Detection of Biofilm and Siderophore Encoding Genes Implicated in the Pathogenesis of Klebsiella pneumoniae Isolated from Different Clinical Specimens

2021 ◽  
Vol 30 (1) ◽  
pp. 101-108
Author(s):  
Iman S. Naga ◽  
Abeer Ghazal ◽  
Saif S. Alomari ◽  
Ahmed Gaballah
Keyword(s):  
Immune System ◽  
Klebsiella Pneumoniae ◽  
Crystal Violet ◽  
Biofilm Formation ◽  
Virulence Genes ◽  
Siderophore Production ◽  
Clinical Specimens ◽  
String Test ◽  
Encoding Genes ◽  
System Objective

Background: Biofilm and siderophores protect K. pneumoniae, making them resistant to antimicrobials and increase the ability to evade immune system. Objective: The aim of the present study was to detect some of the virulence genes responsible for biofilm formation and siderophore production implicated in the pathogenesis of K. pneumoniae. Methodology: Crystal violet method was used to detect biofilm phenotypically, hypermucoviscosity by string test and virulence genes were detected by PCR. Results: Phenotypically, biofilm formation was positive in 76% and negative in 24%. Genotypically, FimH and ycfm were detected in 100% of isolates while mrkD in 98%. Kfu, entB and iutA were found in 100%, 68% and 34% respectively. Phenotypically by string test 66% of isolates were classified as hypermucoviscous, while 34% as classical. rmpA gene was detected in 52% of isolates. Conclusion: FimH, mrkD, ycfm, Kfu and iutA genes are implicated in multisystemic infection of K. pneumoniae. Enterobactin gene is more predominant than other siderophore genes.

scholarly journals The Determination and Correlation of Various Virulence Genes, ESBL, Serum Bactericidal Effect and Biofilm Formation of Clinical Isolated Classical Klebsiella pneumoniae and Hypervirulent Klebsiella pneumoniae from Respiratory Tract Infected Patients

2017 ◽  
Vol 66 (4) ◽  
pp. 501-508 ◽  
Author(s):  
Rambha K. Shah ◽  
Zhao H. Ni ◽  
Xiao Y. Sun ◽  
Guo Q. Wang ◽  
Fan Li
Keyword(s):  
Klebsiella Pneumoniae ◽  
Respiratory Tract ◽  
Biofilm Formation ◽  
Virulence Genes ◽  
Virulence Gene ◽  
Bactericidal Effect ◽  
Biofilm Production ◽  
Significant Difference ◽  
String Test

Klebsiella pneumoniae strains that are commonly recognized by clinicians and microbiologists are termed as classical K. pneumoniae (cKP). A strain with capsule-associated mucopolysaccharide web is known as hypervirulent K. pneumoniae (hvKP) as it enhances the serum resistant and biofilm production. Aim is to determine and correlate various virulence genes, ESBL, serum bactericidal effect and biofilm formation of clinical isolated cKP and hvKP from respiratory tract infected patients. A total of 96 K. pneumoniae strains were isolated from sputum of respiratory tract infected patients. The isolates were performed string test, AST, ESBL virulence gene, serum bactericidal and biofilm assays. Out of 96 isolates, 39 isolates (40.6%) were identified with hypervirulent phenotypes. The number of cKP exhibiting resistance to the tested antimicrobials and ESBLs were significantly higher than that of the hvKP strains. The virulence genes of K. pneumoniae such as K1, K2, rmpA, uge, kfu and aerobactin were strongly associated with hvKP than cKP. However, no significant difference was found in FIM-1 and MrKD3 genes. ESBL producing cKP and hvKP were significantly associated with strong biofilm formation (both P < 0.05) and highly associated with bactericidal effect of serum (both P < 0.05) than cKP strains. However, neither biofilm formation nor bactericidal effect of serum was found with significant difference in between ESBL producing cKP and ESBL producing hvKP strains (both P > 0.05). Although the hvKP possess more virulence gene, but they didn’t show any significant difference between biofilm formation and bactericidal effect of serum compared with ESBL producing cKP strains.

scholarly journals Comparative analysis of Clinial Carbapenem- Resistant (CR), Hypermucoviscous (HM) and CR-HM Klebsiella pneumoniae isolates in China

2020 ◽  
Author(s):  
Jun-Ying Zhu ◽  
Guang-Yu Wang ◽  
Qing Wei ◽  
Zhen Shen ◽  
Qiong Li ◽  
...  
Keyword(s):  
Klebsiella Pneumoniae ◽  
Virulence Genes ◽  
Carbapenem Resistance ◽  
Resistance Rate ◽  
Molecular Characteristics ◽  
Carbapenem Resistant ◽  
Recent Emergence ◽  
String Test

