Comparison of biofilm formation and efflux pumps in ESBL and carbapenemase producing Klebsiella pneumoniae

Introduction: Klebsiella pneumoniae is an opportunistic pathogen that causes a range of diseases. The appearance of extended-spectrum β-lactamase -and carbapenemase-producing strains, in addition to the biofilm-forming phenotype, is a major problem in the clinical environment. Methodology: A total of 33 clinical K. pneumoniae isolates were used in this study. Antimicrobial susceptibilities were assessed by a disc diffusion assay. Biofilm formation was determined by a microtiter plate assay, staining with 1% crystal violet and measuring absorbance after destaining. Moreover, expression of acrA, kdeA, ketM, kpnEF, and kexD efflux associated genes was measured by qRT-PCR. Results: Isolates displayed high resistance to β-lactams such as cefazolin, cefuroxime, ceftriaxone, cefepime, piperacillin-tazobactam, imipenem, and meropenem and decreased resistance to gentamicin, amikacin, ciprofloxacin, and levofloxacin. ESBL-producing isolates formed more biofilm than carbapenemase-producing isolates. The mRNA expression levels in KPC isolates for acrA (2-fold), kdeA (2.7-fold), ketM (2.2-fold), and kpnEF (3.4-fold) were significantly increased compared to ESBL-producing isolates. There was no significant difference in kexD expression level. Conclusions: Under the conditions used here ESBL-producing isolates formed more biofilm than KPC postive isolates; this was associated with virulence determinants which were also transferred by plasmids together with ESBLs enzymes. Moreover, the upregulation of acrA, kdeA, ketM, and kpnEF efflux pumps was seen in carbapenemase-producing isolates demonstrating that high expression of efflux pumps alone could not confer resistance but may act as a physiological determinant such as bacterial pathogenicity and virulence, and cell-to-cell communication for bacteria.

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Cellobiose-Specific Phosphotransferase System of Klebsiella pneumoniae and Its Importance in Biofilm Formation and Virulence

Infection and Immunity ◽

10.1128/iai.06247-11 ◽

2012 ◽

Vol 80 (7) ◽

pp. 2464-2472 ◽

Cited By ~ 29

Author(s):

Meng-Chuan Wu ◽

Ying-Chun Chen ◽

Tzu-Lung Lin ◽

Pei-Fang Hsieh ◽

Jin-Town Wang

Keyword(s):

Klebsiella Pneumoniae ◽

Biofilm Formation ◽

Type Strain ◽

Minimal Medium ◽

Wild Type Strain ◽

Microtiter Plate ◽

Wild Type ◽

Phosphotransferase System ◽

Microtiter Plate Assay ◽

Content Type

ABSTRACTKlebsiella pneumoniaeis a Gram-negative bacillus belonging to the familyEnterobacteriaceae. In the past 20 years,K. pneumoniaehas become the predominant pathogen causing community-acquired pyogenic liver abscess (PLA). The formation of biofilm facilitates bacterial colonization and has been implicated in reduced susceptibility to the host immune response. To investigate genes related to biofilm formation in a PLA-associatedK. pneumoniaestrain, a transposon mutant library was screened by microtiter plate assay to identify isolates impaired for biofilm formation. One of the mutants was disrupted incelB, encoding the putative cellobiose-specific subunit IIC of enzyme II (EIIC) of a carbohydrate phosphotransferase system (PTS). This transmembrane protein is responsible for recognizing and binding specific sugars and transporting them across the cell membrane into the cytoplasm. Deletion and chromosomal complementation ofcelBconfirmed, by microtiter plate and slide culture assays, thatcelBwas indeed responsible for biofilm formation. Cellobiose-specific PTS activities of deletion mutants grown in LB broth and 0.005% cellobiose minimal medium were markedly lower than that of the wild-type strain grown under the same conditions, thereby confirming the involvement ofcelBin cellobiose transport. In 0.005% cellobiose minimal medium, thecelBmutant showed a delay in growth compared to the wild-type strain. In a mouse model of intragastric infection, deletion of thecelBgene increased the survival rate from 12.5% to 87.5%, which suggests that thecelBdeletion mutant also exhibited reduced virulence. Thus, thecelBlocus ofK. pneumoniae may contribute to biofilm formation and virulence through the metabolism of cellobiose.

