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| (A) Colony morphology of Streptomyces sp. ASK2 on ISP2 agar medium; (B) Antagonistic activity against K. pneumoniae by colony overlay assay; (C) Scanning electron micrograph of Streptomyces sp. ASK2 displaying branched ribbon like spores with smooth surface.
Source publication
The emergence and spread of multi-drug resistant (MDR) especially carbapenem-resistant Klebsiella pneumoniae is a major emerging threat to public health, leading to excess in mortality rate as high as 50–86%. MDR K. pneumoniae manifests all broad mechanisms of drug resistance, hence development of new drugs to treat MDR K. pneumoniae infection has...
Contexts in source publication
Context 1
... isolate Streptomyces sp. ASK2 obtained from Solanum trilobatum soil sample displayed promising antagonistic activity with ZOI of 33 ± 0.5 mm by colony over lay assay (Figure 2B). Similarly the culture supernatant exhibited strongest activity against drug resistant K. pneumoniae and thus the strain ASK2 was subjected for taxonomical studies. ...
Context 2
... the culture supernatant exhibited strongest activity against drug resistant K. pneumoniae and thus the strain ASK2 was subjected for taxonomical studies. The strain ASK2 was characterized by wrinkled, rough, irregular, dry and white aerial mycelia on ISP2 agar medium (Figure 2A). The scanning electron micrograph of ASK2 was found to have branched ribbon like spores with smooth surface (Figure 2C). ...
Context 3
... strain ASK2 was characterized by wrinkled, rough, irregular, dry and white aerial mycelia on ISP2 agar medium (Figure 2A). The scanning electron micrograph of ASK2 was found to have branched ribbon like spores with smooth surface (Figure 2C). The sequence (1053 base sequences) similarity search using BLAST tool reveals that ASK2 (Gen Bank Accession Number: KR187109) belongs to a distinct phyletic line in Streptomyces sp. ...
Context 4
... observed a similar kind of pathological consequences in zebrafish planktonic and biofilm model. The infectious dose, severity of illness, survival rates and bacterial counts in muscle homogenates were similar for biofilm and planktonic zebrafish infection models (Tables 2, 3). One possible reason for similar pathological observation is the gene expression for biofilm formation on solid support is different from those required to establish in vivo ( Lavender et al., 2004). ...
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Raoultella planticola is an emerging pathogen causing several infections in humans, and its roles in the propagation of antibiotic resistance genes (ARGs) remain uncharacterized. In this study, a carbapenem and tigecycline-resistant R. planticola isolate was recovered from hospital sewage. It carried nine plasmids, bearing 30 ARGs, including one bl...
Background
The purpose of our study is to analyze the microbiological and clinical characteristics of carbapenem‐resistant hypervirulent Klebsiella pneumoniae (CR‐hvKP) that causes nosocomial infection.
Methods
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Two types of Klebsiella pneumoniae (KP) strains are currently emerging: hypervirulent (hvKP) strains and carbapenem-resistant (CR-KP) strains. To date, these two strain types rarely overlap. Recent reports, however, suggest that CR-KP strains are increasing in virulence. hvKP strains frequently present as highly invasive infections, such as necroti...
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Citations
... In addition, zebrafish can be used for high-throughput drug screening. As a result, Lalitha et al. in 2017 used zebrafish to screen active molecular drugs for the treatment of K. pneumoniae infection [16]. ...
... Five-monthold adult female zebrafish were infected with the isolated K. pneumoniae. The detailed infection method was based on a previous study [16]. Infected zebrafish were cultured in a 10 L glass tank, and fed brine shrimp in the morning and evening; the uneaten food and dead zebrafish were removed in time. ...
Background
Klebsiella pneumoniae is a pathogen that often infects patients in clinical practice. Due to its high virulent and drug resistance, infected patients are difficult to treat. In clinical practice, Klebsiella pneumoniae can infect patients' intestines, intestines, blood, etc., causing pathological changes. However, there is relatively little information on the impact of Klebsiella pneumoniae on intestinal inflammation and microbial populations. Zebrafish is an excellent biomedical model that has been successfully applied to the virulence assessment of Klebsiella pneumoniae.
