Difference in biofilm formation between carbapenem-resistant and carbapenem-sensitive Klebsiella pneumoniae based on analysis of mrkH distribution

A total of 157 clinical samples were collected from different clinical specimens (urine, sputum, blood, swabs, and cannula) from several hospitals in Iraq. Among the samples, 51 isolates (32.48%) of Klebsiella pneumoniae were identified according to morphologicaland cultural characteristics as well as the Enterosystem 18R test. Higher numbers of K. pneumoniae isolates were observed in urine samples (26, 52%) than the other samples, and in females (70.6%) than males (29.4%) (female: male ratio of about 2.4:1). Antibiotic susceptibility of K. pneumoniae against 12 commonly used antibiotics was determined through the disc-diffusion method. The results revealed a higher resistance rate in 51 isolates (100%) against Cephalexin, followed by Ceftazidime (50, 98%), while the lowest resistance rate (24, 47%) was against each of Imipenem and Meropenem. Also, the investigation of the minimum inhibitory concentration (MIC) of Colistin using E-test (strips) demonstrated that 33 isolates were resistance, as compared to 31 using the disk diffusion assay. DNA was extracted from K. pneumoniae isolates and molecularly tested using polymerase chain technique (PCR) with a specific primer and 108 bp product to detect the rpoB gene that represents this bacteria . Also, all of the 51 isolates of K. pneumoniae identified by the rpoB gene were detected for the expression of the Colistin drug resistance gene mgr-B , which was amplified (347 bp) using a specific primer. Colistin resistance gene mgr-B was amplified and sequenced from the twenty isolates. Only 6 isolates appeared with a single nucleotide substitution; G instead A, A instead G, C instead G and G instead C. Also, this study tested biofilm formation from K. pneumoniae isolates , using the microtiter plate method, in association with Colistin and Carbapenem resistant. The Colistin and Carbapenem resistance pattern was compared to the ability of biofilm-formation as weak formation versus strong and also, Multi-drug resistant isolates were more common among weak versus strong biofilm formers.

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699. Relationship Between Klebsiella pneumoniae Antimicrobial Resistance and Biofilm Formation

Open Forum Infectious Diseases ◽

10.1093/ofid/ofy210.706 ◽

2018 ◽

Vol 5 (suppl_1) ◽

pp. S252-S252

Author(s):

Jaclyn Cusumano ◽

Kathryn Daffinee ◽

Megan Luther ◽

Vrishali Lopes ◽

Aisling Caffrey ◽

...

Keyword(s):