Abstract Background: Although carbapenem-resistant Klebsiella pneumoniae (CRKP) and hypermucoviscous K. pneumoniae (HMKP) were largely non-overlapping, the recent emergence of CR-HMKP has raised great alarm in the world. We compared the molecular characteristics of CRKP, HMKP and CR-HMKP isolates.Results: 220 cases of K. pneumoniae isolates was collected and identified between Jan 2015 and Dec 2016 from Renji Hospital. Carbapenem resistance test and string test were performed to screen CRKP, HMKP and CR-HMKP isolates. All the CRKP, HMKP and CR-HMKP isolates were investigated for capsular genotyping, virulence genes and resistance genes by PCR and DNA sequencing. Multilocus sequence typing (MLST) was used to characterize isolates sequence types (STs). Serum killing assay and mouse lethality assay were respectively performed to confirm the virulence of the isolates in vitro and in vivo. Of 220 K. pneumoniae，71 HMKP, 84 CRKP and 8 CR-HMKP were identified. Resistance rate to carbapenems was significantly higher in CRKP than HMKP and CR-HMKP. For MLST and serotyping, ST23 (26.8%)，K1 (33.8%) and K2 (23.9%) serotypes were the most common in HMKP isolates while ST11 (84.5%, 100%) and K-nontypable (91.6%, 100%) were the predominant types in CRKP and CR-HMKP isolates. The existence of virulence genes rmpA, magA and iutA was significantly higher in HMKP while the prevalence of resistance gene blaKPC-2 was higher in CRKP and CR-HMKP. Virulence test in vivo and in vitro both showed the lower virulence of CRKP and CR-HMKP compared to HMKP.Conclusions: In spite of low virulence, the emergence of CR-HMKP indicates a confluence of hypermucoviscous phenotype and carbapenem resistance. Furthermore, the similar molecular characteristics between CRKP and CR-HMKP suggested that CR-HMKP might evolve from CRKP.

scholarly journals Identification of a Chromosomal Integrated DNA Fragment Containing the rmpA2 and iucABCDiutA Virulence Genes in Klebsiella pneumoniae

mSphere ◽  
2020 ◽  
Vol 5 (6) ◽  
pp. e01179-20
Author(s):  
Xuemei Yang ◽  
Lianwei Ye ◽  
Yi Li ◽  
Edward Wai-Chi Chan ◽  
Rong Zhang ◽  
...  
Keyword(s):  
Klebsiella Pneumoniae ◽  
Virulence Genes ◽  
Antibiotic Resistance Genes ◽  
Content Type ◽  
Carbapenem Resistant ◽  
Transmission Mechanisms ◽  
Genetic Features ◽  
Severe Infections ◽  
First Time ◽  
Encoding Genes

ABSTRACTKlebsiella pneumoniae is increasingly being regarded as a reservoir of diverse antibiotic resistance genes and a pathogen that causes severe infections in both the hospital and the community. In this study, we performed molecular characterization of a carbapenem-resistant and highly virulent K. pneumoniae strain recovered from a hospital patient. The virulence-encoding genes rmpA2 and iucABCDiutA were found to be located in the chromosome and are flanked by IS26 elements. These chromosomally located virulence genes, if not incurring fitness costs, are expected to be much more stable than plasmid-located genes. Detailed analysis of the fragment in which these virulence genes are located showed that the fragment can readily form a circular intermediate that may promote integration of this virulence-encoding fragment into various plasmid backbones and other chromosomal regions. Findings in this work provide new insights into mechanisms of transmission of virulence-encoding genes in K. pneumoniae.IMPORTANCE This study reported for the first time and characterized in detail the genetic features of a mobile virulence-encoding fragment located in the chromosome of a clinical virulent K. pneumoniae strain and revealed the occurrence of a transposition event mediated by IS26. This genetic structure could mediate the transposition of the virulence-encoding fragment into various plasmid backbones and chromosomes through formation of a circular intermediate. Therefore, findings in this work provide important insights into the transmission mechanisms of mobile virulence profiles in K. pneumoniae strains and lay the foundation for devising effective intervention approaches aimed at preventing the dissemination of these virulence-encoding elements.

scholarly journals Comparison and Detection of biofilms layers in Staphylococcus aureus and Escherichia coli Isolated from Clinical Specimens

