Therapeutic efficacy of a K5-specific phage and depolymerase against Klebsiella pneumoniae in a mouse model of infection.

Klebsiella pneumoniae has become one of the most intractable gram-negative pathogens infecting humans and animals due to its severe antibiotic resistance. Bacteriophages and protein products derived from them are receiving increasing amounts of attention as potential alternatives to antibiotics. In this study, we isolated and investigated the characteristics of a new lytic phage, P1011, which lyses K5 K. pneumoniae specifically among 26 serotypes. The K5-specific capsular polysaccharide-degrading depolymerase dep1011 was identified and expressed. By establishing murine infection models using bovine strain B16 (capable of supporting phage proliferation) and human strain KP181 (incapable of sustaining phage expansion), we explored the safety and efficacy of phage and dep1011 treatments against K5 K. pneumoniae. Phage P1011 resulted in a 60% survival rate of the mice challenged with K. pneumoniae supporting phage multiplication, concurrently lowering the bacterial burden in their blood, liver, and lungs. Unexpectedly, even when confronted with bacteria impervious to phage multiplication, phage therapy markedly decreased the number of viable organisms. The protective efficacy of the depolymerase was significantly better than that of the phage. The depolymerase achieved 100% survival in both treatment groups regardless of phage propagation compatibility. These findings indicated that P1011 and dep1011 might be used as potential antibacterial agents to control K5 K. pneumoniae infection.

Prevalence of antimicrobial resistance, biofilm formation, efflux pump activity, and virulence capabilities in multi-drug-resistant Klebsiella pneumoniae isolated from freshwater fish farms.

The present study aimed to determine the antibiotic resistance, underlying mechanisms, antibiotic residues, and virulence genes involved in 32 multi-drug-resistant Klebsiella pneumoniae isolates from freshwater fishes in Andhra Pradesh, India. Antibiogram studies revealed that all isolates were multi-drug-resistant, harbored tetA (96.8%), tetC (59.3%), tetD (71.9%), nfsA (59.3%), nfsB (53.1%), sul2 (68.7%), qnrC (43.7%), qnrD (50%), blaSHV (75%), blaTEM (68.7%), and blaCTX-M (93.7%) genes. Multiple antibiotic resistance index was calculated as 0.54. Sixteen isolates were confirmed to be hyper-virulent and harbored magA and rmpA genes. In total, 46.9, 31.2, and 21.9% of the isolates were categorized as strong, moderate, or weak biofilm formers, respectively. All isolates possessed an active efflux pump and harbored acrA, acrB, acrAB, and tolC genes in 94% of the isolates, followed by mdtK (56.2%). Porins such as ompK35 and ompK36 were detected in 59.3 and 62.5% of the isolates, respectively. Virulence genes fimH-1, mrkD, and entB were present in 84.3, 81.2, 87.5% of the isolates, respectively. These findings imply a potential threat that multi-drug-resistant bacterial pathogens could transmit to surrounding environments and humans through contaminated water and the aquaculture food chain.

Investigating the effects of zinc oxide and titanium dioxide nanoparticles on the formation of biofilm and persister cells in Klebsiella pneumoniae.

The biofilm formation in klebsiella pneumoniae isolates poses a significant problem as it can result in treatment failure and the development of chronic infections. These biofilms act as protective barriers, rendering the bacteria resistant to antibiotics. Additionally, persister cells, which make up a small fraction of the bacterial population, have the ability to enter a dormant state after treatment with high doses of antibiotics. These persister cells play a crucial role in the high level of biofilm-mediated tolerance to antibiotics. The present study aimed to investigate the impact of Zinc oxide (ZnO) and titanium dioxide (TiO2) nanoparticles on the formation of biofilm and persister cells in K. pneumoniae. The minimum inhibitory concentration (MIC) of colistin in K. pneumoniae ATCC 13883 was determined using the microdilution method. The formation of persister cells was evaluated by introducing sub-MIC of colistin. Subsequently, the MIC of ZnO NPs and TiO2 NPs in these persister cells was assessed using the microdilution method. Furthermore, the effects of nanoparticles on the expression levels of biofilm-associated genes were analyzed using real-time polymer chain reaction (PCR). The MIC values for colistin, ZnO, and TiO2 were determined at 2, 12.5, and 6.25 µg/mL, respectively. In the presence of nanoparticles, biofilm formation decreased. Real-time PCR results showed the messenger RNA (mRNA) level of mrkH and fimH were decreased and the expression of luxS and mazF were increased. Biofilm formation of K. pneumoniae ATCC 1383 was inhibited in response to nanoparticles. According to the results of the present study use of nanoparticles may help control multidrug-resistant (MDR) infections in hospitalized patients.