Methods
In this study, three clinically isolated representative strains of Klebsiella pneumoniae (high virulence non-resistant, high virulence resistant, and low virulence resistant) were used to infect zebrafish, and their effects on intestinal colonization, inflammation, pathology, and microbial diversity were tested.
Results
Enzyme-linked immunoassay (ELISA) showed that Klebsiella pneumoniae significantly increased levels of the cytokines interleukin-1α (Il-1α), interleukin-1β (Il-1β), and tumor necrosis factor-α (Tnf-α), which increased inflammatory symptoms. Hematoxylin eosin staining(H&S) showed that Klebsiella pneumoniae treatment caused intestinal lesions in zebrafish, in which KP1053 exposure significantly decreased the number of goblet cells, KP1195 caused epithelial dissolution and exfoliation. In addition, Klebsiella pneumoniae disturbed the composition of intestinal microbiota, and the Shannon index increased, which increased the number of harmful bacteria.
Conclusions
Klebsiella pneumoniae infection can lead to intestinal colonization, inflammation, pathological changes, and changes in microbial biodiversity. This study provides a reference for the intestinal pathology of clinical Klebsiella pneumoniae infection.
... The urine isolates K. pneumoniae (n = 3) were obtained from Dr. D. Y. Patil Medical College, Hospital and Research Center, Pune, India. The presumptive identi cation of the isolates was performed using HiCrome UTI agar medium (Cheepurupalli et al., 2017). The environmental strain, K. variicola MTCC 4030 was purchased from Microbial Type Culture Collection (MTCC) at Chandigarh, India. ...
... The infected sh were placed on the iced wax plate and dissected muscle tissue to estimate the K. pneumoniae load in the sh. To estimate the K. pneumoniae load, 100 mg of dissected muscle was homogenized, followed by serial dilutions (Cheepurupalli et al., 2017). The sample for histopathology was prepared as mentioned in Cheepurupalli et al., 2017. ...
... To estimate the K. pneumoniae load, 100 mg of dissected muscle was homogenized, followed by serial dilutions (Cheepurupalli et al., 2017). The sample for histopathology was prepared as mentioned in Cheepurupalli et al., 2017. The cut sections were 5 mM and stained with hematoxylin and eosin for histological examination. ...
In recent times K. pneumoniae , a common nosocomial pathogen, tends to evolve more virulent and multidrug-resistant. Urinary tract infections by antibiotic resistant and virulent K. pneumoniae are a growing concern due to the challenging treatment strategies. Hence understanding the genome and validating the genomic profile along with pan-genome analysis will facilitate surveillance of high risk-clones of K. pneumoniae to underpin management strategies toward early detection. Thus, the present study aimed to correlate genotypic and phenotypic antimicrobial resistance and pathogenicity associated with sequence types and virulence factors in Klebsiella spp. The complete genome profile of 3 K. pneumoniae and a K. variicola were analysed in the study which was assigned to ST200, ST45, ST147 and ST of K. variicola are not known. The accessory genome comprised 66% of genes consisting of shell and cloud genes in K. pneumoniae . The phenotypic antimicrobial resistance identified kp3 to have maximum resistance and the susceptibility of environment isolate K. variicola was less resistant to the tested antibiotics. The in vitro and genomic antimicrobial resistance profiling showed concordance with all the tested antibiotics against the 4 study strains. Hypermucoviscosity was not observed for any of the test isolates; this phenotypic character matches perfectly with the absence of rmpA , rmpA2 and magA . Though the genes encoding for major virulence factors; hypermucoviscosity, yersiniabactin and allantoin metabolism were absent, significant pathogenicity and mortality were observed for kp3 isolate in the Zebrafish infection model. In contrast, the isolates predicted with the aerobactin gene were observed with least mortality rate. In the present study virulome and in vivo study illustrate the discrepancies in the virulence. To the best of our knowledge, this is the first report on the presence of ste, stf , stc and sti major fimbrial operons of Salmonella enteric serotype Typhimurium in K. pneumoniae genome, that might contribute to the virulence of kp3. Our study highlights the further necessity of genomic and phenotypic virulence markers for timely diagnosis and immediate treatment for the management of high risk K. pneumoniae clones.
... Zone of inhibition was measured after 24 h incubation. The increase in zone of inhibition in the extracts was considered as positive for b-lactamase inhibitory activity (Cheepurupalli et al., 2017). ...