2020 ◽  
pp. 09-12
Author(s):  
Saptarshi Pal
Keyword(s):  
Escherichia Coli ◽  
Staphylococcus Aureus ◽  
Extracellular Matrix ◽  
Cell Adhesion ◽  
Crystal Violet ◽  
Environmental Conditions ◽  
Biofilm Formation ◽  
Model Organism ◽  
Clinical Specimens ◽  
Specific Strain

Biofilms are the community that probably represents the mode of growth for microbes in most environments. Biofilms are typically surrounded by an extracellular matrix that provides structure and protection to the community. The mechanisms that different bacteria employ to form biofilms vary, frequently depending on environmental conditions and specific strain attributes. Biofilm formation begins with attachment of bacteria to biotic surface such as host cell. After attachment, aggregation of bacteria is started by cell-cell adhesion. Dispersion is started by certain conditions such as phenol-soluble modulinos. For studying and comparing the different layers of biofilm formation in our model organism like Staphylococcus and Escherichia we used slide detection technique and the extent of biofilm formation is measured using the crystal violet (CV) dye. Using these bacteria as examples, we compared the key sizes of 3 layers of the biofilms as well as effect of antibiotics on these layers. Keywords: Biofilm; Staphylococcus aureus; Escherichia coli

scholarly journals Prevalence of virulence genes and their association with biofilm formation in VRE faecium isolates from Ahvaz, Iran

2018 ◽  
Vol 12 (11) ◽  
pp. 970-977
Author(s):  
Saber Soltani ◽  
Maniya Arshadi ◽  
Muhammad Ibrahim Getso ◽  
Farzaneh Aminharati ◽  
Mahmood Mahmoudi ◽  
...  
Keyword(s):  
Antibiotic Susceptibility ◽  
Biofilm Formation ◽  
Virulence Genes ◽  
Positive Association ◽  
Microtiter Plate ◽  
Clinical Specimens ◽  
Microbiological Methods ◽  
Antibiotic Susceptibility Patterns ◽  
Pcr Method ◽  
Susceptibility Patterns

Introduction: Vancomycin-resistant Enterococcus faecium (VREfm) is a common cause of nosocomial infections. Biofilm formation is an important factor in recurrence of infections, facilitating transfer of genetic elements, leading to treatment failures. The aim of this study was to investigate the virulence genes in biofilm producing isolates and to determine possible association between biofilm formation and the presence of these genes; also to determine association between antibiotic susceptibility patterns of VREfm isolates and their biofilm formation ability. Methodology: A total of 57 isolates of VREfm were recovered from different sources of hospitals under Ahvaz University, Iran. The isolates were examined by conventional microbiological methods and molecular test using PCR. The antibiotic susceptibility patterns of the isolates were determined by disk-diffusion and E-test. The biofilm formation ability of the isolates was investigated by Modified Congo red agar and microtiter plate techniques. The presence of virulence genes was examined using Multiplex-PCR method. Results: Out of 57 VREfm isolates, 63.15% of isolates were biofilm producers. The frequency of biofilm producing isolates from clinical specimens, colonized patients and environmental sources were 78.26%, 60%, and 42.85%, respectively. The prevalence of acm, esp and hyl genes among biofilm producing isolates was 86.10%, 55.56% and 52.77%, respectively. There was statistically significant association between esp gene and biofilm formation among isolates from the clinical specimens. Conclusion: Clinical isolates producing biofilms showed a positive association with the presence of the esp. Our study further suggests that the link between virulence genes and biofilms is affected by the environmental context.

scholarly journals Different effects of sub-minimum inhibitory concentrations of gentamicin on the expression of genes involved in alginate production and biofilm formation of Pseudomonas aeruginosa

2021 ◽  
Author(s):  
Fateme Davarzani ◽  
Zahra Yousefpour ◽  
Navid Saidi ◽  
Parviz Owlia
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Biofilm Formation ◽  
Virulence Genes ◽  
Microtiter Plate ◽  
Inhibitory Effect ◽  
Clinical Isolates ◽  
Minimum Inhibitory Concentrations ◽  
Alginate Production ◽  
Broth Microdilution Method ◽  
Encoding Genes