Protein Synthesis ◽

Antimicrobial Resistance ◽

Klebsiella Pneumoniae ◽

Rhode Island ◽

Biofilm Formation ◽

Multidrug Resistant ◽

Resistant Organism ◽

Protein Synthesis Inhibitors ◽

Carbapenem Resistant ◽

Research Grant

Abstract Background Klebsiella pneumoniae is a frequently multidrug-resistant organism with a high propensity to form biofilm. K. pneumoniae is the most common carbapenem-resistant Enterobacteriaceae (CRE), and labeled an urgent threat by the CDC. The relationship between K. pneumoniae biofilm formation and specific antimicrobial resistance patterns has not been well defined. Methods K. pneumoniae isolates (n = 139) were evaluated for antimicrobial resistance and biofilm formation (CDC, Providence VA Med. Ctr., Rhode Island Hosp., BEI, and ATCC). Susceptibility was based predominantly on 2017 CLSI (Clinical and Laboratory Standards Institute) breakpoints. Isolates were categorized as multidrug-resistant (MDR: resistant to ≥ 1 antimicrobial in ≥ 3 out of 16 antimicrobial categories) or extensively drug-resistant (XDR: resistant to ≥ 1 antimicrobial in all but ≤ 2 out of 16 antimicrobial categories) based on expert consensus criteria for Enterobacteriaceae (European CDC (ECDC)/CDC, 2012). We collapsed antimicrobial categories described by the ECDC/CDC consensus group into nine categories: penicillins, cephalosporins, monobactam, carbapenems, protein synthesis inhibitors, fluoroquinolones, folate pathway inhibitors, fosfomycin, and colistin. Biofilm formation was assessed using a modified crystal violet method (OD570) and defined by tertile cut-points. Antimicrobial resistance was compared for weak (n = 47) vs. strong (n = 46) biofilm formation by chi-square or Fisher’s exact test. Predictors of strong biofilm formation were identified using logistic regression. Results MDR isolates were more common among weak (n = 46/47, 97.9%) vs. strong biofilm formers (n = 35/46, 76.1%; P = 0.002), whereas XDR was similar between groups (n = 12/47, 25.5% vs. n = 13/46, 28.3% P = 0.77). Resistance to penicillins, cephalosporins, monobactams, carbapenems, protein synthesis, or fluoroquinolones was more common among weak biofilm formers (P < 0.05). Carbapenem resistance was inversely associated with strong biofilm formation (odds ratio 0.09; 95% confidence interval 0.02–0.33). Conclusion Carbapenem-resistant K. pneumoniae was 91% less likely to form strong biofilm. Potential trade-off mechanisms between antimicrobial resistance and biofilm formation require further exploration. Disclosures A. Caffrey, Merck: Grant Investigator, Research grant. The Medicine’s Company: Grant Investigator, Research grant. Pfizer: Grant Investigator, Research grant. K. LaPlante, Merck: Grant Investigator, Research grant. Pfizer Pharmaceuticals: Grant Investigator, Research grant. Allergan: Scientific Advisor, Honorarium. Ocean Spray Cranberries, Inc.: Grant Investigator and Scientific Advisor, Honorarium and Research grant. Achaogen, Inc.: Scientific Advisor, Honorarium. Zavante Therapeutics, Inc.: Scientific Advisor, Honorarium.

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Acquisition of the Conjugative Virulence Plasmid From a CG23 Hypervirulent Klebsiella pneumoniae Strain Enhances Bacterial Virulence

Frontiers in Cellular and Infection Microbiology ◽

10.3389/fcimb.2021.752011 ◽

2021 ◽

Vol 11 ◽

Author(s):

Dongxing Tian ◽

Weiwen Wang ◽

Meng Li ◽

Wenjie Chen ◽

Ying Zhou ◽

...

Keyword(s):

Klebsiella Pneumoniae ◽

Biofilm Formation ◽

Evidence Base ◽

Serum Resistance ◽

Virulence Plasmid ◽

E Coli ◽

Virulence Plasmids ◽

Carbapenem Resistant ◽

High Level

The emergence of hypervirulent and carbapenem-resistant Klebsiella pneumoniae (hv-CRKP) has become a hot topic and confounding problem for clinicians and researchers alike. Conjugative virulence plasmids have the potential to cause more threatening dissemination of hv-CRKP strains. We previously identified K2606, a CG23 clinical hypervirulent strain of Klebsiella pneumoniae harboring a conjugative virulence plasmid designated pK2606. In this study we examined hypervirulence levels using assays of biofilm formation, serum resistance, and wax larvae and mouse in vivo infection models. Moreover, to define the transfer ability of pK2606 and whether this confers hypervirulence to other strains we performed plasmid transconjugation experiments between K2606 and the ST11 CRKP strain HS11286 along with E. coli J53. We found that although biofilm formation and serum resistance were not significantly increased, the transconjugants acquired the ability of produce high level of siderophores and also caused high mortality of wax larvae and mice. Furthermore, we identified pK2606-like conjugative virulence plasmids in GenBank, providing evidence that such plasmids may have begun to spread throughout China. These findings provide an evidence base for the possible mechanisms of the emergence of hv-CRKP strains and highlight the potential of pK2606-like conjugative virulence plasmids to spread worldwide.

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Reduced virulence and enhanced host adaption during antibiotics therapy: A story of a within-host carbapenem-resistant Klebsiella pneumoniae sequence type 11 evolution in a fatal scrotal abscess patient

10.1101/2021.05.06.443049 ◽

2021 ◽

Author(s):

Meiping Ye ◽

Chunjie Liao ◽

Mengya Shang ◽

Danyang Zou ◽

Jingmin Yan ◽

...

