Genetic Characterization of Extended-Spectrum Beta-Lactamase (ESBL) and Metallo-Beta-Lactamase (MBL) Producing Klebsiella pneumoniae from Diabetic Foot Ulcer (DFU).

Background: Antibiotic resistance in common pathogenic bacteria is linked with the genetic makeup. The genetic basis of antibiotic resistance may vary in different species or pathophysiological conditions. Objectives: We studied the antibiotic resistance in Klebsiella pneumonia isolates from DFU in the western Indian population. We also studied the presence of ESBL and MBL mechanisms of antibiotic resistance along with the prevalence of the genes involved in ESBL (TEM ESBL , SHV ESBL , and CTX-M ESBL ) and MBL (NDM-1 bla , KPC bla , OXA-48 bla , and VIM bla ) production. Results: A total of 161 K. pneumoniae isolates were analyzed; among which 50.93% were positive for ESBL and 45.96% were positive for MBL production. Most of the isolates were resistant to antibiotics used in the present study and partially resistant to Imipenem and Amikacin. There was no relation between the antibiotic resistance of the isolates and the production of ESBL or MBL mechanism of antibiotic resistance. Further, TEM ESBL was the most prevalent gene in K. pneumoniae isolates followed by CTX-M ESBL , NDM-1 bla , SHV ESBL , and KPC bla . VIM bla was the least prevalent gene found in K. pneumoniae isolates. There was no difference in the prevalence of the genes with respect to the presence or absence of ESBL and MBL mechanism of resistance. Further, there was no relation between the prevalence of the genes and antibiotic resistance in K. pneumoniae isolates. Conclusion: These results along with the literature review suggest that the prevalence of the genes involved in antibiotic resistance mechanisms are widespread in India and their distribution varies in different studies.

Metastatic community acquired Klebsiella pneumonia infection, secondary to skin and soft tissue infection: A case report.

Klebsiella pneumonia is known to cause hospital-acquired infections, primarily in immunocompromised patients. Recently, a distinct syndrome of community-acquired invasive Klebsiella pneumonia infection has been observed, mainly in the Southeast Asian population. This syndrome is associated with disseminated infection and the formation of multiple organ abscesses. Affected organs include the liver, the meninges, the brain, the eyes, and rarely the skin and soft tissue. Most of the affected patients suffer from diabetes mellitus. We present a case of invasive community-acquired Klebsiella pneumonia infection with the skin as the primary source. The patient was found to have multiple abscesses involving the skin, the liver, the right lung, and the brain. Cultures from the wound, the liver abscess, and the blood all revealed Klebsiella pneumonia. The liver abscess was drained, and the patient received a prolonged course of antibiotics based on the sensitivity. One month later, the patient achieved full recovery. Our report highlights the emerging syndrome of invasive community-acquired Klebsiella pneumonia infection and the need for timely diagnosis and treatment to achieve favorable outcomes.

Molecular characterization of NDM and OXA-48-like-producing Klebsiella pneumoniae ST16 and hypervirulent ST337 clone among two patients; a case report.

Carbapenem-resistant Klebsiella pneumoniae (CRKP) infections are a major public health problem, requiring the use of last-resort antibiotics such as colistin. However, there is concern regarding the emergence of isolates resistant to this agent. The report describes two patients with urinary tract infection (UTI) and ventilator-associated pneumonia (VAP) infection caused by CRKP strains. The first case was a 23-year-old male with UTI caused by a strain of ST16 co-harboring blaCTX-M, blaTEM, blaSHV, blaNDM, blaOXA-48-like genes. The second case was a 39-year-old woman with VAP due to hypervirulent ST337-K2 co-harboring blaSHV, blaNDM, blaOXA-48-like, iucA, rmpA2 and rmpA. The patients' general condition improved after combination therapy with colistin (plus meropenem and rifampin, respectively) and both of them recovered and were discharged from the hospital. This study highlights the necessary prevention and control steps to prevent the further spread of CRKP strains should be a priority in our hospital.

Pulmonary abscess secondary to epididymitis caused by extended spectrum ß-lactamase-producing hypervirulent Klebsiella pneumoniae: a case report.

