Search Results (28)

Carbapenem-resistant hypervirulent Klebsiella pneumoniae (CR-hvKp), a pathogen causing severe nosocomial infections and high mortality rates, is increasingly becoming a serious global public health threat. Capsular polysaccharide (CPS), a major virulence factor of hvKp, can be enzymatically degraded by bacteriophage-derived depolymerases. However, to our knowledge, depolymerases targeting K. pneumoniae K54-type strains have rarely been identified. Here, we identified and characterized three novel capsule depolymerases, Dep_C, Dep_Y, and Dep_Z, derived from three different K. pneumoniae phages, which retained robust activity across a broad pH range (pH 3.0–12.0) and demonstrated thermal stability up to 50 °C. These depolymerases could efficiently digest the CPS of K. pneumoniae K54-serotype strains, significantly inhibit biofilm formation, and remove their mature biofilms. Although no bactericidal activity was detected, these depolymerases rendered host bacteria susceptible to serum complement-mediated killing. We further demonstrate that Dep_C, Dep_Y, and Dep_Z can effectively and significantly prolong the survival time of mice in a pneumonia model infected with K54-type K. pneumoniae and reduce the colonization and virulence of the bacteria in the mice. These findings indicate that depolymerases Dep_C, Dep_Y, and Dep_Z could increase bacterial susceptibility to host immune responses of hvKp to the host through their degradation effect on the CPS. In conclusion, our study demonstrates that the three capsule depolymerases are promising antivirulent agents to combat CR-hvKp infections. Full article

Background: Dithiolopyrrolones (DTPs), such as holomycin and thiolutin, exhibit potent antibacterial activities. DTPs contain a disulfide within a unique bicyclic scaffold, which may chelate metal ions and disrupt metal-dependent cellular processes once the disulfide is reductively transformed to thiols. However, the contribution of the intrinsic redox mechanism of DTPs to their antibacterial activity remains unclear. Herein we used pyrroloformamide (Pyf) A, a DTP with a unique formyl substituent, as a prototype to study the antibacterial potential and mechanism against ESKAPE pathogens, in particular carbapenem-resistant Klebsiella pneumoniae (CRKP). Methods: The antibacterial and anti-biofilm activities of Pyf A were mainly assessed against clinical CRKP isolates. Propidium iodide staining, scanning electron microscopy, glutathione (GSH) quantification, and reactive oxygen species (ROS) analysis were utilized to infer its anti-CRKP mechanism. The synergistic antibacterial effects of Pyf A and AgNO₃ were evaluated through checkerboard and time-kill assays, as well as in vivo murine wound and catheter biofilm infection models. Results: Pyf A exhibited broad-spectrum antibacterial activity against ESKAPE pathogens with minimum inhibitory concentrations ranging from 0.25 to 4 μg/mL. It also showed potent anti-biofilm effects against CRKP. Pyf A disrupted the cell membranes of CRKP and markedly depleted intracellular GSH without triggering ROS accumulation. Pyf A and AgNO₃ showed synergistic anti-CRKP activities in vitro and in vivo, by disrupting both GSH- and thioredoxin-mediated redox homeostasis. Conclusions: Pyf A acts as a GSH-depleting agent and, when combined with AgNO₃, achieves dual-targeted disruption of bacterial thiol redox systems. This dual-targeting strategy enhances antibacterial efficacy of Pyf A and represents a promising therapeutic approach to combat CRKP infections. Full article

