The influence of biofilm formation on carbapenem resistance in clinical<i>Klebsiella pneumoniae</i>infections: phenotype vs genome-wide analysis

Klebsiella pneumoniae is one of the leading causes of nosocomial infections. Carbapenem-resistant K. pneumoniae are on the rise globally. The biofilm forming ability of K. pneumoniae further complicates patient management. There is still a knowledge gap on the association of biofilm formation with patient outcome and carbapenem susceptibility, which is investigated in present study. K. pneumoniae isolates from patients admitted in critical care units with catheters and ventilators were included. K. pneumoniae (n = 72) were subjected to 96-well plate biofilm formation assay followed by MBEC assay for subset of strong biofilm formers. Whole genome sequencing and a core genome phylogenetic analysis in comparison with global isolates were performed. Phenotypic analyses showed a positive correlation between biofilm formation and carbapenem resistance. Planktonic cells observed to be susceptible in vitro exhibited higher MICs in biofilm structure, hence MICs cannot be extrapolated for treatment. The biofilm forming ability had a significant association with morbidity/mortality. Infections by stronger biofilm forming pathogens significantly (p < 0.05) resulted in fewer “average days alive” for the patient (3.33 days) in comparison to those negative for biofilms (11.33 days). Phylogenetic analysis including global isolates revealed clear association of sequence types with genes for biofilm formation and carbapenem resistance. Known hypervirulent clone-ST23 with wcaG, magA, rmpA, rmpA2, and wzc with lack of mutation for hyper-capsulation might be poor biofilm formers. ST15, ST16, ST307, and ST258 (reported global high-risk clones) were wcaJ negative indicating the high potential of biofilm forming capacity. Genes wabG and treC for CPS, bcsA and pgaC for adhesins, luxS for quorum sensing were common in all clades in addition to genes for aerobactin (iutA), allantoin (allS), type I and III fimbriae (fimA, fimH, and mrkD) and pili (pilQ and ecpA). This study is the first of its kind to compare genetic features of antimicrobial resistance with a spectrum covering most of the genetic factors for K. pneumoniae biofilm. These results highlight the importance of biofilm screening to effectively manage nosocomial infections by K. pneumoniae. Further, data obtained on epidemiology and associations of biofilm and resistance genetic factors will serve to enhance our understanding on biofilm mechanisms in K. pneumoniae.

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“…: 11940 dt 27-03-2019). This manuscript has been released as a pre-print at bioRxiv (Naveen Kumar et al, 2020).…”

Section: Data Availability Statementmentioning

confidence: 99%

The Influence of Biofilms on Carbapenem Susceptibility and Patient Outcome in Device Associated K. pneumoniae Infections: Insights Into Phenotype vs Genome-Wide Analysis and Correlation

et al. 2020

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“…Furthermore, in-silico screening of the clinical K. pneumoniae genomes available in the public database revealed that 98%, 99.3% and 34.6% of the genomes carried mrkD, pgaC and wcaJ genes 13 . It was also found that mrkD and pgaC genes were present in all bio lm forming K. pneumoniae isolates in the present study.…”

Section: Discussionmentioning

confidence: 99%

Evaluation of genomic markers mrkD, pgaC and wcaJ involved in biofilm formation to aid in rapid screening of K. pneumoniae biofilms from endotracheal aspirates and bronchoalveolar lavage

Ragupathi,

Sethuvel,

et al. 2024

Preprint

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Klebsiella pneumoniae has been identified as one of the most important opportunistic pathogens responsible for nosocomial infections. Antibiotic resistance and ability to form biofilms are the two main factors involved in the persistence of infections. Conventional detection methods involve culture isolation and identification followed by biofilm assay that takes 48–72 hrs. Timely detection of biofilm-forming resistant pathogens is essential to appropriately treat the infection with the right dose and combinations. The present study focusses on evaluating an RT-PCR panel using mrkD, pgaC and wcaJ genes to screen for biofilm-forming K. pneumoniae from ETA/BAL specimens. The assay accurately identified K. pneumoniae harboring samples with a limit of detection (LOD) of 1 ng/µl total RNA. Representative culture-negative-PCR-positive samples were subjected to metagenomics which identified K. pneumoniae reads in these samples confirming the specificity of RT-PCR. mrkD and pgaC acts as K. pneumoniae specific identification, whereas wcaJ act as negative marker for biofilm-forming K. pneumoniae. In addition, RT-PCR results correlated well with the phenotypic biofilm forming assay. This RT-PCR assay is the first of its kind for rapid identification of biofilm-forming K. pneumoniae. The result of this study highlights that the rapid detection of K. pneumoniae biofilms based on the RT-PCR results coupled with clinical conditions would be appropriate to treat emerging infections or to prevent re-infections in the clinical settings.

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“…pneumoniae has a significant association with morbidity/mortality. The infections caused by clinical isolates of this species, strong biofilm formers, resulted in fewer ‘average days to live’ for the patient (3.33 days) compared to non-biofilm-forming isolates (11.33 days) [38].…”

Section: Discussionmentioning

confidence: 99%

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

Stallbaum

Pruski

Amaral

et al. 2021

Journal of Medical Microbiology

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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.

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The influence of biofilm formation on carbapenem resistance in clinicalKlebsiella pneumoniaeinfections: phenotype vs genome-wide analysis

Cited by 4 publications

References 40 publications

The Influence of Biofilms on Carbapenem Susceptibility and Patient Outcome in Device Associated K. pneumoniae Infections: Insights Into Phenotype vs Genome-Wide Analysis and Correlation

The Influence of Biofilms on Carbapenem Susceptibility and Patient Outcome in Device Associated K. pneumoniae Infections: Insights Into Phenotype vs Genome-Wide Analysis and Correlation

Evaluation of genomic markers mrkD, pgaC and wcaJ involved in biofilm formation to aid in rapid screening of K. pneumoniae biofilms from endotracheal aspirates and bronchoalveolar lavage

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

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