... The phylogenetic tree was constructed by using the Mega 5.2 version software program. Kimura's two-parameter model was used for the neighbor-joining method (Cheepurupalli et al., 2017). ...
The increase in drug resistance over the last two decades is a big threat in health care settings. More importantly, the dissemination of carbapenem-resistant Enterobacteriaceae is the major threat to public health with an increase in morbidity and mortality. β-lactamase is known to confer enteric bacteria with nearly complete resistance to all β-lactam antibiotics including the late-generation carbapenems. The commercially available β-lactamase inhibitors, clavulanic acid, sulbactam, and tazobactam are being met with an increasing number of resistant phenotypes and are ineffective against pathogens harbouring New Delhi metallo-β-lactamase (NDM-1). Inhibition of New Delhi metallo-β-lactamase-1 activity is one potential way to treat metallo β-lactamase (MBL) producing multi drug resistant (MDR) pathogen. The present study focused on screening of Klebsiella pneumoniae New Delhi metallo-β-lactamase-1 (BLIs) from endophytic Streptomyces spp. using in vitro and in silico methods. The study identified three potential inhibitors of New Delhi metallo-β-lactamase-1, namely dodecanoic acid, dl-alanyl-l-leucine and phenyl propanedioic acid. These molecules were found to bind to other MBLs namely, IMP-1 and VIM-2. To the best of our knowledge, this is the first kind of study reporting the binding mode of these molecules with New Delhi metallo-β-lactamase-1.
Communicated by Ramaswamy H. Sarma
... The survival rate remained unaltered in the case of E. coli-and DPBS-injected embryos over the time course of infection. Recent studies report that high neutrophil recruitment and zebrafish lethality is observed with K. pneumoniae if directly injected into the blood (34,36). We anticipate two possibilities for the sensitivity of the K. pneumoniae MGH78578 DsoxS strain in zebrafish larvae. ...
Antimicrobial resistance is a global health challenge. Few new antibiotics have been developed for use over the years, and preserving the efficacy of existing compounds is an important step to protect public health. This paper describes a study that examines the effects of exogenously induced oxidative stress on
K. pneumoniae
... It has been shown that only certain K. pneumoniae strains were able to infect zebrafish and cause mortality. In another study, it was shown that a bioactive molecule from Streptomyces sp. was able to reduce mortality of zebrafish infected with K. pneumoniae [14]. However, there are no publications on phage therapy against K. pneumoniae using the zebrafish larvae model although this has been studied for other pathogens such as Vibrio anguillarum [15] and Enterococcus faecalis [16]. ...
... In a study by Marcoleta et al. [9], three types of K. pneumoniae were used to infect zebrafish larvae, but only one (Kp RYC492) was found to be highly virulent and caused 75% mortality after 72 hours post-infection (h.p.i.). In another study by Cheepurupalli et al. [14], clinical MDR K. pneumoniae at 10 7 CFU/ml was used to infect the zebrafish resulting in 100% mortality at 24 h.p.i. In comparison with the current study, K. pneumoniae 2146 was extremely virulent to the zebrafish larvae; causing 100% mortality at 10 h.p.i. after 1 hour and 30 minutes infection at 10 3 CFU/ml. ...
Klebsiella pneumoniae are opportunistic bacteria found in the gut. In recent years they have been associated with nosocomial infections. The increased incidence of multiple drug-resistant K . pneumoniae makes it necessary to find new alternatives to treat the disease. In this study, phage UPM2146 was isolated from a polluted lake which can lyse its host K . pneumoniae ATCC BAA-2146. Observation from TEM shows that UPM2146 belongs to Caudoviriales (Order) based on morphological appearance. Whole genome analysis of UPM2146 showed that its genome comprises 160,795 bp encoding for 214 putative open reading frames (ORFs). Phylogenetic analysis revealed that the phage belongs to Ackermannviridae (Family) under the Caudoviriales . UPM2146 produces clear plaques with high titers of 10 ¹⁰ PFU/ml. The phage has an adsorption period of 4 min, latent period of 20 min, rise period of 5 min, and releases approximately 20 PFU/ bacteria at Multiplicity of Infection (MOI) of 0.001. UPM2146 has a narrow host-range and can lyse 5 out of 22 K . pneumoniae isolates (22.72%) based on spot test and efficiency of plating (EOP). The zebrafish larvae model was used to test the efficacy of UPM2146 in lysing its host. Based on colony forming unit counts, UPM2146 was able to completely lyse its host at 10 hours onwards. Moreover, we show that the phage is safe to be used in the treatment against K . pneumoniae infections in the zebrafish model.