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Genetic Diversity, Antimicrobial Resistance Pattern, and Biofilm Formation in Klebsiella pneumoniae Isolated from Patients with Coronavirus Disease 2019 (COVID-19) and Ventilator-Associated Pneumonia

BioMed Research International ◽

10.1155/2021/2347872 ◽

2021 ◽

Vol 2021 ◽

pp. 1-11

Author(s):

Asma Ghanizadeh ◽

Maede Najafizade ◽

Somaye Rashki ◽

Zeynab Marzhoseyni ◽

Mitra Motallebi

Keyword(s):

Genetic Diversity ◽

Antimicrobial Resistance ◽

Klebsiella Pneumoniae ◽

Biofilm Formation ◽

Assay Method ◽

Diffusion Method ◽

Resistance Pattern ◽

Ventilator Associated Pneumonia ◽

Microtiter Plate Assay ◽

Significant Difference

Introduction. Patients with acute respiratory distress syndrome caused by coronavirus disease 2019 (COVID-19) are at risk for superadded infections, especially infections caused by multidrug resistant (MDR) pathogens. Before the COVID-19 pandemic, the prevalence of MDR infections, including infections caused by MDR Klebsiella pneumoniae (K. pneumoniae), was very high in Iran. This study is aimed at assessing the genetic diversity, antimicrobial resistance pattern, and biofilm formation in K. pneumoniae isolates obtained from patients with COVID-19 and ventilator-associated pneumonia (VAP) hospitalized in an intensive care unit (ICU) in Iran. Methods. In this cross-sectional study, seventy K. pneumoniae isolates were obtained from seventy patients with COVID-19 hospitalized in the ICU of Shahid Beheshti hospital, Kashan, Iran, from May to September, 2020. K. pneumoniae was detected through the ureD gene. Antimicrobial susceptibility testing was done using the Kirby-Bauer disc diffusion method, and biofilm was detected using the microtiter plate assay method. Genetic diversity was also analyzed through polymerase chain reaction based on enterobacterial repetitive intergenic consensus (ERIC-PCR). The BioNumerics software (v. 8.0, Applied Maths, Belgium) was used for analyzing the data and drawing dendrogram and minimum spanning tree. Findings. K. pneumoniae isolates had varying levels of resistance to antibiotics meropenem (80.4%), cefepime-aztreonam-piperacillin/tazobactam (70%), tobramycin (61.4%), ciprofloxacin (57.7%), gentamicin (55.7%), and imipenem (50%). Around 77.14% of isolates were MDR, and 42.8% of them formed biofilm. Genetic diversity analysis revealed 28 genotypes (E1–E28) and 74.28% of isolates were grouped into ten clusters (i.e., clusters A–J). Clusters were further categorized into three major clusters, i.e., clusters E, H, and J. Antimicrobial resistance to meropenem, tobramycin, gentamicin, and ciprofloxacin in cluster J was significantly higher than cluster H, denoting significant relationship between ERIC clusters and antimicrobial resistance. However, there was no significant difference among major clusters E, H, and J respecting biofilm formation. Conclusion. K. pneumoniae isolates obtained from patients with COVID-19 have high antimicrobial resistance, and 44.2% of them have genetic similarity and can be clustered in three major clusters. There is a significant difference among clusters respecting antimicrobial resistance.

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Investigation on Antibiotic-Resistance, Biofilm Formation and Virulence Factors in Multi Drug Resistant and Non Multi Drug Resistant Staphylococcus pseudintermedius

Microorganisms ◽

10.3390/microorganisms7120702 ◽

2019 ◽

Vol 7 (12) ◽

pp. 702 ◽

Cited By ~ 1

Author(s):

Gabriele Meroni ◽

Joel F. Soares Filipe ◽

Lorenzo Drago ◽

Piera A. Martino

Keyword(s):

