Colistin-resistant Klebsiella Research Articles

Molecular characterization of colistin-resistant Klebsiella pneumoniae isolates and their conjugative mcr-carrying plasmids

BackgroundThis study aimed to characterize colistin-resistant K. pneumoniae (CoRKp) strains isolated from patients with urinary tract infections and bacteremia between 1999 and 2022 at a tertiary teaching hospital in Taiwan. MethodsA total of 1,966K. pneumoniae isolates were collected, among which 21 strains were identified as CoRKp. The antimicrobial susceptibility of these CoRKp strains to 19 antibiotics was assessed. The genome characteristics of 21 CoRKp strains were determined by Nanopore-Illumina hybrid whole genome sequencing. Additionally, conjugation assays were conducted to determine the transferability of plasmids carrying mcr genes to K. pneumoniae ATCC BAA-1706 and E. coli C600. The larvae infection model was used to analyze the differences in virulence between transconjugants and recipient strains. ResultsAmong the 21 CoRKp, 12 were multidrug-resistant, and four were extensively drug-resistant. The distribution of sequence types (STs) and K types among the CoRKp strains was quite diverse, and ST307 (5 strains) and K64 (3 strains) dominated in CoRKp. The insertion elements IS903B and ISVsa5, were found to inactivate mgrB of 1 and 2 CoRKp isolates, respectively. Moreover, 1, 4, 6, and 1 missense mutations of PhoQ, PmrA, PmrB, and MgrB, were identified in 21 CoRKp. Only two isolates SC-KP169 and SC-KP585 carried mcr-1 and mcr-8, respectively. The plasmid pSC-KP169-1 could be transferred inter- and intra-genus and contributed to the virulence of K. pneumoniae to larvae. In contrast, the plasmid pSC-KP585-1 could be transferred to E. coli but could not affect its virulence to larvae. ConclusionsWe identified 21 CoRKp from 1,966 isolates and found a conjugative plasmid carrying mcr-1 gene that contributed to the virulence of K. pneumoniae to larvae.

Biogenic silver nanoparticles synthesized using rose petals: Potential to inhibit multidrug-resistant bacterial pathogens in disabled patients

BackgroundDrug resistant bacterial infections can be fatal for disabled people needing multiple medications and new effective antibacterial drugs such as biosynthesized nanoparticles are needed. A cost-effective and green method was studied for the biosynthesis of silver nanoparticles using pink rose petals. Materials and methodsSilver nanoparticles (AgNP) were synthesized using the petals of pink rose flowers. The AgNPs were characterized using FITR, SEM, XRD, and EDS analyses. The antibacterial activity of the AgNPs against multidrug-resistant bacterial pathogens Staphylococcus aureus, Escherichia coli, Acinetobacter baumannii, and colistin-resistant Klebsiella pneumoniae were studied using the microplate method. ResultsTh AgNPs had a spherical shape and an average size of 26 nm. XRD analysis showed that the nanoparticles were Ag0. Antibacterial activity was the highest against the multidrug-resistant S. aureus the minimum inhibitory concentration (MIC) being 400 µg/ml AgNPs. The multidrug-resistant E. coli, multidrug-resistant A. baumannii, and colistin-resistant K. pneumoniae MICs were 800 µg/ml, 3200 µg/ml, and 1600 µg/ml, respectively. ConclusionThe green synthesized of AgNPS using pink rose petals and their antibacterial effects offer material for the treatments of common multidrug-resistant bacterial pathogens.

Adjuvants restore colistin sensitivity in mouse models of highly colistin-resistant isolates, limiting bacterial proliferation and dissemination.

Antimicrobial resistance (AMR) has led to a marked reduction in the effectiveness of many antibiotics, representing a substantial and escalating concern for global health. Particularly alarming is resistance in Gram-negative bacteria due to the scarcity of therapeutic options for treating infections caused by these pathogens. This challenge is further compounded by the rising incidence of resistance to colistin, an antibiotic traditionally considered a last resort for the treatment of multi-drug resistant (MDR) Gram-negative bacterial infections. In this study, we demonstrate that adjuvants restore colistin sensitivity in vivo. We previously reported that the salicylanilide kinase inhibitor IMD-0354, which was originally developed to inhibit the human kinase IKKβ in the NFκB pathway, is a potent colistin adjuvant. Subsequent analog synthesis using an amide isostere approach led to the creation of a series of novel benzimidazole compounds with enhanced colistin adjuvant activity. Herein, we demonstrate that both IMD-0354 and a lead benzimidazole effectively restore colistin susceptibility in mouse models of highly colistin-resistant Klebsiella pneumoniae and Acinetobacter baumannii-induced peritonitis. These novel adjuvants show low toxicity in vivo, significantly reduce bacterial load, and prevent dissemination that could otherwise result in systemic infection.