Due to the involvement of biofilms in the pathogenesis of bone and joint infections (BJI), the treatment of these infections is often challenging, especially when multidrug- or extensively drug-resistant (MDR/XDR) pathogens are involved. Intravenous fosfomycin (FOS) is a phosphoenolpyruvate analogue with a unique mode of action and broad-spectrum activity against both Gram-positive (GP) and Gram-negative (GN) pathogens. It is used in various severe and deep-seated infections, including BJIs. This review article focuses on preclinical and clinical data surrounding the use of FOS for biofilm-related BJIs. Data from several in vitro and animal models of infection demonstrated that FOS, especially in combination with other antibiotics, is effective against biofilms of (methicillin-resistant) Staphylococcus spp., (vancomycin-resistant) Enterococcus spp., carbapenem-resistant and extended-spectrum beta-lactamase-producing Enterobacterales, and MDR Pseudomonas aeruginosa. Data from clinical studies, mostly retrospective observational studies and case reports/case series, revealed that FOS was typically used in combination with other antibiotics for the treatment of various BJI, including acute and chronic osteomyelitis, prosthetic joint infections, and fracture-related infections, in adult and pediatric patients. Success rates often exceeded 80%. FOS exhibits good and fast penetration into bone tissue and is generally well tolerated, with only a few adverse drug reactions, such as gastrointestinal disorders and electrolyte imbalances. Collectively, the data indicate that FOS is a valuable option as part of combination regimens for the treatment of BJIs caused by both GP and GN bacteria. Full article

Background/Objectives. One of the pressing challenges in global public health is the rise in infections caused by carbapenem-resistant Enterobacteriaceae. Growing bacterial drug resistance, coupled with the slow development of new antibiotics, highlights the critical need to explore and develop new broad-spectrum antimicrobial agents able to inhibit bacterial growth efficiently. In recent years, antimicrobial peptides (AMPs) have gained significant attention as a promising alternative to conventional drugs, owing to their antimicrobial potency, low toxicity, and reduced propensity for fostering resistance. Our research aims to investigate the antibacterial ability of three amphibian AMPs, namely Hylin-a1, AR-23, and RV-23, against both antibiotic-sensitive and carbapenem-resistant strains of Escherichia coli and Klebsiella pneumoniae. Methods. A 3-(4,5 dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay (MTT) was performed to identify non-cytotoxic concentrations of peptides. A microdilution assay evaluated the antibacterial effect, determining the peptides’ minimum inhibitory concentration (MIC). In addition, the checkerboard test analyzed the compounds’ synergistic effect with meropenem. Results. We demonstrated that peptides with low toxicity profile and resistance to proteolytic activity exhibited strong antibacterial activity, with MIC ranging from 6.25 to 25 μM. The antibiofilm mechanism of action of peptides was also investigated, suggesting that they had a crucial role during the biofilm formation step by inhibiting it. Finally, we highlighted the synergistic effects of peptides with meropenem. Conclusions. Our study identifies Hylin-a1, AR-23, and RV-23 as promising candidates against Gram-negative bacterial infections with a favorable therapeutic profile. This effect could be related to their great flexibility, as evidenced by circular dichroism data, confirming that the peptides could assume an α-helical conformation interacting with bacterial membranes. Full article

Background/Objectives: Ventilator-associated pneumonia (VAP) remains a critical challenge in ICU settings, often driven by the biofilm-mediated bacterial colonization of endotracheal tubes (ETTs). This study investigates antimicrobial resistance patterns and biofilm dynamics in ICU patients, focusing on microbial colonization and resistance trends in tracheal aspirates and endotracheal tube biofilms at a county emergency hospital in Romania. Methods: We conducted a longitudinal analysis of ICU patients requiring mechanical ventilation for more than 48 h. Tracheal aspirates and ETT biofilms were collected at three key time points: T1 (baseline), T2 (48 h post-intubation with ETT replacement), and T3 (92–100 h post-T2); these were analyzed using sonication and microbiological techniques to assess microbial colonization and antimicrobial resistance patterns. Results: In a total of 30 patients, bacteria from the ESKAPEE group (e.g., Klebsiella pneumoniae, Acinetobacter baumannii, Staphylococcus aureus) dominated the microbiota, increasing their prevalence over time. Resistance to carbapenems, colistin, and vancomycin was notably observed, particularly among K. pneumoniae and A. baumannii. Biofilm analysis revealed high persistence rates and the emergence of multidrug-resistant strains, underscoring the role of ETTs as reservoirs for resistant pathogens. The replacement of ETTs at T2 correlated with a shift in microbial composition and reduced biofilm-associated contamination. Conclusions: This study highlights the temporal evolution of antimicrobial resistance and biofilm-associated colonization in a limited number of ICU patients (30 patients). The findings support implementing routine ETT management strategies, including scheduled replacements and advanced biofilm-disruption techniques, to mitigate VAP risk and improve patient outcomes. Full article