Background and Objectives: Antibiotics at sub-minimum inhibitory concentrations (sub-MIC) may alter bacterial viru- lence factors. The objective of this study was to investigate the effect of gentamicin at sub-MIC concentrations on the expres- sion of genes involved in alginate production and biofilm formation of Pseudomonas aeruginosa. Materials and Methods: The broth microdilution method was used to determine the MIC of gentamicin for three P. aeru- ginosa clinical isolates (P1-P3) and standard strains (PAO1 and 8821M). Alginate production and biofilm formation of the bacteria in the presence and absence of sub-MIC concentrations of gentamicin were measured using microtiter plate and carbazole assay, respectively. The real-time PCR method was used to determine the effect of gentamicin at sub-MIC con- centrations on the expression level of genes involved in biofilm formation (pelA and pslA) and alginate production (algD and algU). Results: Gentamicin at sub-MIC concentrations significantly reduced alginate production, biofilm formation, and the expres- sion of alginate and biofilm-encoding genes in clinical isolate P1. This inhibitory effect was also observed on the alginate production of 8821M strain and biofilm formation of PAO1strain. In clinical isolates, P2 and P3, alginate production, biofilm formation, and the expression of alginate and biofilm-encoding genes were significantly increased in exposure to sub-MIC concentrations of gentamicin. Conclusion: This study showed that different phenotypic changes in clinical isolates and standard strains of P. aeruginosa in exposure to sub-MIC concentrations of gentamicin are associated with changes in the expression of virulence genes. Further researches are required to understand the mechanisms involved in regulating the expression of virulence genes after exposure to sub-MIC concentrations of antibiotics.

Phenotypic and molecular evaluation of biofilm formation in Klebsiella pneumoniae carbapenemase (KPC) isolates obtained from a hospital of Pelotas, RS, Brazil

2021 ◽  
Vol 70 (11) ◽  
Author(s):  
Letícia Roloff Stallbaum ◽  
Beatriz Bohns Pruski ◽  
Suelen Cavalheiro Amaral ◽  
Stella Buchhorn de Freitas ◽  
Daniela Rodriguero Wozeak ◽  
...  
Keyword(s):  
Antibiotic Resistance ◽  
Klebsiella Pneumoniae ◽  
Crystal Violet ◽  
Biofilm Formation ◽  
Staining Method ◽  
Multidrug Resistant ◽  
Crystal Violet Staining ◽  
Content Type ◽  
Klebsiella Pneumoniae Carbapenemase

Introduction. A significant cause of mortality in the intensive care unit (ICU) is multidrug-resistant (MDR) Gram-negative bacteria, such as Klebsiella pneumoniae carbapenemase (KPC). Biofilm production is a key factor in KPC colonization and persistence in the host, making the treatment difficult. Gap Statement. The aim of this study was to evaluate the antibiotic resistance, molecular and phenotypic biofilm profiles of 12 KPC isolates associated with nosocomial infection in a hospital in Pelotas, Rio Grande do Sul, Brazil. Methodology. Clinical isolates were obtained from different sources, identified and characterized by antibiotic resistance and carbapenemase synthesis following the Clinical and Laboratory Standards Institute (CLSI) guidelines. Polymerase chain reaction (PCR) was used to evaluate the presence of carbapenemase (blaKPC ) and biofilm formation-associated genes (fimA, fimH, rmpA, ecpA, mrkD and wabG). Additionally, phenotypic evaluation of in vitro biofilm formation capacity was evaluated by Congo red agar (CRA) assay and the crystal violet staining method. Results. The 12 isolates evaluated in this study presented the blaKPC gene and were positive for synthesizing carbapenemases in vitro. In the carbapenem class, 83.3 % isolates were resistant and 16.7 % intermediately resistant to imipenem and meropenem. Molecular analyses found that the fimA and wabG genes were detected in 75 % of isolates, while fimH and ecpA were detected in 42 % and mrkD were detected in 8.3 % (1). The CRA assay demonstrated that all isolates were slime producers and 91.7 % (11) of isolates were classified as strong and 8.3 % (1) as moderate biofilm producers by the crystal violet staining method. The optical density (OD540nm) for strong biofilm formers ranged from 0.80±0.05 to 2.47±0.28 and was 0.55±0.12 for moderate biofilm formers. Conclusion. Our study revealed a high level of antibiotic resistance and biofilm formation in KPC isolates obtained from a hospital in Pelotas, RS, Brazil.

scholarly journals Virulence Characteristics and Molecular Epidemiology of Pyogenic Liver Abscess Causing Multidrug Resistant Klebsiella pneumoniae in Wenzhou, China

2021 ◽  
Author(s):  
Zhongyong Wang ◽  
Siqin Zhang ◽  
Na Huang ◽  
Shixing Liu ◽  
Ye Xu ◽  
...  
Keyword(s):  
Klebsiella Pneumoniae ◽  
Molecular Epidemiology ◽  
Liver Abscess ◽  
Biofilm Formation ◽  
Growth Curves ◽  
Pyogenic Liver Abscess ◽  
Multidrug Resistant ◽  
Infection Model ◽  
String Test ◽  
Serum Killing