Genome sequence of a ST65 hypervirulent Klebsiella pneumoniae isolate carrying mcr-8 from China

ObjectivesIn this study, we report the complete genome sequence of a ST65 hypervirulent Klebsiella pneumoniae isolate carrying mcr-8 from China. The aim was to investigate its molecular characteristics and resistant mechanism. MethodsA colistin-resistant hvKP was isolated from an inpatient in China. The whole genome was sequenced on Illumina NovaSeq 6000 and long-read ONT platforms. de novo assembly was conducted using SPAdes and Unicycler. S1 nuclease pulsed-field gel electrophoresis, Southern-blot, and antimicrobial susceptibility testing were performed. Sequence type, antimicrobial resistance and virulence-related genes were predicted from the sequence. The circular maps of multiple plasmids comparisons were drawn by the BLAST Ring Image. ResultsThe complete genome sequence of K. pneumoniae ACESH00926 consists of one chromosome and two plasmids. ACESH00926 belongs to K2 ST65 according to the MLST scheme. ACESH00926 showed high resistance to colistin (MIC > 8 µg/mL). Several ARGs were identified, including mobile colistin-resistant gene mcr-8 which was located in an IncFIl(K)/IncFIA(HI1) type plasmid. The bigger plasmid was a pK244-like virulence plasmid. It carrying a series of virulence genes, such as the regulator of the mucoid phenotype (rmpA and rmpA2), salmochelin siderophore biosynthesis (iroB), ABC transporter (iroC), ferric aerobactin receptor (iutA), aerobactin siderophore biosynthesis protein (iucC), and aerobactin synthetase (iucA) encoded genes. And another plasmid carrying mcr-8 with a conserved genetic context (dgkA-sasA-copR-mcr-8-ccdA-ccdB-xerD-repE-parM-umuC-lexA-klcA). ConclusionsIt is necessary to emphasize the necessity of monitoring a combination of colistin-resistant and hypervirulent Klebsiella pneumoniae strains in the future.

Eco-friendly drugs induce cellular changes in colistin-resistant bacteria

Abstract Background and objective As a last option, multidrug-resistant Gram-negative infections (caused by Enterobacteriaceae) are treated with the antibiotic colistin, also known as polymyxin E. Colistin-resistant superbugs predispose people to untreatable infections, possibly leading to a high mortality rate. This project aims to study the effect of Acacia nilotica aqueous extract and zinc oxide nanoparticles (ZnO-NPs) on colistin-resistant Klebsiella pneumonia (CRKP). Materials and methods ZnO-NPs were synthesized using the green method and characterized by UV-vis, Fourier transform infrared spectroscopy, and X-ray diffraction. The zone of inhibition (ZI) was measured using the agar-well diffusion method, and the minimum inhibitory concentration (MIC) and minimum bactericidal concentration were estimated to determine the antimicrobial activity of the tested compound. Scanning electron microscopy (SEM) and transmission electronic microscopy (TEM) were used to investigate the alterations in bacterial cells that were treated with the tested drugs. Results The synthesized ZnO-NPs presented good chemical and physical properties, and the plant extract and ZnO-NPs displayed a large ZI. ZnO-NPs had the lowest MIC (0.2 mg·mL−1). SEM and TEM observations revealed various morphological modifications in CRKP cells, including cell shrinkage, cell damage, cytoplasm loss, cell wall thinning, and cell death. Conclusion A. nilotica aqueous extract and ZnO-NPs could be used as alternative natural products to produce antibacterial drugs and to prevent CRKP infection.