The emergence of hypervirulent and carbapenem-resistant hypermucoviscous Klebsiella pneumoniae strains presents a significant public health challenge due to their increased virulence and resistance to multiple antibiotics. This study evaluates the antibiotic susceptibility patterns and virulence profiles of classical and hypervirulent K. pneumoniae strains isolated from various clinical samples. A total of 500 clinical samples were collected from patients at the Mardan Medical Complex and Ayub Medical Complex in KPK between July 2022 and June 2024. Among these, 64 K. pneumoniae strains were isolated and subsequently subjected to antimicrobial susceptibility testing (AST) and phenotypic virulence detection. Among the 64 isolates, 21 (32.8%) exhibited hypermucoviscosity, a characteristic associated with increased pathogenicity. Hemagglutination was observed in 35 (54.1%) of the isolates, indicating the presence of surface adhesins that facilitate bacterial adherence to host tissues. A high prevalence of biofilm formation was noted, with 54 (84%) isolates capable of forming biofilms, which are known to protect bacteria from antibiotics and the host immune response. Most isolates (59/64, 92.1%) were resistant against ampicillin, highlighting its limited efficacy against these strains. Conversely, the lowest resistance was observed for tigecycline, with only 15% (10/64) of the isolates showing resistance, indicating its potential utility as a treatment option. The study also found that 38 (59.3%) of the isolates were extended-spectrum beta-lactamase (ESBL) producers, 42 (65.6%) were multidrug-resistant (MDR), 32 (50%) were extensively drug-resistant (XDR), and 13 (20.3%) were resistant to carbapenems. The genetic study revealed biofilm producer and enhancer genes (mrkD, pgaABCD, fimH, treC, wzc, pilQ, and luxS) mainly in the hypervirulent strains. These hypervirulent strains also show a high number of resistance genes. The findings of this study underscore the critical need for the active surveillance of antimicrobial resistance and virulence determinants in K. pneumoniae. The coexistence of high levels of antibiotic resistance and virulence factors in these isolates poses a severe threat to public health, as it can lead to difficult-to-treat infections and increased morbidity and mortality. Full article

The transferable genetic elements are associated with the dissemination of virulence determinants amongst Klebsiella pneumoniae. Thus, we assessed the correlated antimicrobial resistance in carbapenem-resistant Klebsiella pneumoniae clinical isolates. Each isolate’s ability to biosynthesize biofilm, carbapenemase, and extended-spectrum β-lactamase were examined. Genotypically, the biofilm-, outer membrane porin-, and some plasmid-correlated antimicrobial resistance genes were screened. About 50% of the isolates were multidrug-resistant while 98.4% were extended-spectrum β-lactamase producers and 89.3% were carbapenem-resistant. Unfortunately, 93.1% of the multidrug-resistant isolates produced different biofilm levels. Additionally, fimD and mrkD genes encoding adhesins were detected in 100% and 55.2% of the tested isolates, respectively. Also, the bla_KPC, bla_OXA-48-like, and bla_NDM-encoding carbapenemases were observed in 16.1%, 53.6%, and 55.4% of the tested isolates, respectively. Moreover, the bla_SHV and bla_CTX-M extended-spectrum β-lactamase-associated genes were detected at 95.2% and 61.3%, respectively. Furthermore, aac(3)IIa, qnrB, and tetB resistance-correlated genes were observed in 38.1%, 46%, and 7.9% of the tested isolates, respectively. Certainly, the tested antimicrobial resistance-encoding genes were concurrently observed in 3.2% of the tested isolates. These findings confirmed the elevated prevalence of various antimicrobial resistance-associated genes in Klebsiella pneumoniae. The concurrent transferring of plasmid-encoding antimicrobial resistance-related genes could be associated with the possible acquisition of multidrug-resistant Klebsiella pneumoniae phenotypes. Full article