Myeloid miR-155 plays a limited role in antibacterial defense during Klebsiella-derived pneumosepsis and is dispensable for lipopolysaccharide- or Klebsiella-induced inflammation in mice.

MicroRNA-155 (miR-155) plays a crucial role in regulating host inflammatory responses during bacterial infection. Previous studies have shown that constitutive miR-155 deficiency alleviates inflammation while having varying effects in different bacterial infection models. However, whether miR-155 in myeloid cells is involved in the regulation of inflammatory and antibacterial responses is largely elusive. Mice with myeloid cell specific miR-155 deficiency were generated to study the in vitro response of bone marrow-derived macrophages (BMDMs), alveolar macrophages (AMs) and peritoneal macrophages (PMs) to lipopolysaccharide (LPS), and the in vivo response after intranasal or intraperitoneal challenge with LPS or infection with Klebsiella (K.) pneumoniae via the airways. MiR-155-deficient macrophages released less inflammatory cytokines than control macrophages upon stimulation with LPS in vitro. However, the in vivo inflammatory cytokine response to LPS or K. pneumoniae was not affected by myeloid miR-155 deficiency. Moreover, bacterial outgrowth in the lungs was not altered in myeloid miR-155-deficient mice, but Klebsiella loads in the liver of these mice were significantly higher than in control mice. These data argue against a major role for myeloid miR-155 in host inflammatory responses during LPS-induced inflammation and K. pneumoniae-induced pneumosepsis but suggest that myeloid miR-155 contributes to host defense against Klebsiella infection in the liver.

The effect of Bdellovibrio bacteriovorus containing dressing on superficial incisional surgical site infections experimentally induced by Klebsiella pneumoniae in mice.

Bdellovibrio bacteriovorus is a bacterial agent that stands out for its ability to act as a predator against gram-negative bacteria and has found application against antibiotic-resistant pathogens. The aim of this study is to determine the efficacy of Bdellovibrio bacteriovorus against antibiotic-resistant pathogens, particularly those causing infections in surgical incision sites. A total of 6 experimental groups were created in mice, and surgical area infections were initiated with Klebsiella pneumoniae in incision sites. The effects of antibiotics and Bdellovibrio bacteriovorus alone or in combination were compared to the control group. In the Bdellovibrio bacteriovorus treatment group, edema and redness were observed in all mice at 24th hours, in 20% of mice at 48th hours, and in none at the 72 nd h. A significant difference was observed in the Bdellovibrio bacteriovorus treatment groups in reducing Klebsiella pneumoniae burden in the incision area compared to antibiotics alone or Bdellovibrio bacteriovorus + antibiotics, (p < 0.001). Likewise, cytokine level determinations indicated that B. bacteriovorus applications generated a therapeutic response without inducing an inflammatory response.

Molecular basis of Klebsiella pneumoniae colonization in host.

Klebsiella pneumoniae (K. pneumoniae) is a common cause of nosocomial infection, which causing disseminated infections such as cystitis, pneumonia and sepsis. K. pneumoniae is intrinsic resistant to penicillin, and members of the population usually have acquired resistance to a variety of antibiotics, which makes it a major threat to clinical and public health. Bacteria can colonize on or within the hosts, accompanied by growth and reproduction of the organisms, but no clinical symptoms are presented. As the "first step" of bacterial infection, colonization in the hosts is of great importance. Colonization of bacteria can last from days to years, with resolution influenced by immune response to the organism, competition at the site from other organisms and, sometimes, use of antimicrobials. Colonized pathogenic bacteria cause healthcare-associated infections at times of reduced host immunity, which is an important cause of clinical occurrence of postoperative complications and increased mortality in ICU patients. Though, K. pneumoniae is one of the most common conditional pathogens of hospital-acquired infections, the mechanisms of K. pneumoniae colonization in humans are not completely clear. In this review, we made a brief summary of the molecular basis of K. pneumoniae colonization in the upper respiratory tract and intestinal niche, and provided new insights for understanding the pathogenesis of K. pneumoniae.