In vitro activity of zidebactam/cefepime (WCK 5222), a β-lactam enhancer/ β-lactam combination against carbapenem- and colistin-resistant Klebsiella pneumoniae isolates

ObjectivesIn vitro activity of β-lactam enhancer/β-lactam combination zidebactam/cefepime was evaluated against carbapenem- and colistin-resistant Klebsiella pneumoniae isolates. MethodsNon duplicate K. pneumoniae (n=185), resistant to colistin as well as non-susceptible to carbapenems were collected (2018-2019) at two large tertiary care hospitals in India. Colistin resistance-conferring genes mcr1 and mcr3 were screened among 123 of 185 randomly-selected isolates. These isolates were also subjected to multi-locus sequence typing (MLST). Additionally, alterations in mgrB were screened in 109 of these 123 isolates. All the study isolates were screened for presence of carbapenemases genes. MICs of zidebactam/cefepime, colistin, carbapenems, ceftazidime/avibactam, imipenem/relebactam, amikacin and piperacillin/tazobactam were determined by reference CLSI broth dilution method. ResultsAmong the isolates, 65.4% (121/185) carried blaOXA-48-like gene and 27.6% isolates (51/185) carried dual carbapenemase genes; blaOXA-48-like and blaNDM. Of the remainder, 8 isolates carried blaNDM and 5 isolates lacked carbapenemases gene despite being carbapenem-resistant. None of the isolates showed presence of mcr1 and mcr3. Out of 109 isolates analysed for mgrB, 36 showed mutational changes. The MLST profile revealed at least 14 unique sequence types with ST231 being the dominant clone. All the isolates showed colistin MICs >2 mg/L and were non-susceptible to carbapenems. Zidebactam/cefepime demonstrated potent activity with MIC50 and MIC90 of 1 and 2 mg/L, respectively. MIC90s of amikacin, ceftazidime/avibactam and imipenem/relebactam were >32 mg/L. ConclusionZidebactam/cefepime combination was highly active against multi-clonal, carbapenem-non-susceptible and colistin-resistant K. pneumoniae isolates producing OXA-48-like (Ambler class D) or/and NDM (Ambler class B) carbapenemases, thus potentially offering a valuable treatment options for infections caused by such pan-drug resistant resistotypes. Though, zidebactam is not an inhibitor of class B and D β-lactamases, potent activity of zidebactam/cefepime combination is attributable to β-lactam enhancer mechanism.

A novel chicken-origin colistin-resistant Klebsiella pneumoniae ST5982 Co-harboring mcr-3.11 and CTX-M-27

Klebsiella pneumoniae (K. pneumoniae) is an important zoonotic opportunistic pathogen of Enterobacteriaceae that has become one of the most common infectious diseases causing Enterobacteriaceae after Escherichia coli. In this study, we identified a colistin-resistant, multidrug-resistant ST5982 K. pneumoniae strain of broiler origin. The isolate carried 35 resistance genes of 10 antibiotics classes, detected by whole genome sequencing (WGS); 11.4 % (4/35) of the resistance genes were distributed in the chromosome, and 88.6 % (31/35) of the resistance genes were located in four different resistance plasmids. Among the four plasmids, we found for the first time that CTX-M-27 and mcr-3.11 simultaneously coexisted in K. pneumoniae, and a resistance plasmid of IncI1 carrying a combination of mcr-3.11 and qnrS1 was identified. We successfully transferred mcr-3.11, qnrS1 and CTX-M-27 genes into E. coli J53 through conjugation experiments. In the present study, the co-occurrence of CTX-M-27 and mcr-3.11 in multidrug-resistant K. pneumoniae strain ST5982 was detected for the first time; its drug resistance was evaluated, and the risk of its transmission was assessed to provide a reference for further prevention and treatment of K. pneumoniae.

INvestigation of the effectiveness of mesenchymal stem cell and resveratrol+pipperine combined treatment in colistin-resistant Klebsiella Pneumoniae sepsis

INvestigation of the effectiveness of mesenchymal stem cell and resveratrol+pipperine combined treatment in colistin-resistant Klebsiella Pneumoniae sepsis

Molecular Characterization of Carbapenem and Colistin Resistance in Klebsiella pneumoniae Isolates Obtained from Clinical Samples at a University Hospital Center in Algeria.