BACKGROUND: Pulmonary abscesses resulting from epididymitis caused by extended spectrum ß-lactamase-producing hypervirulent Klebsiella pneumoniae (ESBL-hvKp) in a nondiabetic patient are extremely uncommon. The infection caused by this disseminated drug-resistant bacteria, which is generally considered an intractable case, poses a potential challenge in clinical practice. CASE PRESENTATION: In this case report, we present the clinical course of a 71-year-old male patient with epididymitis, who subsequently developed cough and dyspnea following anti-infection treatment. Imaging examinations revealed severe pneumonia and pulmonary abscess. The infection of ESBL-hvKp in the epididymis led to bacteremia and subsequent lung lesions. Due to poor response to anti-infection therapy, the patient required an extended duration of anti-infection treatment and ultimately chosed to discontinue treatment. CONCLUSIONS: Acute epididymitis caused by ESBL-hvKP infection can result in the spread of the infection through the bloodstream, leading to severe pneumonia and lung abscess. Given the critical condition of the patient, even with active anti-infection treatment, there is a risk of treatment failure or potentially fatal outcomes.

High prevalence of polymyxin-heteroresistant carbapenem-resistant Klebsiella pneumoniae and its within-host evolution to resistance among critically ill scenarios.

PURPOSE: We aimed to explore the prevalence and within-host evolution of resistance in polymyxin-heteroresistant carbapenem-resistant Klebsiella pneumoniae (PHR-CRKP) in critically ill patients. METHODS: We performed an epidemiological analysis of consecutive patients with PHR-CRKP from clinical cases. Our study investigated the within-host resistance evolution and its clinical significance during polymyxin exposure. Furthermore, we explored the mechanisms underlying the dynamic evolution of polymyxin resistance at both subpopulation and genetic levels, involved population analysis profile test, time-killing assays, competition experiments, and sanger sequencing. Additionally, comparative genomic analysis was performed on 713 carbapenemase-producing K. pneumoniae strains. RESULTS: We enrolled 109 consecutive patients, and PHR-CRKP was found in 69.7% of patients without previous polymyxin exposure. 38.1% of PHR-CRKP isolates exhibited polymyxin resistance and led to therapeutic failure in critically ill scenarios. An increased frequency of resistant subpopulations was detected during PHR-CRKP evolution, with rapid regrowth of resistant subpopulations under high polymyxin concentrations, and a fitness cost in an antibiotic-free environment. Mechanistic analysis revealed that diverse mgrB insertions and pmrB hypermutations contributed to the dynamic changes in polymyxin susceptibility in dominant resistant subpopulations during PHR evolution, which were validated by comparative genomic analysis. Several deleterious mutations (e.g. pmrBLeu82Arg, pmrBSer85Arg) were firstly detected during PHR-CRKP evolution. Indeed, specific sequence types of K. pneumoniae demonstrated unique deletions and deleterious mutations. CONCLUSIONS: Our study emphasizes the high prevalence of pre-existing heteroresistance in CRKP, which can lead to polymyxin resistance and fatal outcomes. Hence, it is essential to continuously monitor and observe the treatment response to polymyxins in appropriate critically ill scenarios.

Failure of ceftazidime/avibactam experimental therapy in the treatment of Klebsiella pneumoniae ST11 co-producing NDM-1 and OXA-48 carbapenemases infection.

Carbapenem-resistant organisms (CRO) represent a significant threat because of their widespread in hospital settings, difficult-to-treat, and association with high morbidity and mortality rates. Data on the efficacy of ceftazidime/avibactam (CAZ-AVI) among patients infected with CRO in Iran are lacking. Herein, we report a case of a 91-year-old man with infection caused by extensively drug-resistant ST11 co-harbouring blaNDM and blaOXA-48-like strain from seven isolates. During ICU hospitalization, 10 different antibiotics were prescribed to the patient, and CAZ-AVI was experimentally prescribed in combination with tobramycin and tigecycline to the patient for the first time in the teaching hospitals of Isfahan City. The patient died on the 56th day of hospitalization. The present study revealed that the use of CAZ-AVI should be limited to targeted therapy after susceptibility results and minimum inhibitory concentration values are available to the treating clinicians and not be used for empirical therapy of patients with an infection caused by CRO, underscoring the urgent need for stringent policies for antibiotic stewardship to preserve the activity of novel ß-lactam/ß-lactamase inhibitors.

Antibiotyping, RAPD- and ERIC-PCR fingerprinting of Klebsiella pneumoniae clinical isolates at a tertiary reference hospital in Denpasar, Bali, Indonesia.