Abstract Background To date, little is known about the virulence characteristics of pyogenic liver abscess (PLA) that cause multidrug resistant (MDR) Klebsiella pneumoniae (K. pneumoniae), which might be due to the rarity of these strains. This study aimed to analyze the virulence characteristics and molecular epidemiology of 12 MDR strains obtained from 163 PLA cases in a tertiary teaching hospital from the perspective of clinical characteristics, virulence phenotypes, and genotypes. Methods The MDR strains were obtained from sterile fluid samples collected from patients with PLA. The antimicrobial susceptibility testing was confirmed by the agar dilution method and microdilution broth method. The virulence phenotypes were analyzed by the growth curves, string test, capsular quantification, serum killing test, biofilm formation assay and infection model. Polymerase chain reaction (PCR) was used to investigate the virulence genotypes. The molecular epidemiology was identified by multilocus sequence typing (MLST). Results The results of growth curves, string test, capsular quantification, serum killing test, biofilm formation assay, and infection model revealed that the virulence phenotypes of the 12 PLA-causing MDR K. pneumoniae were similar to or more obvious than those of the typical hypervirulent K. pneumoniae strains. These MDR strains were mainly non-K1/K2 serotypes and carried multiple virulence genes. The results of MLST illustrated that the MDR strains were categorized into 9 sequence types. Conclusions This is the first study to analyze the virulence characteristics in PLA-causing MDR strains. The data revealed the coexistence of hypervirulence and MDR in PLA-causing MDR K. pneumoniae strains, and the clones of these strains were diverse and scattered. Also, one ST11 carbapenem-resistant hypervirulent strain was identified in PLA.

scholarly journals Phenotypic and Genotypic Features of Klebsiella pneumoniae Harboring Carbapenemases in Egypt: OXA-48-Like Carbapenemases as an Investigated Model

Antibiotics ◽  
2020 ◽  
Vol 9 (12) ◽  
pp. 852
Author(s):  
Suzan Mohammed Ragheb ◽  
Mahmoud Mohamed Tawfick ◽  
Amani Ali El-Kholy ◽  
Abeer Khairy Abdulall
Keyword(s):  
Klebsiella Pneumoniae ◽  
Biofilm Formation ◽  
Profile Analysis ◽  
Diversity Index ◽  
Plasmid Profile ◽  
Control Measures ◽  
Discriminatory Power ◽  
Resistance Pattern ◽  
Carbapenem Resistant ◽  
Encoding Genes

This study aimed at the characterization of carbapenem-resistant Klebsiella pneumoniae isolates focusing on typing of the blaOXA-48-like genes. Additionally, the correlation between the resistance pattern and biofilm formation capacity of the carbapenem-resistant K. pneumoniae isolates was studied. The collected isolates were assessed for their antimicrobial resistance and carbapenemases production by a modified Hodge test and inhibitor-based tests. The carbapenemases encoding genes (blaKPC, blaNDM, blaVIM, blaIMP, and blaOXA-48-like) were detected by PCR. Isolates harboring blaOXA-48-like genes were genotyped by Enterobacterial Repetitive Intergenic Consensus-Polymerase Chain Reaction (ERIC-PCR) and plasmid profile analysis. The discriminatory power of the three typing methods (antibiogram, ERIC-PCR, and plasmid profile analysis) was compared by calculation of Simpson’s Diversity Index (SDI). The transferability of blaOXA-48 gene was tested by chemical transformation. The biofilm formation capacity and the prevalence of the genes encoding the fimbrial adhesins (fimH-1 and mrkD) were investigated. The isolates showed remarkable resistance to β-lactams and non-β-lactams antimicrobials. The coexistence of the investigated carbapenemases encoding genes was prevalent except for only 15 isolates. The plasmid profile analysis had the highest discriminatory power (SDI = 0.98) in comparison with ERIC-PCR (SDI = 0.89) and antibiogram (SDI = 0.78). The transferability of blaOXA-48 gene was unsuccessful. All isolates were biofilm formers with the absence of a significant correlation between the biofilm formation capacity and resistance profile. The genes fimH-1 and mrkD were prevalent among the isolates. The prevalence of carbapenemases encoding genes, especially blaOXA-48-like genes in Egyptian healthcare settings, is worrisome and necessitates further strict dissemination control measures.

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