The current study aimed to determine the molecular mechanisms of carbapenem and colistin resistance among the clinical isolates of Klebsiella pneumoniae from hospitalized patients admitted to a university hospital in Eastern Algeria. In total, 124 non-duplicate isolates of K. pneumoniae were collected from September 2018 to April 2019. Bacterial identification was performed using MALDI-TOF MS. The presence of extended spectrum β-lactamase (ESBL) genes, carbapenemase genes, chromosomal mutation and mcr genes in colistin-resistant K. pneumoniae were evaluated by PCR. ESBLs represented a rate of 49.1% and harbored blaCTX-M, blaTEM and blaSHV genes. Concerning carbapenems, 12 strains (9.6%) were resistant to ertapenem (MIC: 1-32 μg/mL), of which one strain (0.8%) was also resistant to imipenem (MIC: 32 μg/mL). Among these strains, nine (75%) harbored blaOXA-48 gene. Seven strains (5.6%) expressed resistance to colistin (MIC: 2-32 μg/mL), of which two harbored mcr-8 and mgrB genes simultaneously. The existence of a double resistance to colistin in the same strain is new in Algeria, and this could raise concerns about the increase in levels of resistance to this antibiotic (MIC: 32 μg/mL). The mgrB gene alone was observed in five isolates (71.4%), including two strains harboring blaOXA-48. This is the first report revealing the presence of K. pneumoniae strains carrying the blaOXA-48 gene as well as a mutation in the mgrB gene. Large-scale surveillance and effective infection control measures are also urgently needed to prevent the outbreak of various carbapenem- and colistin-resistant isolates.

Thymus algeriensis essential oil: Phytochemical investigation, bactericidal activity, synergistic effect with colistin, molecular docking, and dynamics analysis against Gram-negative bacteria resistant to colistin

Due to the increasing resistance prevalence to the last line of antibiotics, such as colistin, and the rising threat of multi-drug resistant bacteria, it is crucial to find alternative therapeutic options. The current study focuses on evaluating antibacterial activities alone and in combination with colistin of Thymus algeriensis essential oil (TA-EO) against colistin-resistant Klebsiella pneumoniae, Pseudomonas aeruginosa, and Escherichia coli co-harboring mcr-1 gene. GC/MS was used to determine the chemical composition of TA-EO. Disc diffusion and microdilution techniques were used to evaluate the antimicrobial activities of TA-EO. Synergism between colistin and TA-EO was evaluated by checkerboard assay. The major compounds of TA-EO were docked with known enzymes involved in resistance to colistin, as well as the biosynthesis of peptidoglycan and amino acids. GC/MS revealed that TA-EO was of carvacrol chemotype (67.94 %). The TA-EO showed remarkable antibacterial activities against all Gram-negative bacterial strains, with the diameter of inhibition zones varied between 30 and 50 mm and a ratio MBC/MIC equal to 1 for the vast majority of bacterial isolates. Interestingly, the checkerboard showed synergism between TA-EO and colistin against colistin-resistant Escherichia coli co-harboring mcr-1 gene (FICI˂1) and reduced the MIC of colistin by 16- to 512-fold and those of TA-EO by 4- to 16-fold. The docking study demonstrated that carvacrol had high binding free energies against MCR-1, a phosphoethanolamine transferase extracellular domain, and its catalytic domain implicated in resistance to colistin, and undecaprenyl pyrophosphate synthase in complex with magnesium which is involved in bacterial peptidoglycan biosynthesis. The molecular dynamics study for 100-ns also revealed the stability of the MCR-1/carvacrol complex with a constant surface area over the simulation. These results support using carvacrol or TA-EO as a bactericidal agent, either alone or in combination with colistin, to treat infections caused by colistin-resistant Gram-negative bacteria.

Isobavachalcone enhances sensitivity of colistin-resistant Klebsiella pneumoniae: In vitro and in vivo proof-of-concept studies

ObjectiveAntibiotic resistance poses a considerable worldwide concern, particularly in clinical environments where drug-resistant Gram-negative bacteria like Klebsiella pneumoniae (K. pneumoniae) present a major challenge. The objective of this research was to investigate the mechanisms by which isobavachalcone (IBC) restores the sensitivity of K. pneumoniae to colistin in vitro and to validate the synergistic therapeutic effect in vivo. ResultsThe results indicate that the combined administration of colistin and IBC exhibits a potent antibacterial effect both in vitro and in vivo. The in vitro concurrent administration of colistin and IBC resulted in increased membrane permeability, compromised cell integrity, diminished membrane fluidity, and disrupted membrane homeostasis. Additionally, this combination reduced biofilm production, inhibited the synthesis of the autoinducer factor, altered membrane potential, and affected levels of reactive oxygen species and adenosine triphosphate synthesis, ultimately leading to bacterial death. In vivo experiments on Galleria mellonella and mice demonstrated that the co-administration of colistin and IBC increased the survival rate and significantly reduced pathological damage compared to colistin alone. ConclusionThese results suggested that IBC effectively restores the sensitivity of colistin by inducing physical disruption of bacterial membranes and oxidative stress. The combination therapy of colistin and IBC presents a viable and safe strategy to combat drug-resistant K. pneumoniae-associated infections.