Antibiotic Resistance ◽

Virulence Factors ◽

Biofilm Formation ◽

Microtiter Plate ◽

Drug Resistant ◽

Virulence Determinants ◽

Commensal Bacterium ◽

Staphylococcus Pseudintermedius ◽

Microtiter Plate Assay ◽

Human Agent

Staphylococcus pseudintermedius is a commensal bacterium frequently isolated from canine skin and recognized as a zoonotic agent especially for dog-owners. This study focused on (a) the antibiotic-resistance phenotypes; (b) the ability to produce biofilm (slime); and (c) the dissemination of virulence factors in S. pseudintermedius strains. Seventy-three S. pseudintermedius strains were screened for antibiotic-resistance against 22 different molecules by means of Kirby-Bauer assay. The ability to produce biofilm was investigated using the microtiter plate assay (MtP) and the amplification of icaA and icaD genes. Virulence factors such as cytotoxins (lukI), enterotoxins (seC), and exfoliative toxins (siet, expA, and expB) were evaluated. The antibiotic-resistance profiles revealed 42/73 (57%) multi-drug resistant (MDR) strains and 31/73 (43%) not-MDR. All the MDR strains and 8/31 (27%) of not-MDR resulted in biofilm producers. Leukotoxin LukI was found in 70/73 (96%) of the isolates. Moreover, the enterotoxin gene seC was detected in 47/73 (64%) of the strains. All the isolates carried the siet gene, whereas expA and expB were found in 3/73 (4%) and 5/73 (7%), respectively. In conclusion, S. pseudintermedius should be considered a potential zoonotic and human agent able to carry different virulence determinants and capable of producing biofilm which facilitates horizontal gene transfer.

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atpD gene sequencing, multidrug resistance traits, virulence-determinants, and antimicrobial resistance genes of emerging XDR and MDR-Proteus mirabilis

Scientific Reports ◽

10.1038/s41598-021-88861-w ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Abdelazeem M. Algammal ◽

Hany R. Hashem ◽

Khyreyah J. Alfifi ◽

Helal F. Hetta ◽

Norhan S. Sheraba ◽

...

Keyword(s):

Antimicrobial Resistance ◽

Resistance Genes ◽

Proteus Mirabilis ◽

Biofilm Formation ◽

Gene Sequencing ◽

Opportunistic Pathogen ◽

Severe Illness ◽

Virulence Determinants ◽

Antimicrobial Resistance Genes ◽

Port Said

AbstractProteus mirabilis is a common opportunistic pathogen causing severe illness in humans and animals. To determine the prevalence, antibiogram, biofilm-formation, screening of virulence, and antimicrobial resistance genes in P. mirabilis isolates from ducks; 240 samples were obtained from apparently healthy and diseased ducks from private farms in Port-Said Province, Egypt. The collected samples were examined bacteriologically, and then the recovered isolates were tested for atpD gene sequencing, antimicrobial susceptibility, biofilm-formation, PCR detection of virulence, and antimicrobial resistance genes. The prevalence of P. mirabilis in the examined samples was 14.6% (35/240). The identification of the recovered isolates was confirmed by the atpD gene sequencing, where the tested isolates shared a common ancestor. Besides, 94.3% of P. mirabilis isolates were biofilm producers. The recovered isolates were resistant to penicillins, sulfonamides, β-Lactam-β-lactamase-inhibitor-combinations, tetracyclines, cephalosporins, macrolides, and quinolones. Using PCR, the retrieved strains harbored atpD, ureC, rsbA, and zapA virulence genes with a prevalence of 100%, 100%, 94.3%, and 91.4%, respectively. Moreover, 31.4% (11/35) of the recovered strains were XDR to 8 antimicrobial classes that harbored blaTEM, blaOXA-1, blaCTX-M, tetA, and sul1 genes. Besides, 22.8% (8/35) of the tested strains were MDR to 3 antimicrobial classes and possessed blaTEM, tetA, and sul1genes. Furthermore, 17.1% (6/35) of the tested strains were MDR to 7 antimicrobial classes and harbored blaTEM, blaOXA-1, blaCTX-M, tetA, and sul1 genes. Alarmingly, three strains were carbapenem-resistant that exhibited PDR to all the tested 10 antimicrobial classes and shared blaTEM, blaOXA-1, blaCTX-M, tetA, and sul1 genes. Of them, two strains harbored the blaNDM-1 gene, and one strain carried the blaKPC gene. In brief, to the best of our knowledge, this is the first study demonstrating the emergence of XDR and MDR-P.mirabilis in ducks. Norfloxacin exhibited promising antibacterial activity against the recovered XDR and MDR-P. mirabilis. The emergence of PDR, XDR, and MDR-strains constitutes a threat alarm that indicates the complicated treatment of the infections caused by these superbugs.