Background and Objectives: Klebsiella pneumoniae is a healthcare-associated infections agent and could be an extended spectrum ß-lactamase (ESBL) producer. Understanding the transmission of this bacterium in a hospital setting needs accurate typing methods. An antibiogram is used to detect the resistance pattern of the isolates. Random Amplified Polymorphic DNA (RAPD) and Enterobacterial Repetitive Intergenic Consensus (ERIC)-PCR are rapid, technically simple, and easy-to-interpret DNA typing methods. This study aimed to evaluate the use of antibiotyping, RAPD-, and ERIC-PCR to investigate the heterogeneity of K. pneumoniae isolated from clinical specimens. Materials and Methods: The antibiograms of 46 K. pneumoniae clinical isolates were determined by Vitek® 2 Compact. All isolates underwent RAPD-PCR using AP4 primer and ERIC-PCR using ERIC-2 primer. The dendrogram was generated using the GelJ software and analyzed to determine its similarity. The analysis of antibiogram and the molecular typing diversity index was calculated using the formula of the Simpson's diversity index. Results: About 71.7% of the isolates were ESBL-producers, and more than 80% of isolates were susceptible to amikacin, ertapenem, and meropenem. The antibiotyping produced 32 diverse types with DI = 0.964. In addition, the RAPD-PCR produced 47 different types with DI = 1, while ERIC-PCR was 46 (DI=0.999). Conclusion: Antibiotyping, RAPD- and ERIC-PCR showed powerful discrimination power among the isolates, supported the diversity of K. pneumoniae isolates in current study. These combination could be promising tools for clonal relationship determination, including in tracking the transmission of the outbreak's agent in hospital setting.

Secondary bacterial infections in post-COVID-19 mucormycosis cases: a retrospective study at Imam Khomeini Hospital Complex, March 2020-April 2021.

Background and Objectives: To explore the prevalence and characteristics of secondary bacterial infections among patients suffering from mucormycosis following COVID-19 infection. Materials and Methods: We conducted a cross-sectional, retrospective analysis from March 2020 to April 2022 at Imam Khomeini Hospital Complex in Tehran. The study included patients with histopathologically confirmed mucormycosis and documented secondary bacterial infections. We extracted and analyzed data from hospital records using SPSS software, version 26. Results: The study comprised 27 patients, with a predominance of females (70.4%) and an average age of 56 years. The majority of these patients (63%) had pre-existing diabetes mellitus. The severity of their COVID-19 infections varied. Treatment regimens included immunosuppressive drugs and antibiotics. Rhinocerebral mucormycosis was the most common form observed. The predominant secondary infections involved the urinary tract, respiratory system, bloodstream (bacteremia), and soft tissues, with resistant strains of Acinetobacter baumannii, Escherichia coli, and Klebsiella pneumoniae being the most frequently identified microorganisms. Notably, cases of bacteremia and pneumonia exhibited a higher mortality rate. Ultimately, 55.6% of patients were discharged, while 44.4% succumbed to their infections. Conclusion: Patients recovering from COVID-19 with mucormycosis are significantly susceptible to secondary bacterial infections, particularly those with diabetes mellitus or those undergoing immunosuppressive therapy. Such infections compound the morbidity and mortality risks in this vulnerable patient cohort.

Identification of coumarin - benzimidazole hybrids as potential antibacterial agents: Synthesis, in vitro and in vivo biological assessment, and ADMET prediction.

The direct-linked coumarin-benzimidazole hybrids, featuring aryl and n-butyl substituents at the N1-position of benzimidazole were synthesized through a Knoevenagel condensation reaction. This reaction involved the condensation of 1,2-diaminobenzene derivatives with coumarin-3-carboxylic acids in the presence of polyphosphoric acid (PPA) at 154 °C. The in vitro antibacterial potency of the hybrid molecules against different gram-positive and gram-negative bacterial strains led to the identification of the hybrids 6m and 6p with a MIC value of 6.25 µg/mL against a gram-negative bacterium, Klebsiella pneumonia ATCC 27736. Cell viability studies on THP-1 cells demonstrated that the compounds 6m and 6p were non-toxic at a concentration of 50 µM. Furthermore, in vivo efficacy studies using a murine neutropenic thigh infection model revealed that both compounds significantly reduced bacterial (Klebsiella pneumonia ATCC 27736) counts (more than 2 log) compared to the control group. Additionally, both compounds exhibited favorable physicochemical properties and drug-likeness characteristics. Consequently, these compounds hold promise as lead candidates for further development of effective antibacterial drugs.