Evaluation of bacteriophage cocktail on urinary tract infection caused by colistin-resistant Klebsiella pneumoniae in mice model

ObjectiveThe colistin-resistant Klebsiella pneumoniae causes complicated urinary tract infections (UTIs). Of them, 73% of strains of K. pneumoniae formed moderate to strong biofilm. Multidrug-resistant (MDR)/Pandrug-resistant (PDR) bacteria causing UTIs are very challenging to conventional antibiotic therapy. However, bacteriophages may be a promising alternative as they easily disrupt the biofilm and act on receptors unrelated to antibiotic resistance mechanisms. This preclinical study evaluated the efficacy of a phage cocktail with different routes and dosages (in quantity and frequency) to eradicate the K. pneumoniae-associated UTI in the mice model. MethodsThe three lytic phages with the broadest spectrum activity (ΦKpnBHU1, ΦKpnBHU2 and ΦKpnBHU3) were meticulously characterized using SEM and sequencing. The cocktails were administered to mice through urethral, rectal, subcutaneous and oral routes after establishing the UTI with 1 × 108 colony-forming unit/mouse (CFU/mouse) of K. pneumoniae (KpnBHU09) resistant to both the drugs carbapenem and colistin. The efficacy of different routes with varying dosages and frequency of administration was thoroughly optimized. ResultsWe observed that two doses of a phage cocktail containing 1 × 105 Plaque-Forming Unit (PFU/mouse) and a single dose of 1 × 109 PFU/mouse per urethra could eradicate KpnBHU09. Intriguingly, the non-invasive administration through oral and rectal routes required higher concentration and many dosages of phages to eliminate KpnBHU09 at any stage of acute UTI. The subcutaneous route was found unsatisfactory in curing the infection. ConclusionBacteriophage cocktails administered through transurethral, oral and rectal routes may cure UTIs.

Genomic analysis of carbapenem- and colistin-resistant Klebsiella pneumoniae complex harbouring mcr-8 and mcr-9 from individuals in Thailand

The surge in mobile colistin-resistant genes (mcr) has become an increasing public health concern, especially in carbapenem-resistant Enterobacterales (CRE). Prospective surveillance was conducted to explore the genomic characteristics of clinical CRE isolates harbouring mcr in 2015–2020. In this study, we aimed to examine the genomic characteristics and phonotypes of mcr-8 and mcr-9 harbouring carbapenem-resistant K. pneumoniae complex (CRKpnC). Polymerase chain reaction test and genome analysis identified CRKpnC strain AMR20201034 as K. pneumoniae (CRKP) ST147 and strain AMR20200784 as K. quasipneumoniae (CRKQ) ST476, harbouring mcr-8 and mcr-9, respectively. CRKQ exhibited substitutions in chromosomal-mediated colistin resistance genes (pmrB, pmrC, ramA, and lpxM), while CRKP showed two substitutions in crrB, pmrB, pmrC, lpxM and lapB. Both species showed resistance to colistin, with minimal inhibitory concentrations of 8 µg/ml for mcr-8-carrying CRKP isolate and 32 µg/ml for mcr-9-carrying CRKQ isolate. In addition, CRKP harbouring mcr-8 carried blaNDM, while CRKQ harbouring mcr-9 carried blaIMP, conferring carbapenem resistance. Analysis of plasmid replicon types carrying mcr-8 and mcr-9 showed FIA-FII (96,575 bp) and FIB-HI1B (287,118 bp), respectively. In contrast with the plasmid carrying the carbapenemase genes, the CRKQ carried blaIMP-14 on an IncC plasmid, while the CRKP harboured blaNDM-1 on an FIB plasmid. This finding provides a comprehensive insight into another mcr-carrying CRE from patients in Thailand. The other antimicrobial-resistant genes in the CRKP were blaCTX-M-15, blaSHV-11, blaOXA-1, aac(6′)-Ib-cr, aph(3′)-VI, ARR-3, qnrS1, oqxA, oqxB, sul1, catB3, fosA, and qacE, while those detected in CRKQ were blaOKP-B-15, qnrA1, oqxA, oqxB, sul1, fosA, and qacE. This observation highlights the importance of strengthening official active surveillance efforts to detect, control, and prevent mcr-harbouring CRE and the need for rational drug use in all sectors.