... Antibiotic resistance is an ever growing problem for all bacteria in the ESKAPE category and one of the very crucial bacteria of great medical concern is Klebsiella pneumoniae (Effah et al. 2020). It is considered as an opportunistic pathogen causing a wide variety of diseases (Cheepurupalli et al. 2017) and is responsible for one-thirds of all Gram-negative infections affecting humans (Navon-Venezia, Kondratyeva, and Carattoli 2017). One of the major consequences of Klebsiella infection is the long duration of hospitalization and high morbidity (Giske et al. 2008)..There are plenty of studies on the emergence of MDR and extremely drug-resistant (XDR) forms of this bacteria and this is a major concern for researchers and clinicians worldwide. ...
Klebsiella pneumonia, gram-negative bacilli, fits in the family ‘Enterobacteriaceae’ and is considered as the normal flora of human beings. It is commonly found in healthcare and community related infections. The frequent occurrence of resistance towards the antimicrobials amongst the isolates of K. pneumoniae is an important health issue. The emergence of newly emerged K. pneumoniae strains show resistance to drugs of choice. Therefore, novel strategies are required to prevent the transmission and contamination of K. pneumoniae strains. Rapid diagnosis and treatment are necessary to manage resistant bacteria such as carbapenemase producing K. pneumoniae (Cp-Kp). For the control of Cp-Kp, the triple-combination remedy is being regarded as best option for treatment. In such treatment the polymyxin based remedy is the mainstay antibiotic. The emergence of resistant bacterial strains has extremely constrained our capability for treating the diseases and therefore the development of new antibiotics is presently desirable. Traditional medicinal plants are credible to offer additional novel antibiotics in impending health concerns. The usage of plants extracts or pure natural substances in combination with conservative drugs might grip better promise for immediate and reasonable options for treatment. Certainly, some antibiotic remedies are now clinically accessible. For controlling the K. pneumoniae infections, the alternate strategies are employed like, vaccinations (capsular-polysaccharides, lipo-polysaccharides), traditional medications, bacteriophage remedy, plant derivative therapies of antibiotics, combinational chemotherapies of antimicrobial and plant extracts/compounds synergistic blends with conservative antibiotics.
Keywords: Klebsiella pneumoniae, Enterobacteriaceae, Antibiotics, Capsular-polysaccharides.
... https://doi.org/10.1101/2020.08.21.262022 doi: bioRxiv preprint in the case of E. coli and DPBS injected embryos over the time course of infection. Recent studies report that high neutrophil recruitment and zebrafish lethality is observed with K. pneumoniae if directly injected into the blood 34,36 . We anticipate two possibilities for the sensitivity of K. pneumoniae MGH78578 ΔsoxS in zebrafish larvae. ...
In bacteria, the defense system deployd to counter oxidative stress is orchestrated by three transcriptional factors − SoxS, SoxR, and OxyR. Although the regulon that these factors control is known in many bacteria, similar data is not available for Klebsiella pneumoniae . To address this data gap, oxidative stress was artificially induced in K. pneumoniae MGH78578 using paraquat and the corresponding oxidative stress regulon recorded using RNA−seq. The soxS gene was significantly induced during oxidative stress and a knock-out mutant was constructed, to explore its functionality. The wild-type and mutant were grown in the presence of paraquat and subjected to RNA−seq to elucidate the soxS regulon in K. pneumoniae MGH78578. Genes that are commonly regulated both in the oxidative stress regulon and soxS regulon were identified and denoted as the ′oxidative SoxS regulon′ − these included a stringent group of genes specifically regulated by SoxS. Efflux pump encoding genes such as acrAB−tolC, acrE , and global regulators such as marRAB were identified as part of this regulon. Consequently, the isogenic soxS mutant was found to exhibit a reduction in the minimum bactericidal concentration against tetracycline compared to that of the wild type. Impaired efflux activity, allowing tetracycline to be accumulated in the cytoplasm to bactericidal levels, was further evaluated using a tetraphenylphosphonium (TPP ⁺ ) accumulation assay. The soxS mutant was also susceptible to tetracycline in vivo, in a zebrafish embryo model. We conclude that the soxS gene could be considered as a genetic target against which an inhibitor could be developed in the future and used in combinatorial therapy with tetracycline to combat infections associated with multi-drug resistant K. pneumoniae .