Whole-genome sequencing of Klebsiella pneumoniae isolated from clinical specimens in Chattogram, Bangladesh.

The emergence of multidrug-resistant Klebsiella pneumoniae (Kpn) is a global concern due to the increasing rate of mortality and hospital cost burden in the affected population. This study reports the whole-genome sequences of nine multidrug-resistant Kpn from a hospital in Chattogram city of Bangladesh.

Tracking intra-species and inter-genus transmission of KPC through global plasmids mining.

Klebsiella pneumoniae carbapenemase (KPC) poses a major public health risk. Understanding its transmission dynamics requires examining the epidemiological features of related plasmids. Our study compiled 15,660 blaKPC-positive isolates globally over the past two decades. We found extensive diversity in the genetic background of KPC, with 23 Tn4401-related and 341 non-Tn4401 variants across 163 plasmid types in 14 genera. Intra-K. pneumoniae and cross-genus KPC transmission patterns varied across four distinct periods. In the initial periods, plasmids with narrow host ranges gradually established a survival advantage. In later periods, broad-host-range plasmids became crucial for cross-genera transmission. In total, 61 intra-K. pneumoniae and 66 cross-genus transmission units have been detected. Furthermore, phylogenetic reconstruction dated the origin of KPC transmission back to 1991 and revealed frequent exchanges across countries. Our research highlights the frequent and transient spread events of KPC mediated by plasmids across multiple genera and offers theoretical support for high-risk plasmid monitoring.

Molecular mechanisms responsible KPC-135-mediated resistance to ceftazidime-avibactam in ST11-K47 hypervirulent Klebsiella pneumoniae.

Ceftazidime-avibactam resistance attributable to the blaKPC-2 gene mutation is increasingly documented in clinical settings. In this study, we characterized the mechanisms leading to the development of ceftazidime-avibactam resistance in ST11-K47 hypervirulent Klebsiella pneumoniae that harboured the blaKPC-135 gene. This strain possessed fimbriae and biofilm, demonstrating pathogenicity. Compared with the wild-type KPC-2 carbapenemase, the novel KPC-135 enzyme exhibited a deletion of Glu168 and Leu169 and a 15-amino acid tandem repeat between Val262 and Ala276. The blaKPC-135 gene was located within the Tn6296 transposon truncated by IS26 and carried on an IncFII/IncR-type plasmid. Compared to the blaKPC-2-positive cloned strain, only the MIC of ceftazidime increased against blaKPC-135-positive K. pneumoniae and wasn't inhibited by avibactam (MIC 32 µg/mL), while clavulanic acid and vaborbactam demonstrated some inhibition. Kinetic parameters revealed that KPC-135 exhibited a lower Km and kcat/Km with ceftazidime and carbapenems, and a higher (∼26-fold) 50% inhibitory concentration with avibactam compared to KPC-2. The KPC-135 enzyme exerted a detrimental effect on fitness relative to the wild-type strain. Furthermore, this strain possessed hypervirulent determinants, which included the IncHI1B/FIB plasmid with rmpA2 and expression of type 1 and 3 fimbriae. In conclusion, we reported a novel KPC variant, KPC-135, in a clinical ST11-K47 hypervirulent K. pneumoniae strain, which conferred ceftazidime-avibactam resistance, possibly through increased ceftazidime affinity and decreased avibactam susceptibility. This strain simultaneously harboured resistance and virulence genes, posing an elevated challenge in clinical treatment.

Co-production of 1,3-propanediol and phage phiKpS2 from the glycerol fermentation by Klebsiella pneumoniae.

As an alternative to antibiotics in response to antimicrobial-resistant infections, bacteriophages (phages) are garnering renewed interest in recent years. However, the massive preparation of phage is restricted using traditional pathogens as host cells, which incurs additional costs and contamination. In this study, an opportunistic pathogen, Klebsiella pneumoniae used to convert glycerol to 1,3-propanediol (1,3-PDO), was reused to prepare phage after fermentation. The phage infection showed that the fed-batch fermentation broth containing 71.6 g/L 1,3-PDO can be directly used for preparation of phage with a titer of 1 × 108 pfu/mL. Then, the two-step salting-out extraction was adopted to remove most impurities, e.g. acetic acid (93.5%), ethanol (91.5%) and cells (99.4%) at the first step, and obtain 1,3-PDO (56.6%) in the top phase as well as phage (97.4%) in the middle phase at the second step. This integrated process provides a cheap and environment-friendly manner for coproduction of 1,3-PDO and phage.