Genetic and structural basis of colistin resistance in Klebsiella pneumoniae: Unravelling the molecular mechanisms

ObjectiveAntimicrobial resistance (AMR), together with multidrug resistance (MDR), mainly among Gram-negative bacteria, has been on the rise. Colistin (polymyxin E) remains one of the primary available last resorts to treat infections caused by MDR bacteria during the rapid emergence of global resistance. As the exact mechanism of bacterial resistance to colistin remains undetermined, this study warranted elucidation of the underlying mechanisms of colistin resistance and heteroresistance among carbapenem-resistant Klebsiella pneumoniae isolates. MethodsMolecular analysis was carried out on the resistant isolates using a genome-wide characterisation approach, as well as MALDI-TOF mass spectrometry, to identify lipid A. ResultsAmong the 32 carbapenem-resistant K. pneumoniae isolates, several isolates showed resistance and intermediate resistance to colistin. The seven isolates with intermediate resistance exhibited the “skip-well” phenomenon, attributed to the presence of resistant subpopulations. The three isolates with full resistance to colistin showed ions using MALDI-TOF mass spectrometry at m/z of 1840 and 1824 representing bisphosphorylated and hexa-acylated lipid A, respectively, with or without hydroxylation at position C’-2 of the fatty acyl chain. Studying the genetic environment of mgrB locus revealed the presence of two insertion sequences that disrupted the mgrB locus in the three colistin-resistant isolates: IS1R and IS903B. ConclusionsOur findings show that colistin resistance/heteroresistance was inducible with mutations in chromosomal regulatory networks controlling the lipid A moiety and insertion sequences disrupting the mgrB gene, leading to elevated minimum inhibitory concentration values and treatment failure. Different treatment strategies should be employed to avoid colistin heteroresistance-linked treatment failures, mainly through combination therapy using colistin with carbapenems, aminoglycosides, or tigecycline.

Artificial Intelligence-Clinical Decision Support System in Infectious Disease Control: Combatting Multidrug-Resistant Klebsiella pneumoniae with Machine Learning.

The World Health Organization has identified Klebsiella pneumoniae (KP) as a significant threat to global public health. The rising threat of carbapenem-resistant Klebsiella pneumoniae (CRKP) leads to prolonged hospital stays and higher medical costs, necessitating faster diagnostic methods. Traditional antibiotic susceptibility testing (AST) methods demand at least 4 days, requiring 3 days on average for culturing and isolating the bacteria and identifying the species using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), plus an extra day for interpreting AST results. This lengthy process makes traditional methods too slow for urgent clinical situations requiring rapid decision-making, potentially hindering prompt treatment decisions, especially for fast-spreading infections such as those caused by CRKP. This research leverages a cutting-edge diagnostic method that utilizes an artificial intelligence-clinical decision support system (AI-CDSS). It incorporates machine learning algorithms for the swift and precise detection of carbapenem-resistant and colistin-resistant strains. We selected 4307 KP samples out of a total of 52,827 bacterial samples due to concerns about multi-drug resistance using MALDI-TOF MS and Vitek-2 systems for AST. It involved thorough data preprocessing, feature extraction, and machine learning model training fine-tuned with GridSearchCV and 5-fold cross-validation, resulting in high predictive accuracy, as demonstrated by the receiver operating characteristic and area under the curve (AUC) scores, laying the groundwork for our AI-CDSS. MALDI-TOF MS analysis revealed distinct intensity profiles differentiating CRKP and susceptible strains, as well as colistin-resistant Klebsiella pneumoniae (CoRKP) and susceptible strains. The Random Forest Classifier demonstrated superior discriminatory power, with an AUC of 0.96 for detecting CRKP and 0.98 for detecting CoRKP. Integrating MALDI-TOF MS with machine learning in an AI-CDSS has greatly expedited the detection of KP resistance by approximately 1 day. This system offers timely guidance, potentially enhancing clinical decision-making and improving treatment outcomes for KP infections.