Keyword(s):

Klebsiella Pneumoniae ◽

Binding Site ◽

Biofilm Formation ◽

Capsular Polysaccharide ◽

Human Life ◽

Sequence Type ◽

Genomic Diversity ◽

Rna Seq ◽

Carbapenem Resistant ◽

Virulence Attenuation

Carbapenem-resistant Klebsiella pneumoniae (CRKP) has disseminated globally and become a major threat to human life. The sequence type (ST) 11 CRKP is a dominant clone in Asia, especially China, but how this clone evolves in vivo, then adapts to host and facilitates dissemination remain largely unknown. We analyzed the genomic dynamics of 4 ST11-CRKP isolates sequencially isolated from the urine of a patient with initial fatal scrotal abscess and finally recovered without effective medication. Genomic differences were identified and their implications for pathogenesis and host adaptation were investigated. The related transcriptional pathways were further explored by RNA-Seq. Genomic analysis identified 4-24 mutations and 94%-100% were synonymous or intergenic. The mutation rate of ST11-CRKP was 2.1×10-6-1.7×10-5 substitutions/site/year over 47 days of antibiotics therapy. During this period, CRKP underwent several adaptive changes including tigecycline resistance and virulence attenuation. Tigecycline resistance was caused by ramR ribosomal binding site (RBS) deletion, which has been described by us previously. In this study, we demonstrated that mutations associated with acyltransferase (act) and ompK26 caused the virulence attenuation of ST11-CRKP. act deletion reduced the production of capsular polysaccharide and enhanced biofilm formation. RNA-Seq analysis revealed that act influenced the expression of ldhA, bglX, mtnK and metE which likely participate in capsular synthesis and biofilm formation. ompK26 affected the virulence by its overexpression caused by the deletion of upstream repressor binding site. Our finding suggested that the broad genomic diversity, high evolutionary capacity and rapid within-host adaptability of ST11-CRKP might contribute to the worldwide dissemination of this clone.

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Outbreak of KPC-2 Carbapenem-Resistant Klebsiella pneumoniae ST76 and Carbapenem-Resistant K2 Hypervirulent Klebsiella pneumoniae ST375 Strains in Northeast China: Molecular and Virulent Characteristics

10.21203/rs.2.9470/v1 ◽

2019 ◽

Author(s):

Shanshan Su ◽

Yongxin Zhao ◽

Lan Yu ◽

Chunjiang Li ◽

Yong Wang ◽

...

Keyword(s):

Antibiotic Resistance ◽

Klebsiella Pneumoniae ◽

Biofilm Formation ◽

Northeast China ◽

Capsular Polysaccharide ◽

Molecular Characteristics ◽

Circular Chromosome ◽

Carbapenem Resistant ◽

Positive Isolate ◽

Serotype K2

Abstract Background Carbapenem-resistant hypervirulent Klebsiella pneumoniae strains have recently come into existence worldwide; however, researchers in northeast China are not aware of their clinical features and molecular characteristics. Methods Here, the molecular and virulent characteristics of 44 carbapenem-resistant K. pneumoniae (CRKP) isolates collected from January 2015 to December 2017 were studied. Multilocus sequence typing (MLST) and pulsed-field gel electrophoresis (PFGE) were carried out to define the clonal relatedness among the isolates. PCR and capsular serotyping of the virulence-associated genes, as well as biofilm formation and serum complement-mediated killing assays, were employed to determine the virulent potential. The genomic features and associated mobile genetic elements of JmsCRE57 were detected by whole genome sequencing. Results The only positive isolate was JmsCRE57, which belonged to the ST375 serotype K2 that expressed uge, mrkD, fimH, kpn, aerobactin and rmpA virulence-associated genes and showed strong biofilm formation and serum sensitivity. Sequencing results showed that the JmsCRE57 genome mainly consisted of a circular chromosome, three antibiotic resistance plasmids and a virulent plasmid. The antibiotic resistance plasmid expressing blaKPC-2, blaCTX-M-15, aph(3'')-Ib, aph(6)-Id, qnrB1, aac(3)-IIa, aac(6')-Ib-cr, blaOXA-1, blaTEM-1B, catB4, sul2, dfrA14 and blaSHV-99. The virulent plasmid belonged to the IncHI1B group, which is mainly composed of capsular polysaccharide genes and siderophore-associated genes. The remaining CRKP strains that expressed uge, fimH, mrkD and kpn virulence-associated genes were not successfully typed. We also identified an isolate that expressed the alls gene. Conclusion Our results provide new insights on the epidemiology of carbapenem-resistant K2 hypervirulent K. pneumoniae ST375 and CRKP ST76 strains in northeast China, which may help control their future outbreaks.