(1) Background: Carbapenem-resistant (CR) bacteria pose a significant global public health challenge due to their ability to evade treatment with beta-lactam antibiotics, including carbapenems. This study investigates the biofilm-forming capabilities of CR clinical bacterial isolates and examines the impact of serum on biofilm formation. Additionally, the study evaluates the resistance profiles and genetic markers for carbapenemase production. (2) Methods: Bacterial isolates were collected from the microbiology laboratory of Mures County Clinical Hospital between October 2022 and September 2023. Pharyngeal and rectal swabs were screened for carbapenem-resistant bacteria using selective media. Lower respiratory tract samples were also analyzed for CR Gram-negative bacteria. The isolates were tested for their ability to form biofilms in the presence and absence of fetal bovine serum at 24 and 48 h. Carbapenemase production was detected phenotypically and confirmed via PCR for relevant genes. (3) Results: Out of 846 screened samples, 4.25% from pharyngeal swabs and 6.38% from rectal swabs tested positive for CR bacteria. Acinetobacter baumannii and Klebsiella pneumoniae were the most common species isolated. Biofilm formation varied significantly between clinical isolates and standard strains, with clinical isolates generally showing higher biofilm production. The presence of serum had no significant effect on biofilm formation in Klebsiella spp., but stimulated biofilm formation for Acinetobacter spp. Carbapenemase genes bla_KPC, bla_OXA-48-like, and bla_NDM were detected in various isolates, predominantly in Klebsiella spp., but were not the main determinants of carbapenem resistance, at least in screening isolates. (4) Conclusions: This study highlights the variability in biofilm formation among CR clinical isolates and underscores the differences between the bacteria found as carriage versus infection. Both bacterial species and environmental factors variably influence biofilm formation. These insights are crucial for the development of effective treatment and infection control strategies in clinical settings. Full article

Klebsiella pneumoniae, a member of the ESKAPE pathogen group, is a prominent cause of hospital-acquired infections. The WHO has recognized carbapenem-resistant K. pneumoniae as a critical-one priority pathogen. These resilient superbugs have the ability to form biofilms and present a significant global threat. In the present study, we isolated and characterized a bacteriophage SAKp02, from hospital sewage, infectious to carbapenem-resistant K. pneumoniae patient isolates. SAKp02 could infect 43 of 72 clinical isolates, indicating a broad host spectrum. Whole genome analysis classified SAKp02 within the family Casjensviridae, with a 59,343 bp genome encoding 82 ORFs. Comparative genomic analysis revealed significant differences between SAKp02 and its closest viruses, indicating a distinct genetic makeup positioning it as a novel phage strain within the lineage. The SAKp02 genome comprises bacteriolytic enzymes, including holin, endolysin, and phage depolymerase, crucial for bacterial lysis and biofilm disruption. It reduced biofilm biomass by over threefold compared to the control and eradicated 99% of viable cells within a 4 h treatment period. Scanning electron microscopy corroborated the ability of the phage to dismantle biofilm matrices and lyse bacterial cells. Safe and effective treatments are warranted, and hence, the fully characterized lytic phages with therapeutic potential against drug-resistant clinical isolates of bacteria are needed. Our study is the first to report the antibacterial and antibiofilm activity of Casjensviridae phages, and our discovery of a novel K. pneumoniae phage broadens the arsenal against the bacteria. Full article

Both honey and fish oil have been historically used in medicine and identified as having antimicrobial properties. Although analyses of the substances have identified different components within them, it is not fully understood how these components interact and contribute to the observed effect. With the increase in multi-drug resistant strains of bacteria found in infections, new treatment options are needed. This study aimed to assess the antimicrobial abilities of fish oil components, including docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA), and derived resolvins (RvE1, RvD2, and RvD3), as well as two varieties of manuka honey, against a panel of medically relevant microorganisms and antimicrobial resistant organisms, such as Methicillin Resistant Staphylococcus aureus (MRSA) and carbapenem-resistant Escherichia coli. Minimum inhibitory concentrations (MIC) and minimum bactericidal concentrations (MBC) were identified; further minimum biofilm eradication concentrations (MBEC) were investigated for responsive organisms, including S. aureus, E. coli, Staphylococcus epidermidis, Klebsiella pneumoniae, and Pseudomonas aeruginosa. Concurrent with the existing literature, manuka honey was found to be a broad-spectrum antimicrobial with varied potency according to methylglyoxal content. DHA and EPA were both effective against Gram-positive and negative bacteria, but some drug-resistant strains or pathogens were not protected by a capsule. Only E. coli was inhibited by the resolvins. Full article