Global prevalence of mutation in the mgrB gene among clinical isolates of colistin-resistant Klebsiella pneumoniae: a systematic review and meta-analysis.

Colistin is used as a last resort for managing infections caused by multidrug-resistant bacteria. However, the high emergence of colistin-resistant strains has restricted the clinical use of this antibiotic in the clinical setting. In the present study, we evaluated the global prevalence of the mutation in the mgrB gene, one of the most important mechanisms of colistin resistance in Klebsiella pneumoniae. Several databases, including Scopus, Medline (via PubMed), and Web of Science, were searched (until August 2023) to identify those studies that address the mgrB mutation in clinical isolates of K. pneumoniae. Using Stata software, the pooled prevalence of mgrB mutation and subgroup analyses for the year of publication, country, continent, mgrB mutation types, and detection methods of mgrB mutation were analyzed. Out of the 115 studies included in the analysis, the prevalence of mgrB mutations in colistin-resistant K. pneumoniae isolates was estimated at 65% of isolates, and mgrB variations with insertional inactivation had the highest prevalence among the five investigated mutations with 69%. The year subgroup analysis indicated an increase in mutated mgrB from 46% in 2014 to 61% in 2022. Europe had the highest prevalence of mutated mgrB at 73%, while Africa had the lowest at 54%. Mutations in the mgrB gene are reported as one of the most common mechanisms of colistin resistance in K. pneumoniae, and the results of the present study showed that 65% of the reported colistin-resistant K. pneumoniae had a mutation in this gene.

Lipid A modification of colistin-resistant Klebsiella pneumoniae does not alter innate immune response in a mouse model of pneumonia.

Polymyxin resistance in carbapenem-resistant Klebsiella pneumoniae bacteria is associated with high morbidity and mortality in vulnerable populations throughout the world. Ineffective antimicrobial activity by these last resort therapeutics can occur by transfer of mcr-1, a plasmid-mediated resistance gene, causing modification of the lipid A portion of lipopolysaccharide (LPS) and disruption of the interactions between polymyxins and lipid A. Whether this modification alters the innate host immune response or carries a high fitness cost in the bacteria is not well established. To investigate this, we studied infection with K. pneumoniae (KP) ATCC 13883 harboring either the mcr-1 plasmid (pmcr-1) or the vector control (pBCSK) ATCC 13883. Bacterial fitness characteristics of mcr-1 acquisition were evaluated. Differentiated human monocytes (THP-1s) were stimulated with KP bacterial strains or purified LPS from both parent isolates and isolates harboring mcr-1. Cell culture supernatants were analyzed for cytokine production. A bacterial pneumonia model in WT C57/BL6J mice was used to monitor immune cell recruitment, cytokine induction, and bacterial clearance in the bronchoalveolar lavage fluid (BALF). Isolates harboring mcr-1 had increased colistin MIC compared to the parent isolates but did not alter bacterial fitness. Few differences in cytokines were observed with purified LPS from mcr-1 expressing bacteria in vitro. However, in a mouse pneumonia model, no bacterial clearance defect was observed between pmcr-1-harboring KP and parent isolates. Consistently, no differences in cytokine production or immune cell recruitment in the BALF were observed, suggesting that other mechanisms outweigh the effect of these lipid A mutations in LPS.

Genomic characterization of carbapenem and colistin-resistant Klebsiella pneumoniae isolates from humans and dogs.

