance and biofilm formation capacity, and the majority of isolates were clonally unrelated. These findings highlight the urgent need for implementing strict infection control measures in clinical settings.

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Detection of Biofilm Producing Staphylococci among Different Clinical Isolates and Its Relation to Methicillin Susceptibility

Open Access Macedonian Journal of Medical Sciences ◽

10.3889/oamjms.2018.246 ◽

2018 ◽

Vol 6 (8) ◽

pp. 1335-1341 ◽

Cited By ~ 4

Author(s):

Rania M. Abdel Halim ◽

Nevine N. Kassem ◽

Basma S. Mahmoud

Keyword(s):

Tissue Culture ◽

Congo Red ◽

Biofilm Formation ◽

Methicillin Resistance ◽

Screening Method ◽

Culture Plate ◽

Tissue Culture Plate ◽

Blood Specimens ◽

Phenotypic Methods

AIMS: To evaluate three in vitro phenotypic methods; tissue culture plate, tube method, and Congo red agar for detection of biofilm formation in staphylococci and assess the relation of biofilm formation with methicillin resistance and anti-microbial resistance. METHODS: The study included 150 staphylococcal isolates. Biofilm detection in staphylococci was performed using tissue culture plate, tube method, and Congo red agar. RESULTS: Tissue culture plate, tube method, and Congo red agar detected 74%, 42.7%, and 1.3% biofilm producing staphylococci respectively. S. aureus isolates were more common biofilm producers (53.2%) than CONS (46.8%). Biofilm production in CONS species was highest in S. hemolyticus (57.7%). Tube method was 51.4% sensitive, 82.1% specific. As for Congo red agar, sensitivity was very low (0.9%), but specificity was 97.4%. Biofilm producers were mostly; isolated from blood specimens (82.6%) and detected in methicillin-resistant strains 96/111 (86.5%). They were resistant to most antibiotics except vancomycin and linezolid. CONCLUSIONS: Tissue culture plate is a more quantitative and reliable method for detection of biofilm producing staphylococci compared to tube method and Congo red agar. Hence, it can still be used as a screening method for biofilm detection. Vancomycin and Linezolid are the most sensitive antibiotics among biofilm producing staphylococci.

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The effects of U.V light on mrkA, mrkD genes in local isolates of Klebsiella pneumoniae

Al-Mustansiriyah Journal of Science ◽

10.23851/mjs.v27i4.32 ◽

2017 ◽

Vol 27 (4) ◽

Author(s):

Ali Hussein Alwan ◽

Sura Mouaid Abass

Keyword(s):

Tissue Culture ◽

Klebsiella Pneumoniae ◽

Genetic Study ◽

Biofilm Production ◽

Culture Plate ◽

Tissue Culture Plate

Klebsiella pneumoniae is dangerous pathogens that can cause severe diseases. This study included isolation of 50 isolates of K.pneumoniaefrom different clinical sources from different hospitals in Baghdad city, the number and percentage of isolates according to the sources (urine, blood, sputum, burns, ear swabs, pus, wounds and stool ) were 22(44%), 11(22%), 4(8%), 4(8%), 3(6%), 3(6%), 2(4%) and 1(2%) respectively. The ability to form biofilm was carried out using Tissue culture plate methods (-TCP-). The results showed that 80% of the isolates were producer biofilm; the genetic study was used to detect the presence of mrkA, mrkD genes that are believed to be responsible of biofilm production. The ratio was mrkA 87.5% and mrkD 50% before exposure to U.V.light to reduce to 43.7% mrkA and 18.7% mrkD in isolates after exposure to U.V.light source.

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Comparative study of antibiofilm activity of lime juice and lithium dioxide nanoparticles against Salmonella isolated from local cheese

Journal of Biotechnology Research Center ◽

10.24126/jobrc.2018.12.2.542 ◽

2018 ◽

Vol 12 (2) ◽

pp. 82-94

Author(s):

Zainab Zamel Khalaf

Keyword(s):

Tissue Culture ◽

Synergistic Effect ◽

Comparative Study ◽

Congo Red ◽

Microbial Contamination ◽

Antibiofilm Activity ◽

Plate Method ◽

Lime Juice ◽

White Cheese ◽

Tissue Culture Plate

Thirty specimens of fresh white cheeses, presented for sale in different markets at different cities of Iraq were analyzed microbiologically in this study. Isolates of Salmonella, included in the specimens collected from November 2017 to January 2018, were investigated. Capacity for biofilm producing was demonstrated by two method, Tissue culture plate method (TCP) and Congo red agar (CRA).After that the antibiofilm activity of lime extract and LiO2NPs was studied as each one of them alone and then the synergistic effect was done by TCP method. The results showed that all Salmonella isolates produce biofilm but in different degrees. The results also displayed that Lime extract and LiO2NPs had antibiofilm effect against Salmonella when used alone and when the combination done between each one of these materials. In conclusion, it was observed that the specimens of fresh white cheese included in this study contained microbial contamination at a health-threatening level but we can eliminate this contamination by using lime extract and LiO2NPs.

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