... Therefore, it has become an urgent problem to find a more suitable host animal model to study intestinal colonization. In recent years, a large number of classical infection models of mammals have been established (Paczosa and Mecsas, 2016), such as Galleria mellonella (Wand et al., 2013), Caenorhabditis elegans (Kamaladevi and Balamurugan, 2017), and zebrafish (Cheepurupalli et al., 2017). Those models have been successfully applied for virulence testing, infection-pathogenesis, and antimicrobial/antiviral drugs screening. ...
... In recent years, studies on clinical and subclinical infections have been successfully carried out by optimizing intramuscular injection in adult zebrafish; the effectiveness of antibiotics in vivo has also been investigated (Cheepurupalli et al., 2017). It is feasible to evaluate the virulence, neutrophil recruitment, bacterial clearance, and colonization of the bacteria by different methods of infection (injection/immersion) (Marcoleta et al., 2018). ...
The intestine is the main reservoir of bacterial pathogens in most organisms. Klebsiella pneumoniae is an important opportunistic pathogen associated with nosocomial bacterial infections. Intestinal colonization with K. pneumoniae has been shown to be associated with an increased risk of subsequent infections. However, not all K. pneumoniae strains in the intestine cause further infection, and the distinction of the difference among strains that cause infection after colonization and the ones causing only asymptomatic colonization is unclear. In this study, we report a case of a hospitalized patient from the ICU. We screened out two intestine colonization strains (FK4111, FK4758) to analyze the subsequent infection conditions. We set up infection models of zebrafish and Galleria mellonella to establish the differences in the potential for causing subsequent infection and the immunological specificities after K. pneumoniae intestine colonization. Sudan Black B and neutral red staining results indicated that FK4758 was more responsive to neutrophil recruitment and phagocytosis of macrophages than FK4111. The results of the assessment of the organ bacterial load revealed that FK4111 and FK4758 both had the highest bacterial loads in the zebrafish intestine compared to those in other organs. However, in the zebrafish spleen, liver, and heart, the FK4758 load was significantly higher than that of FK4111. The ST37 strain FK4111, which does not produce carbapenemase, did not cause infection after colonization, whereas the ST11 strain FK4758, which produces carbapenemase, caused infection after intestinal colonization. Our finding demonstrated that not all intestinal colonization of K. pneumoniae subsequently caused infections, and the infections of K. pneumoniae after colonization are different. Therefore, the infection models we established provided possibility for the estimation of host-microbial interactions.
... to explore ability of natural products to interact synergistically with colistin and augment bactericidal effect of colistin in clinical isolates of Enterobacteriaceae (especially E. coli and Klebsiella pneumoniae.) both in vitro and in vivo in a zebrafish infection model that we and others have developed (Christena et al. 2016;Cheepurupalli et al. 2017). In addition, we were also interested in exploring mechanism of action of plant metabolites that displays synergistic interaction with colistin. ...
Colistin resistance in Enterobacteriaceae especially Klebsiella pneumoniae and Escherichia coli is driving the evolution of pan drug resistant strains. Screening a library of 13 plant nutraceuticals led to the identification of acetyl shikonin and ursolic acid, which exhibited synergy with colistin against extremely drug resistant (XDR) clinical strains of E. coli (U3790) and K. pneumoniae (BC936). Ursolic acid caused a significant colistin MIC reversal of 16-fold in U3790 and 4-fold in BC936 strains. Ursolic acid also potentiated the bactericidal effect of colistin against both U3790 and BC936 by causing ~ 4 to 4.5 log fold decline in CFU of both clinical isolates in a time kill assay. At 2× minimum effective concentration, ursolic acid was non-toxic to zebrafish as evidenced by brain and liver enzyme profiles and by histopathology studies. In combination with colistin, ursolic acid reduced bacterial bioburden of U3790/BC936 by 1–1.58 log fold from the infected muscle tissue of zebrafish. Mechanistic explorations via studies on real time efflux, membrane potential and intracellular accumulation of dansyl chloride tagged colistin revealed that colistin efflux is inhibited by ursolic acid. In addition, ursolic acid also enhanced outer membrane permeability which probably facilitates colistin’s attack on outer and inner membranes. Our study shows that ursolic acid synergizes with colistin by inhibiting colistin efflux in Enterobacteriaceae that helps to curtail colistin resistant Enterobacteriaceae.