The worldwide increase of multidrug-resistant Gram-negative bacteria is a global threat. The emergence and global spread of Klebsiella pneumoniae carbapenemase- (KPC-) producing Klebsiella pneumoniae represent a particular concern. This pathogen has increased resistance and abilities to persist in human reservoirs, in hospital environments, on medical devices, and to generate biofilms. Mortality related to this microorganism is high among immunosuppressed oncological patients and those with multiple hospitalizations and an extended stay in intensive care. There is a severe threat posed by the ability of biofilms to grow and resist antibiotics. Various nanotechnology-based strategies have been studied and developed to prevent and combat serious health problems caused by biofilm infections. The aim of this review was to evaluate the implications of nanotechnology in eradicating biofilms with KPC-producing Klebsiella pneumoniae, one of the bacteria most frequently associated with nosocomial infections in intensive care units, including in our department, and to highlight studies presenting the potential applicability of TiO₂ nanocomposite materials in hospital practice. We also described the frequency of the presence of bacterial biofilms on medical surfaces, devices, and equipment. TiO₂ nanocomposite coatings are one of the best long-term options for antimicrobial efficacy due to their biocompatibility, stability, corrosion resistance, and low cost; they find their applicability in hospital practice due to their critical antimicrobial role for surfaces and orthopedic and dental implants. The International Agency for Research on Cancer has recently classified titanium dioxide nanoparticles (TiO₂ NPs) as possibly carcinogenic. Currently, there is an interest in the ecological, non-toxic synthesis of TiO₂ nanoparticles via biological methods. Biogenic, non-toxic nanoparticles have remarkable properties due to their biocompatibility, stability, and size. Few studies have mentioned the use of nanoparticle-coated surfaces as antibiofilm agents. A literature review was performed to identify publications related to KPC-producing Klebsiella pneumoniae biofilms and antimicrobial TiO₂ photocatalytic nanocomposite coatings. There are few reviews on the antibacterial and antibiofilm applications of TiO₂ photocatalytic nanocomposite coatings. TiO₂ nanoparticles demonstrated marked antibiofilm activity, but being nano in size, these nanoparticles can penetrate cell membranes and may initiate cellular toxicity and genotoxicity. Biogenic TiO₂ nanoparticles obtained via green, ecological technology have less applicability but are actively investigated. Full article

Infections caused by carbapenem-resistant Klebsiella pneumoniae (CRKP) are becoming increasingly common within clinical settings, requiring the development of alternative therapies. In this study, we isolated, characterized, and sequenced the genome of a CRKP phage, Phage168. The total genomic DNA of Phage168 was 40,222 bp in length, encoding 49 predicted proteins. Among these proteins, Dep40, the gene product of ORF40, is a putative tail fiber protein that exhibits depolymerase activity based on the result of bioinformatics analyses. In vitro, we confirmed that the molecular weight of the Phage168 depolymerase protein was about 110 kDa, the concentration of the produced phage 168 depolymerase protein was quantified as being 1.2 mg/mL, and the depolymerase activity was still detectable after the dilution of 1.2 µg/mL. This recombinant depolymerase exhibited enzyme activity during the depolymerization of the formed CRKP biofilms. We also found that depolymerase, when combined with polymyxin B, was able to enhance the bactericidal effect of polymyxin B on CRKP strains by disrupting their biofilm. When recombinant depolymerase was used in combination with human serum, it enhanced the sensitivity of the CRKP strain UA168 to human serum, and the synergistic bactericidal effect reached the strongest level when the ratio of depolymerase to human serum was 3:1. Our results indicated that depolymerase encoded by Phage168 may be a promising strategy for combating infections caused by drug-resistant CRKP formed within the biofilm. Full article