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Effect of S-PRG Eluate on Biofilm Formation and Enzyme Activity of Oral Bacteria

International Journal of Dentistry ◽

10.1155/2012/814913 ◽

2012 ◽

Vol 2012 ◽

pp. 1-6 ◽

Cited By ~ 18

Author(s):

Masahiro Yoneda ◽

Nao Suzuki ◽

Yosuke Masuo ◽

Akie Fujimoto ◽

Kosaku Iha ◽

...

Keyword(s):

Biofilm Formation ◽

Composite Resin ◽

Fusobacterium Nucleatum ◽

Microtiter Plate ◽

Oral Bacteria ◽

Gelatin Film ◽

Glass Ionomer ◽

Microtiter Plate Assay ◽

Substrate Hydrolysis ◽

Adherence Ability

Recently, the antibacterial activity of a composite resin containing prereacted glass ionomer (S-PRG) filler was revealed. We examined the effect of an S-PRG eluate on various biologic activities ofStreptococcus mutansandPorphyromonas gingivalis. Adherence ability ofS. mutanswas evaluated by microtiter plate assay; protease and gelatinase activities ofP. gingivaliswere examined by synthetic substrate hydrolysis and gelatin film spot assay, respectively. Coaggregation ofP. gingivaliswithFusobacterium nucleatumwas also examined. S-PRG eluate was found to suppress streptococcal adherence. S-PRG eluate inhibited the protease and gelatinase activities ofP. gingivalisand the coaggregation betweenP. gingivalisandF. nucleatum. These results indicate that S-PRG eluate suppresses streptococcal adherence and inhibits the protease and coaggregation activities ofP. gingivalis. These findings may prompt research into novel strategies for preventing caries and periodontitis.

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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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Effect of Silver Nanoparticles on Biofilm Formation and EPS Production of Multidrug-Resistant Klebsiella pneumoniae

BioMed Research International ◽

10.1155/2020/6398165 ◽

2020 ◽

Vol 2020 ◽

pp. 1-9 ◽

Cited By ~ 1

Author(s):

Muhammad Hussnain Siddique ◽

Bilal Aslam ◽

Muhammad Imran ◽

Asma Ashraf ◽

Habibullah Nadeem ◽

...

Keyword(s):

Silver Nanoparticles ◽

Biofilm Formation ◽

Extracellular Polymeric Substances ◽

Microtiter Plate ◽

Cellular Protein ◽

Antibiofilm Activity ◽

Biofilm Inhibition ◽

Microtiter Plate Assay ◽

Protein Leakage ◽

Hela Cell Lines

Antibiotic resistance against present antibiotics is rising at an alarming rate with need for discovery of advanced methods to treat infections caused by resistant pathogens. Silver nanoparticles are known to exhibit satisfactory antibacterial and antibiofilm activity against different pathogens. In the present study, the AgNPs were synthesized chemically and characterized by UV-Visible spectroscopy, scanning electron microscopy, and X-ray diffraction. Antibacterial activity against MDR K. pneumoniae strains was evaluated by agar diffusion and broth microdilution assay. Cellular protein leakage was determined by the Bradford assay. The effect of AgNPs on production on extracellular polymeric substances was evaluated. Biofilm formation was assessed by tube method qualitatively and quantitatively by the microtiter plate assay. The cytotoxic potential of AgNPs on HeLa cell lines was also determined. AgNPs exhibited an MIC of 62.5 and 125 μg/ml, while their MBC is 250 and 500 μg/ml. The production of extracellular polymeric substance decreased after AgNP treatment while cellular protein leakage increased due to higher rates of cellular membrane disruption by AgNPs. The percentage biofilm inhibition was evaluated to be 64% for K. pneumoniae strain MF953600 and 86% for MF953599 at AgNP concentration of 100 μg/ml. AgNPs were evaluated to be minimally cytotoxic and safe at concentrations of 15-120 μg/ml. The data evaluated by this study provided evidence of AgNPs being safe antibacterial and antibiofilm compounds against MDR K. pneumoniae.