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

Antibiotics ◽

10.3390/antibiotics9120852 ◽

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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Antibiogram and Biofilm Formation among Carbapenem Resistant Klebsiella pneumoniae

Tribhuvan University Journal of Microbiology ◽

10.3126/tujm.v6i0.26586 ◽

2019 ◽

Vol 6 ◽

pp. 63-69

Author(s):

Roshani Nhuchhen Pradhan ◽

Surendra Kumar Madhup ◽

Shyam Prasad Pant

Keyword(s):

Klebsiella Pneumoniae ◽

Congo Red ◽

Biofilm Formation ◽

Microtitre Plate ◽

Sensitivity Testing ◽

Clinical Specimens ◽

Plate Assay ◽

Carbapenem Resistant ◽

Microtitre Plate Assay ◽

Agar Method

Objectives: This cross-sectional study was designed to detect the carbapenemase producing K. pneumoniae along with biofilm producers from different clinical specimens and to compare antibiotic susceptibility pattern of biofilm producing carbapenem resistant Klebsiella pneumoniae and biofilm non-producing carbapenem resistant Klebsiella pneumoniae. Methods: A total of 1475 non-repetitive clinical samples were included on this study. Antibiotic Sensitivity Testing (AST), Modified Hodge Test (MHT) and Modified Carbapenem inactivation method (mCIM) were performed for detection of carbapenemase production and Congo red agar method (CRA) along with Microtitre plate method were performed for detecting biofilm production. Results: Among the clinical specimens cultured, growth positivity was 62.71%. E. coli was most predominant organism followed by K. pneumoniae (17.89%). Among the 110 K. pneumoniae, 57 were found to be carbapenemase producer. Majority of the carbapenemase producing K. pneumoniae were isolated from sputum (45.61%), in the specimen collected from age group 61-70 (28.07%) and in out-patient department (50.88%). Similarly, 65.45% K. pneumoniae out of 110 were found to be biofilm producer by Congo red agar method while among those 72, 73.59% isolates were found to be quantitatively biofilm producer in Microtitre plate assay. Out of 57 carbapenemase producer, 35.08% were strongly biofilm producer while among 53 carbapenemase non-producer 30.18% were strongly biofilm producer from Congo red agar method. Moreover, Microtitre plate assay evidenced that, out of 57 carbapenemase producer, 40.35% were highly biofilm producing and among the 15 carbapenemase nonproducer 66.66% were highly biofilm producer. Conclusion: Biofilm formation is highly prevalent with varying degree of resistance among different antibiotics including carbapenems that further augments antibiotic resistance. The study showed carbapenemase producers are stronger biofilm producer than the non-carbapenemase producer. Therefore, it is recommended to identify biofilm formation among carbapenemase producers for effective choice of antibiotics.

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Correlation between biofilm formation and carbapenem resistance among clinical isolates of Klebsiella pneumoniae

Ethiopian Journal of Health Sciences ◽

10.4314/ejhs.v29i6.11 ◽

1970 ◽

Vol 29 (6) ◽

Author(s):

Hossein Ali Rahdar ◽

Ebadallah Shiri Malekabad ◽

Ali-Reza Dadashi ◽

Elahe Takei ◽

Masuod Keikha ◽

...