Klebsiella pneumoniae (K. pneumoniae) is an opportunistic bacterium that has drawn attention due to its resistance to carbapenem antibiotics. The treatment of patients with severe infections has been challenging. Thus, silver nanoparticles (AgNPs) have been applied for their antimicrobial effects. This work aims to analyze the synergistic effect of the carbapenem antibiotic Imipenem with AgNPs against different susceptibility clinical profiles of K. pneumoniae. The silver nanoparticles were synthesized by bottom-up methodology and capped with alpha-lipoic acid. Susceptibility tests were performed using four K. pneumoniae strains with different susceptibility profiles to Imipenem. The strains were induced to form a biofilm for 48 h. Crystal violet and Resazurin assays were performed to determine biofilm formation and minimal inhibitory concentration, respectively. The reduction in Imipenem concentration with the association of nanoparticles was found in all strains studied in planktonic form, and the synergism between silver nanoparticles and Imipenem was demonstrated through the analysis of the fractional inhibitory concentration index. The viability percentage was reduced at rates ≥80% in the biofilm analysis, characterized by the minimal biofilm inhibitory concentration. The study’s proposed association resulted in inhibitory effects on different K. pneumoniae profiles, both in planktonic forms and biofilm, with peculiar behavior in the Imipenem-resistant profile. Full article

Klebsiella pneumoniae is an opportunistic pathogen that frequently causes nosocomial and community-acquired (CA) infections. Until now, a limited number of studies has been focused on the analyses of changes affecting the virulence attributes. Genotypic and phenotypic methods were used to characterise the 39 clinical K. pneumoniae isolates; all belonged to the pan-drug resistant, widespread clone ST 15 and expressed the K24 capsule. PFGE has revealed that the isolates could be divided into three distinct genomic clusters. All isolates possessed allS and uge genes, known to contribute to the virulence of K. pneumoniae and 10.25% of the isolates showed hypermucoviscosity, 94.87% produced type 1 fimbriae, 92.3% produced type 3 fimbriae, and 92.3% were able to produce biofilm. In vivo persistence could be supported by serum resistance 46.15%, enterobactin (94.87%) and aerobactin (5.12%) production and invasion of the INT407 and T24 cell lines. Sequence analysis of the whole genomes of the four representative strains 11/3, 50/1, 53/2 and 53/3 has revealed high sequence homology to the reference K. pneumoniae strain HS11286. Our results represent the divergence of virulence attributes among the isolates derived from a common ancestor clone ST 15, in an evolutionary process that occurred both in the hospital and in the community. Full article

Klebsiella pneumoniae (K. pneumoniae) colonizes the human gut and is a causative factor of pyogenic liver abscess (PLA). Retrospective studies conducted on K. pneumoniae PLA patients revealed subsequent CRC development in later years of their life with increasing prevalence of these strains harbouring polyketide synthase (PKS) genes. To our knowledge there are no known studies directly implicating K. pneumoniae with CRC to date. Our aims are to characterize K. pneumoniae isolates from CRC patients and investigate its effects on cell proliferation in vitro. K. pneumoniae isolates were characterized by screening virulence genes including polyketide synthase (PKS), biofilm assay, antibiotic susceptibility, and string test to determine hypervirulent (hvKp) strains. Solubilised antigens of selected K. pneumoniae isolates were co-cultured with primary colon cell lines and CRC cell lines (Stage I-IV) for 48 h. The enhancement of proliferation was measured through MTT and ECIS assay. Twenty-five percent of K. pneumoniae isolates were PKS-positive out of which 50% were hvKp strains. The majority of the isolates were from the more virulent serotype of K1 (30%) and K2 (50%). PKS-positive K. pneumoniae isolates did not possess genes to confer carbapenem resistance but instead were more highly associated with siderophore genes (aerobactin, enterobactin, and yersiniabactin) and allantoin metabolism genes (allS, allS2). Cell proliferation in primary colon, SW1116 (Stage I), and SW480 (Stage II) CRC cell lines were enhanced when co-cultured with PKS-positive K. pneumoniae antigens. ECIS revealed enhanced cell proliferation upon recurrent antigen exposure. This demonstrates the possible role that PKS-positive K. pneumoniae has in exacerbating CRC progression. Full article