Electronic supplementary material
The online version of this article (10.1186/s13568-019-0750-4) contains supplementary material, which is available to authorized users.
... In previous work, we successfully used zebrafish larvae to study intestinal colonization and lethality by Salmonella Typhimurium, using two methods of infection: static immersion and microinjection (Varas et al., 2017a,b). In this regard, a recent work used adult zebrafish to test the safety and the efficacy in vivo of molecules with antibacterial activity against multiresistant K. pneumoniae, indicating that this pathogen can infect zebrafish when administered by intramuscular injection (Cheepurupalli et al., 2017). However, it remained unknown if K. pneumoniae can infect zebrafish in its larval stage, which harbors advantageous features compared to adult zebrafish as a host model, permitting live-cell imaging of the infection, an easier manipulation of the individuals, and larger population sizes that allow more robust statistical analyses. ...
... To our knowledge, this is the first report using zebrafish larvae to study K. pneumoniae virulence. A recent work used adult zebrafish to test the safety and the efficacy in vivo of Streptomyces-derived molecules with antibacterial activity against multiresistant K. pneumoniae (Cheepurupalli et al., 2017). An intramuscular injection protocol was optimized to produce clinical and sub-clinical infections with this pathogen, and to successfully test the efficacy of the antimicrobials in vivo. ...
... Remarkably, immersion assays showed that zebrafish larvae could be easily infected with K. pneumoniae through its natural route of entry, leading to the colonization of different zones of the gastrointestinal tract. Noteworthy, Cheepurupalli et al. (2017) observed that immersion in doses of K. pneumoniae up to 10 12 CFU/mL did not lead to clinical symptoms of adult fish, which only were achieved after intramuscular inoculation of 10 12 CFU. In our assays, infection by immersion was achieved using 5-8 10 8 CFU/mL, while 15,000-18,000 CFU were used for injections. ...
Multiresistant and invasive hypervirulent Klebsiella pneumoniae strains have become one of the most urgent bacterial pathogen threats. Recent analyses revealed a high genomic plasticity of this species, harboring a variety of mobile genetic elements associated with virulent strains, encoding proteins of unknown function whose possible role in pathogenesis have not been addressed. K. pneumoniae virulence has been studied mainly in animal models such as mice and pigs, however, practical, financial, ethical and methodological issues limit the use of mammal hosts. Consequently, the development of simple and cost-effective experimental approaches with alternative host models is needed. In this work we described the use of both, the social amoeba and professional phagocyte Dictyostelium discoideum and the fish Danio rerio (zebrafish) as surrogate host models to study K. pneumoniae virulence. We compared three K. pneumoniae clinical isolates evaluating their resistance to phagocytosis, intracellular survival, lethality, intestinal colonization, and innate immune cells recruitment. Optical transparency of both host models permitted studying the infective process in vivo, following the Klebsiella-host interactions through live-cell imaging. We demonstrated that K. pneumoniae RYC492, but not the multiresistant strains 700603 and BAA-1705, is virulent to both host models and elicits a strong immune response. Moreover, this strain showed a high resistance to phagocytosis by D. discoideum, an increased ability to form biofilms and a more prominent and irregular capsule. Besides, the strain 700603 showed the unique ability to replicate inside amoeba cells. Genomic comparison of the K. pneumoniae strains showed that the RYC492 strain has a higher overall content of virulence factors although no specific genes could be linked to its phagocytosis resistance, nor to the intracellular survival observed for the 700603 strain. Our results indicate that both zebrafish and D. discoideum are advantageous host models to study different traits of K. pneumoniae that are associated with virulence.