Keyword(s):

Klebsiella Pneumoniae ◽

Biofilm Formation ◽

Severe Infection ◽

Carbapenem Resistance ◽

Clinical Isolates ◽

P Value ◽

Public Health Services ◽

Carbapenem Resistant ◽

Amplification Assay ◽

Hospital Equipment

Background: Klebsiella pneumoniae is a Gram-negative enteric bacterium that causes nosocomial infections; this bacterium has survived from harsh condition using biofilm formation in hospital equipment and cause severe infection. In the other hand, the emergence and extension of carbapenem resistance burden among K. pneumonia producing biofilm is the current concern of public health services. There are controversial findings about this subject. The aim of this study was to evaluate the correlation between biofilm formation and resistance to carbapenem among clinical isolates of K. pneumoniae.Methods: A total of 160 K. pneumoniae isolates were collected from various infections of hospitalized patients. The Carba NP test and molecular methods were used for detection of carbapenem resistance isolates of K. pneumonia. Subsequently, the ability for biofilm production was performed from all isolates. Finally, Correlation of biofilm formation among carbapenem resistant isolates was calculated using χ2 and Fisher’s exact tests.Results: Among K. pneumoniae isolates 42.5% have carbapenemase activity by Carba NP test, while carbapenemase genes were detected in 35.6% of isolates in amplification assay. Moreover, there are 52.5% (n= 84) of all isolates were formed a strong biofilm, while 38.1% (n= 61) and 9.3% (n= 15) of isolates were middle and weak biofilm producer, respectively. Among carbapenem resistant cases (n= 68), there are 77.9% (n= 53) and 22% (n= 15) of isolates were reported as strong and middle biofilm producer, respectively. We see a significant correlation was seen between biofilm formation ability and carbapenem resistant isolates (p-value < 0.00001).Conclusion: The increase of carbapenem resistance burden in biofilm producing isolates of K. pneumoniae is considered as serious alert and the basic measures to combat this phenomenon is imperative.

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Outbreak of KPC-2 Carbapenem-Resistant Klebsiella pneumoniae ST76 and Carbapenem-Resistant K2 Hypervirulent Klebsiella pneumoniae ST375 Strains in Northeast China: Molecular and Virulent Characteristics

10.21203/rs.2.12529/v1 ◽

2019 ◽

Author(s):

Shanshan Su ◽

Yongxin Zhao ◽

Lan Yu ◽

Chunjiang Li ◽

Yong Wang ◽

...

Keyword(s):

Antibiotic Resistance ◽

Klebsiella Pneumoniae ◽

Biofilm Formation ◽

Northeast China ◽

Molecular Characteristics ◽

Circular Chromosome ◽

Carbapenem Resistant ◽

Resistance Plasmids ◽

Positive Isolate ◽

Serotype K2

Abstract Background Carbapenem-resistant hypervirulent Klebsiella pneumoniae strains have recently come into existence worldwide; however, researchers in northeast China are not aware of their clinical features and molecular characteristics. Methods Here, the molecular and virulent characteristics of 44 carbapenem-resistant K. pneumoniae (CRKP) isolates collected from January 2015 to December 2017 were studied. Multilocus sequence typing (MLST) and pulsed-field gel electrophoresis (PFGE) were carried out to define the clonal relatedness among the isolates. PCR and capsular serotyping of the virulence-associated genes, as well as biofilm formation and serum complement-mediated killing assays, were employed to determine the virulent potential. The genomic features and associated mobile genetic elements of JmsCRE57 were detected by whole genome sequencing. Results The only positive isolate was JmsCRE57, which belonged to the ST375 serotype K2 that expressed uge, mrkD, fimH, kpn, aerobactin and rmpA virulence-associated genes and showed strong biofilm formation and serum sensitivity. Sequencing results showed that the JmsCRE57 genome mainly consisted of a circular chromosome, three antibiotic resistance plasmids and a virulent plasmid. The antibiotic resistance plasmid expressing blaKPC-2, blaCTX-M-15, aph(3'')-Ib, aph(6)-Id, qnrB1, aac(3)-IIa, aac(6')-Ib-cr, blaOXA-1, blaTEM-1B, catB4, sul2, dfrA14 and blaSHV-99. The virulent plasmid belonged to the IncHI1B group, which is mainly composed of mucoid phenotype genes and siderophore-associated genes. The remaining CRKP strains that expressed uge, fimH, mrkD and kpn virulence-associated genes were not successfully typed. Conclusion Our results provide new insights on the epidemiology of carbapenem-resistant K2 hypervirulent K. pneumoniae ST375 and CRKP ST76 strains in northeast China, which may help control their future outbreaks.

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