Carbapenem and colistin-resistant Enterobacteriaceae, including Klebsiella pneumoniae, have become a growing global concern, posing a significant threat to public health. Currently, there is limited information about the genetic background of carbapenem and colistin-resistant K. pneumoniae isolates infecting humans and dogs in Thailand. This study aimed to characterize carbapenem and colistin-resistant genes in six resistant K. pneumoniae clinical isolates (three from humans and three from dogs) which differed in their pulse field gel electrophoresis profiles. Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS), antimicrobial susceptibility testing, and whole-genome sequencing were employed to identify and analyze the isolates. All six isolates were carbapenemase-producing K. pneumoniae isolates with chromosomally carried blaSHV, fosA, oqxA and oqxB genes, as well as nine to 21 virulence genes. The isolates belonged to five multilocus sequence types (STs): one isolate from a human and one from a dog belonged to ST16, with the other two human isolates being from ST340 and ST1269 and the other two dog isolates were ST147 and ST15. One human isolate and two dog isolates harbored the same blaOXA-232 gene on the ColKP3 plasmid, and one dog isolate carried the blaOXA-48 gene on the IncFII plasmid. Notably, one human isolate exhibited resistance to colistin mediated by the mcr-3.5 gene carried on the IncFII plasmid, which co-existed with resistance determinants to other antibiotics, including aminoglycosides and quinolones. In conclusion, this study provides a comprehensive characterization of both chromosome- and plasmid-mediated carbapenem and colistin resistance in a set of K. pneumoniae clinical isolates from unrelated humans and dogs in Thailand. The similarities and differences found contribute to our understanding of the potential widescale dissemination of these important resistance genes among clinical isolates from humans and animals, which in turn may contribute to outbreaks of emerging resistant clones in hospital settings.

Nanoemulsion of cinnamon oil to combat colistin-resistant Klebsiella pneumoniae and cancer cells

This study aimed to investigate the potential of cinnamon oil nanoemulsion (CONE) as an antibacterial agent against clinical strains of colistin-resistant Klebsiella pneumoniae and its anticancer activity. The prepared and characterized CONE was found to have a spherical shape with an average size of 70.6 ± 28.3 nm under TEM and a PDI value of 0.076 and zeta potential value of 6.9 mV using DLS analysis. The antibacterial activity of CONE against Klebsiella pneumoniae strains was investigated, and it was found to have higher inhibitory activity (18.3 ± 1.2–30.3 ± 0.8 mm) against the tested bacteria compared to bulk cinnamon oil (14.6 ± 0.88–20.6 ± 1.2) with MIC values ranging from 0.077 to 0.31 % v/v which equivalent to 0.2–0.82 ng/ml of CONE. CONE inhibited the growth of bacteria in a dose and time-dependent manner based on the time-kill assay in which Klebsiella pneumoniae B-9 was used as a model among the bacterial strains under investigation. The study also investigated the expression of the mcr-1 gene in the Klebsiella pneumoniae strains and found that all strains were positive for the gene expression and subsequently its presence. The level of mcr-1 gene expression among the B-2, B-4, B-9, and B-11 control strains and that treated with colistin was similar, but it was different in both B-5 and B-2. However, all strains exhibited a significant downregulation in gene expression (ranging from 3.97 to 8.7-fold) after their treatment with CONE. Additionally, the CONE-treated bacterial cells appeared with a great deformation compared with control cells under TEM. Finally, CONE exhibited selective toxicity against different cancer cell lines depending on comparison with the normal cell lines.

Occurrence and mechanisms of tigecycline resistance in carbapenem- and colistin-resistant Klebsiella pneumoniae in Thailand

Tigecycline has been regarded as one of the most important last-resort antibiotics for the treatment of infections caused by extensively drug-resistant (XDR) bacteria, particularly carbapenem- and colistin-resistant Klebsiella pneumoniae (C-C-RKP). However, reports on tigecycline resistance have been growing. Overall, ~ 4000 K. pneumoniae clinical isolates were collected over a five-year period (2017–2021), in which 240 isolates of C-C-RKP were investigated. Most of these isolates (91.7%) were resistant to tigecycline. Notably, a high-risk clone of ST16 was predominantly identified, which was associated with the co-harboring of blaNDM-1 and blaOXA-232 genes. Their major mechanism of tigecycline resistance was the overexpression of efflux pump acrB gene and its regulator RamA, which was caused by mutations in RamR (M184V, Y59C, I141T, A28T, C99/C100 insertion), in RamR binding site (PI) of ramA gene (C139T), in MarR (S82G), and/or in AcrR (L154R, R13Q). Interestingly, four isolates of ST147 carried the mutated tet(A) efflux pump gene. To our knowledge, this is the first report on the prevalence and mechanisms of tigecycline resistance in C-C-RKP isolated from Thailand. The high incidence of tigecycline resistance observed among C-C-RKP in this study reflects an ongoing evolution of XDR bacteria against the last-resort antibiotics, which demands urgent action.