Klebsiella pneumoniae activates the TGF-ß signaling pathway to adhere to and invade intestinal epithelial cells via enhancing TLL1 expression.

Klebsiella pneumoniae is a gram-negative bacterium that can cause many diseases in hospitals and communities. Intestinal K. pneumoniae infections are relatively rare. Most K. pneumoniae infections begin with the colonization of the gastrointestinal system. In this study, clinically isolated K. pneumoniae strains were used to infect intestinal epithelial Caco-2 cells to study the possible intestinal translocation mechanism of K. pneumoniae. We found that of the three K. pneumoniae strains tested, KP1821 exhibited the strongest adhesive and invasive abilities and that the adhesion to Caco-2 intestinal epithelial cells was affected by the acidic environment of the stomach. Transcriptome sequencing revealed the involvement of molecules associated with the extracellular matrix and cell adhesion, inflammatory response, calcium ion and transforming growth factor ß (TGF-ß) signaling pathways, and other abnormalities in biological processes and cell signaling pathways. Additionally, tolloid-like protein 1 (TLL1) was significantly upregulated. Knocking down TLL1 with shRNA significantly reduced KP1821's ability to invade and adhere to intestinal epithelial cells. TLL1 is involved in the activation of the TGF-ß signaling pathway. Inhibition of this pathway using the inhibitor SB431542 induced significantly reduced adhesion and invasion capabilities of KP1821. Our findings demonstrate that TLL1 participates in K. pneumoniae adhesion and invasion of intestinal epithelial cells by activating the TGF-ß signaling pathway.

The Therapeutic Treatment with the GAG-Binding Chemokine Fragment CXCL9(74-103) Attenuates Neutrophilic Inflammation and Lung Dysfunction during Klebsiella pneumoniae Infection in Mice.

Klebsiella pneumoniae is an important pathogen associated with hospital-acquired pneumonia (HAP). Bacterial pneumonia is characterized by a harmful inflammatory response with a massive influx of neutrophils, production of cytokines and chemokines, and consequent tissue damage and dysfunction. Targeted therapies to block neutrophil migration to avoid tissue damage while keeping the antimicrobial properties of tissue remains a challenge in the field. Here we tested the effect of the anti-inflammatory properties of the chemokine fragment CXCL9(74-103) in pneumonia induced by Klebsiella pneumoniae in mice. Mice were infected by intratracheal injection of Klebsiella pneumoniae and 6 h after infection were treated systemically with CXCL9(74-103). The recruitment of leukocytes, levels of cytokines and chemokines, colony-forming units (CFU), and lung function were evaluated. The treatment with CXCL9(74-103) decreased neutrophil migration to the airways and the production of the cytokine interleukin-1ß (IL-1ß) without affecting bacterial control. In addition, the therapeutic treatment improved lung function in infected mice. Our results indicated that the treatment with CXCL9(74-103) reduced inflammation and improved lung function in Klebsiella pneumoniae-induced pneumonia.

A Multiwell-Plate Caenorhabditis elegans Assay for Assessing the Therapeutic Potential of Bacteriophages against Clinical Pathogens.