Demographics and prognosis of patients with pyogenic liver abscess due to Klebsiella pneumonia or other species.

Background: Pyogenic liver abscess (PLA) is a potentially life-threatening intra-abdominal infection. We compared the clinical features, treatments, and prognoses of patients who had Klebsiella pneumonia pyogenic liver abscess (KPPLA) and non-Klebsiella pneumonia pyogenic liver abscess (non-KPPLA). Methods: A retrospective analysis was used to compare the medical records of KPPLA and non-KPPLA patients with positive pus cultures at a single hospital in China from January 2017 to December 2019. Results: We examined 279 patients with definitively established PLA, 207 (74.2 %) with KPPLA, and 72 with non-KPPLA. The non-KPPLA group had a higher Charlson comorbidity index, longer hospital stay, longer duration of fever, and greater hospital costs. Multivariate analysis showed that alcohol intake (OR: 2.44, P = 0.048), cancer (OR: 4.80, P = 0.001), ICU admission (OR: 10.61, P = 0.026), resolution of fever OR: 1.04, P = 0.020), and a positive blood culture (OR: 2.87, P = 0.029) were independent predictors of primary treatment failure. Percutaneous needle aspiration (PNA) and pigtail catheter drainage (PCD) provided satisfactory outcomes, but PNA led to shorter hospital stays and reduced costs, especially in KPPLA patients whose abscesses were smaller than 10 cm. Conclusion: KPPLA and non-KPPLA patients had some differences in comorbidities and treatment strategies, and non-KPPLA patients had a significantly worse prognosis.

Surveillance and Resistance of Community-Onset Extended-Spectrum ß-Lactamase-Producing Escherichia coli and Klebsiella pneumonia in Oral and Maxillofacial Surgery Site Infections.

Background: The prevalence of community-onset infections of extended spectrum ß-lactamase (ESBL)-producing strains has increased globally, yet surveillance and resistance in patients with oral and maxillofacial surgery site infections is less investigated. Patients and Methods: A retrospective cohort study was performed to investigate risk factors and resistance of ESBL-producing Escherichia coli (ESBL-EC) and ESBL-producing Klebsiella pneumonia (ESBL-KP) among community-onset patients with oral and maxillofacial surgery during January 2010 to December 2016. Demographic features, predisposing factors, clinical outcomes, and antibiotic agent costs were analyzed. Antimicrobial susceptibility testing of nine antimicrobial agents against ESBL-KP and ESBL-EC were measured. Results: Among 2,183 cultures from infection sites in patients with oral and maxillofacial surgery site (45 cases [2.06%]) were confirmed with community-onset ESBL-KP (24; 1.10%) or ESBL-EC (21; 0.96%) infection. Multivariable analysis showed the independent risk factors for ESBL-producing bacterial infection were prior history of hospitalization (adjusted odds ratio [aOR], 10.984; 95% confidence interval [CI], 5.965-59.879; p = 0.025) and malignant condition (aOR, 3.373; 95% CI 2.947-7.634; p = 0.024). Based on antimicrobial susceptibility testing, 57.8% ESBL-KP and ESBL-EC were found receiving inappropriate antimicrobial therapy, and antibiotic agent costs were higher than non-ESBL-producing bacterial infections ($493.8 ± $367.3 vs. $304.1 ± $334.7; p = 0.031). Conclusions: Infections caused by ESBL-KP and ESBL-EC among patients in sites with oral and maxillofacial surgery are associated with prior history of hospitalization and malignant conditions. Prompt detection and appropriate antibiotic administration for community-onset infections of ESBLs are necessary for such populations.

Treatment of polluted river water using potential bioflocculant produced by Klebsiella pneumonia UKD24.