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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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Peptide Mix from Olivancillaria hiatula Interferes with Cell-to-Cell Communication in Pseudomonas aeruginosa

BioMed Research International ◽

10.1155/2019/5313918 ◽

2019 ◽

Vol 2019 ◽

pp. 1-12

Author(s):

Edward Ntim Gasu ◽

Hubert Senanu Ahor ◽

Lawrence Sheringham Borquaye

Keyword(s):

Pseudomonas Aeruginosa ◽

Quorum Sensing ◽

Biofilm Formation ◽

Antimicrobial Agents ◽

Cell Communication ◽

Microtiter Plate ◽

Antibiofilm Activity ◽

Dependent Manner ◽

Biofilm Inhibition ◽

Swarming Motility

Bacteria in biofilms are encased in an extracellular polymeric matrix that limits exposure of microbial cells to lethal doses of antimicrobial agents, leading to resistance. In Pseudomonas aeruginosa, biofilm formation is regulated by cell-to-cell communication, called quorum sensing. Quorum sensing facilitates a variety of bacterial physiological functions such as swarming motility and protease, pyoverdine, and pyocyanin productions. Peptide mix from the marine mollusc, Olivancillaria hiatula, has been studied for its antibiofilm activity against Pseudomonas aeruginosa. Microscopy and microtiter plate-based assays were used to evaluate biofilm inhibitory activities. Effect of the peptide mix on quorum sensing-mediated processes was also evaluated. Peptide mix proved to be a good antibiofilm agent, requiring less than 39 μg/mL to inhibit 50% biofilm formation. Micrographs obtained confirmed biofilm inhibition at 1/2 MIC whereas 2.5 mg/mL was required to degrade preformed biofilm. There was a marked attenuation in quorum sensing-mediated phenotypes as well. At 1/2 MIC of peptide, the expression of pyocyanin, pyoverdine, and protease was inhibited by 60%, 72%, and 54%, respectively. Additionally, swarming motility was repressed by peptide in a dose-dependent manner. These results suggest that the peptide mix from Olivancillaria hiatula probably inhibits biofilm formation by interfering with cell-to-cell communication in Pseudomonas aeruginosa.

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Prevalence of Adhesion and Regulation of Biofilm-Related Genes in Different Clones ofStaphylococcus aureus

Journal of Biomedicine and Biotechnology ◽

10.1155/2012/976972 ◽

2012 ◽

Vol 2012 ◽

pp. 1-10 ◽

Cited By ~ 24

Author(s):

Salman Sahab Atshan ◽

Mariana Nor Shamsudin ◽

Zamberi Sekawi ◽

Leslie Than Thian Lung ◽

Rukman Awang Hamat ◽

...

Keyword(s):

Polymerase Chain Reaction ◽

Biofilm Formation ◽

Clinical Information ◽

Microtiter Plate ◽

Rt Pcr ◽

Chain Reaction ◽

Microtiter Plate Assay ◽

High Prevalence ◽

Different Types ◽

Polymerase Chain

Clinical information about genotypically different clones of biofilm-producingStaphylococcus aureusis largely unknown. We examined whether different clones of methicillin-sensitive and methicillin-resistantS. aureus(MSSA and MRSA) differ with respect to staphylococcal microbial surface components recognizing adhesive matrix molecules (MSCRAMMs) in biofilm formation. The study used 60 different types ofspaand determined the phenotypes, the prevalence of the 13 MSCRAMM, and biofilm genes for each clone. The current investigation was carried out using a modified Congo red agar (MCRA), a microtiter plate assay (MPA), polymerase chain reaction (PCR), and reverse transcriptase polymerase chain reaction (RT-PCR). Clones belonging to the samespatype were found to have similar properties in adheringto thepolystyrene microtiter plate surface. However, their ability to produce slime on MCRA medium was different. PCR experiments showed that 60 clones of MSSA and MRSA were positive for 5 genes (out of 9 MSCRAMM genes).icaADBCgenes were found to be present in all the 60 clones tested indicating a high prevalence, and these genes were equally distributed among the clones associated with MSSA and those with MRSA. The prevalence of other MSCRAMM genes among MSSA and MRSA clones was found to be variable. MRSA and MSSA gene expression (MSCRAMM andicaADBC) was confirmed by RT-PCR.

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