Chemically enhanced primary treatment (CEPT) is a rapid wastewater treatment process involving treating wastewater with two chemical-aided processes, coagulation, and flocculation. In the present study, a natural extracellular polymeric substance flocculant (EPSBF) produced by Klebsiella pneumonia UKD24, a bacterium isolated from the sewage treatment plant, and a synthetic polyacrylamide anionic polymer flocculant (PAM) were evaluated to treat polluted river water. The synthetic PAM showed immediate turbidity reduction after agitation, while the EPSBF expressed a rapid decrease in optical density. After 20 min of the settling period, the EPSBF showed reduced rates of turbidity, optical density, and chemical oxygen demand at 74.14 ± 5.2%, 89.37 ± 0.76%, and 87.21 ± 0.73%, respectively, while PAM showed 67.08 ± 4%, 85.68 ± 2%, and 86.57 ± 2%, respectively. EPSBF treatment significantly improved the water quality parameters in terms of total dissolved solids, total suspended solids, conductivity, and oxidation-reduction potential than PAM treatment. However, the EPSBF has shown a more water-holding capacity and relatively weak flock formation, producing more sludge volume than PAM. Furthermore, though the sludge produced by the EPSBF treatment had a higher moisture content, it showed shorter capillary suction time (CST). In contrast, sludge formed in PAM treatment had lower moisture content, but it exhibited prolonged CST value indicating that PAM treatment sludge showed slow dewaterability.

The Clinical Efficacy of Adding Ceftazidime/Avibactam to Standard Therapy in Treating Infections Caused by Carbapenem-Resistant Klebsiella pneumonia with blaOXA-48-like Genes.

Ceftazidime/avibactam (CAZ-AVI) is FDA-approved for managing infections caused by resistant gram-negative bacilli, particularly infections via carbapenem-resistant Enterobacterales pathogens. The clinical data are still limited, particularly those in Saudi Arabia. The present study is a retrospective cohort study that was carried out at the Armed Forces Hospital in the southern region of Saudi Arabia to compare the clinical and microbiological outcomes for CAZ-AVI-treated patients as monotherapy and as an add-on to standard therapy for carbapenem-resistant Klebsiella pneumonia (CRKP) OXA-48 infections to those treated with standard drugs. The study included CRKP OXA-48-like infected patients who were administered antibiotics for more than seven days from 1 August 2018 to May 2023. Patients' baseline characteristics and demography were extracted from the clinical records, and their clinical/microbiology efficiencies were assessed as per the corresponding definitions. Univariate and multivariate logistic regressions were conducted to identify the potential independent variable for CAZ-AVI efficiency. A total of 114 patient files were included for the evaluation. Among these patients, 64 used CAZ-AVI combined with standard therapy and were included in the intervention group, and 50 of them used standard therapy and were included in the comparative group. Following analysis, CAZ-AVI's clinical success was 42.2% (p = 0.028), while the intervention versus comparative groups showed decreased 30-day all-cause mortality (50.0% versus 70.0%; p = 0.036) and infection recurrence (7.8% versus 24.0%; p = 0.019), as well as substantially increased rates of microbial eradication (68.8% versus 42.0%; p = 0.007). CAZ-AVI add-on therapy rather than monotherapy showed statistically significant favored clinical and microbial outcomes over the standard therapy. Furthermore, sex (female %), ICU admission, and fever were negatively associated with patients' 30-day all-cause mortality, serving as independent negative factors. Only fever, CRP bio levels, inotropes, and ICU admissions were significant predictors influencing the CAZ-AVI's clinical efficiency. The duration of CAZ-AVI therapy positively influenced CAZ-AVI's microbial eradication, while both WBC counts and fever experiences were negative predictors. This study shows the effective usage of CAZ-AVI against CRKP OXA-48-like infections. The influencing independent variables depicted here should recommend that clinicians individualize the CAZ-AVI dose based on co-existing risk factors to achieve optimal survival and efficacy. Prospective multicenter and randomized control studies are recommended, with individualized CAZ-AVI precision administration implemented based on patients' characteristics.

Klebsiella pneumoniae K2 capsular polysaccharide degradation by a bacteriophage depolymerase does not require trimer formation.

K2-capsular Klebsiella pneumoniae is a hypervirulent pathogen that causes fatal infections. Here, we describe a phage tailspike protein, named K2-2, that specifically depolymerizes the K2 capsular polysaccharide (CPS) of K. pneumoniae into tetrasaccharide repeating units. Nearly half of the products contained O-acetylation, which was thought crucial to the immunogenicity of CPS. The product-bound structures of this trimeric enzyme revealed intersubunit carbohydrate-binding grooves, each accommodating three tetrasaccharide units of K2 CPS. The catalytic residues and the key interactions responsible for K2 CPS recognition were identified and verified by site-directed mutagenesis. Further biophysical and functional characterization, along with the structure of a tetrameric form of K2-2, demonstrated that the formation of intersubunit catalytic center does not require trimerization, which could be nearly completely disrupted by a single-residue mutation in the C-terminal domain. Our findings regarding the assembly and catalysis of K2-2 provide cues for the development of glycoconjugate vaccines against K. pneumoniae infection. IMPORTANCE: Generating fragments of capsular polysaccharides from pathogenic bacteria with crucial antigenic determinants for vaccine development continues to pose challenges. The significance of the C-terminal region of phage tailspike protein (TSP) in relation to its folding and trimer formation remains largely unexplored. The polysaccharide depolymerase described here demonstrates the ability to depolymerize the K2 CPS of K. pneumoniae into tetrasaccharide fragments while retaining the vital O-acetylation modification crucial for immunogenicity. By carefully characterizing the enzyme, elucidating its three-dimensional structures, conducting site-directed mutagenesis, and assessing the antimicrobial efficacy of the mutant enzymes against K2 K. pneumoniae, we offer valuable insights into the mechanism by which this enzyme recognizes and depolymerizes the K2 CPS. Our findings, particularly the discovery that trimer formation is not required for depolymerizing activity, challenge the current understanding of trimer-dependent TSP activity and highlight the catalytic mechanism of the TSP with an intersubunit catalytic center.

Did algal toxin and Klebsiella infections cause the unexplained 2007 mass mortality event in Danish and Swedish marine mammals?

An unusual mass mortality event (MME) of harbour seals (Phoca vitulina) and harbour porpoises (Phocoena phocoena) occurred in Denmark and Sweden in June 2007. Prior to this incident, the region had experienced two MMEs in harbour seals caused by Phocine Distemper Virus (PDV) in 1988 and 2002. Although epidemiology and symptoms of the 2007 MME resembled PDV, none of the animals examined for PDV tested positive. Thus, it has been speculated that another - yet unknown - pathogen caused the June 2007 MME. To shed new light on the likely cause of death, we combine previously unpublished veterinary examinations of harbour seals with novel analyses of algal toxins and algal monitoring data. All harbour seals subject to pathological examination showed pneumonia, but were negative for PDV, influenza and coronavirus. Histological analyses revealed septicaemia in multiple animals, and six animals tested positive for Klebsiella pneumonia. Furthermore, we detected the algal Dinophysis toxin DTX-1b (1-115 ng g-1) in five seals subject to toxicology, representing the first time DTX-1b has been detected in marine vertebrates. However, no animals tested positive for both Klebsiella and toxins. Thus, while our relatively small sample size prevent firm conclusions on causative agents, we speculate that the unexplained MME may have been caused by a chance incidence of multiple pathogens acting in parallel in June 2007, including Dinophysis toxin and Klebsiella. Our study illustrates the complexity of wildlife MMEs and highlights the need for thorough sampling during and after MMEs, as well as additional research on and monitoring of DTX-1b and other algal toxins in the region.

Insights into the preventive actions of natural compounds against Klebsiella pneumoniae infections and drug resistance.

Klebsiella pneumoniae is a type of Gram-negative bacteria that causes a variety of infections, including pneumonia, bloodstream infections, wound infections, and meningitis. The treatment of K. pneumoniae infection depends on the type of infection and the severity of the symptoms. Antibiotics are generally used to treat K. pneumoniae infections. However, some strains of K. pneumoniae have become resistant to antibiotics. This comprehensive review examines the potential of natural compounds as effective strategies against K. pneumonia infections. The alarming rise in antibiotic resistance underscores the urgent need for alternative therapies. This article represents current research on the effects of diverse natural compounds, highlighting their anti-microbial and antibiofilm properties against K. pneumonia. Notably, compounds such as andrographolide, artemisinin, baicalin, berberine, curcumin, epigallocatechin gallate, eugenol, mangiferin, piperine, quercetin, resveratrol, and thymol have been extensively investigated. These compounds exhibit multifaceted mechanisms, including disruption of bacterial biofilms, interference with virulence factors, and augmentation of antibiotic effectiveness. Mechanistic insights into their actions include membrane perturbation, oxidative stress induction, and altered gene expression. While promising, challenges such as limited bioavailability and varied efficacy across bacterial strains are addressed. This review further discusses the potential of natural compounds as better alternatives in combating K. pneumonia infection and emphasizes the need for continued research to harness their full therapeutic potential. As antibiotic resistance persists, these natural compounds offer a promising avenue in the fight against K. pneumonia and other multidrug-resistant pathogens.