In order to establish phage therapy as a standard clinical treatment for bacterial infections, testing of every phage to ensure the suitability and safety of the biological compound is required. While some issues have been addressed over recent years, standard and easy-to-use animal models to test phages are still rare. Testing of phages in highly suitable mammalian models such as mice is subjected to strict ethical regulations, while insect larvae such as the Galleria mellonella model suffer from batch-to-batch variations and require manual operator skills to inject bacteria, resulting in unreliable experimental outcomes. A much simpler model is the nematode Caenorhabditis elegans, which feeds on bacteria, a fast growing and easy to handle organism that can be used in high-throughput screening. In this study, two clinical bacterial strains of Escherichia coli, one Klebsiella pneumoniae, and one Enterobacter cloacae strain were tested on the model system together with lytic bacteriophages that we isolated previously. We developed a liquid-based assay, in which the efficiency of phage treatment was evaluated using a scoring system based on microscopy and counting of the nematodes, allowing increasing statistical significance compared to other assays such as larvae or mice. Our work demonstrates the potential to use Caenorhabditis elegans to test the virulence of strains of Klebsiella pneumoniae, Enterobacter cloacae, and EHEC/EPEC as well as the efficacy of bacteriophages to treat or prevent infections, allowing a more reliable evaluation for the clinical therapeutic potential of lytic phages. IMPORTANCE Validating the efficacy and safety of phages prior to clinical application is crucial to see phage therapy in practice. Current animal models include mice and insect larvae, which pose ethical or technical challenges. This study examined the use of the nematode model organism C. elegans as a quick, reliable, and simple alternative for testing phages. The data show that all the four tested bacteriophages can eliminate bacterial pathogens and protect the nematode from infections. Survival rates of the nematodes increased from <20% in the infection group to >90% in the phage treatment group. Even the nematodes with poly-microbial infections recovered during phage cocktail treatment. The use of C. elegans as a simple whole-animal infection model is a rapid and robust way to study the efficacy of phages before testing them on more complex model animals such as mice.

Combination of pre-adapted bacteriophage therapy and antibiotics for treatment of fracture-related infection due to pandrug-resistant Klebsiella pneumoniae.

A 30-year-old bombing victim with a fracture-related pandrug-resistant Klebsiella pneumoniae infection after long-term (>700 days) antibiotic therapy is treated with a pre-adapted bacteriophage along with meropenem and colistin, followed by ceftazidime/avibactam. This salvage therapy results in objective clinical, microbiological and radiological improvement of the patient's wounds and overall condition. In support, the bacteriophage and antibiotic combination is highly effective against the patient's K. pneumoniae strain in vitro, in 7-day mature biofilms and in suspensions.

Extensive outbreak of colistin resistant, carbapenemase (blaOXA-48, blaNDM) producing Klebsiella pneumoniae in a large tertiary care hospital, India.

BACKGROUND: Extensive drug resistance in Klebsiella pneumoniae (K. pneumoniae) causing major outbreaks in large hospitals is an emerging challenge. We describe a near fatal outbreak of colistin resistant, carbapenem resistant K. pneumoniae (CRKp) producing metallo beta-lactamases (blaNDM) and blaOXA-48 in the neonatal intensive care unit (NICU) at the background of a larger outbreak involving multiple parts of the hospital and the challenges in its containment. METHODS: Following identification of an outbreak due to colistin resistant CRKp between April to June 2017 in the NICU, a thorough surveillance of similar cases and the hospital environment was performed to trace the source. All the isolated K. pneumoniae were tested for susceptibility to standard antibiotics by disc diffusion and microbroth dilution methods. Molecular detection of extended spectrum beta lactamases (ESBLs) and carbapenemases (classes A, B, D) genes was done. Enterobacterial repetitive intergenic consensus (ERIC) PCR and multi-locus sequence typing (MLST) was done to determine the genetic relatedness of the isolates. Characteristics of different sequence types were statistically compared (Student's t-test). RESULTS: A total of 45 K. pneumoniae isolates were studied from NICU (14 cases of neonatal sepsis), ICU (18 cases), other wards (7 cases) along with 6 isolates from hospital environment and human colonizers. The primary case was identified in the ICU. All the K. pneumoniae from NICU and 94.4% from the ICU were colistin resistant CRKp. Majority (59.37% and 56.25%) harbored blaSHV/blaCTXM and blaOXA-48 genes, respectively. Two distinct sequence types ST5235 and ST5313 were noted with colistin resistance, distribution within the NICU and mortality as significant attributes of ST5235 (p < 0.05). The outbreak was contained with strengthening of the infection control practices and unintended short duration closure of the hospital. CONCLUSION: Large hospital outbreaks with considerable mortality can be caused by non-dominant clones of colistin resistant CRKp harboring blaOXA-48 and blaNDM carbapenemases in endemic regions. The exact global impact of these sequence types should be further studied to prevent future fatal outbreaks.

Klebsiella pneumoniae infection following H9N2 influenza A virus infection contributes to the development of pneumonia in mice.

In this study, whether H9N2 influenza A virus (IAV) infection contributed to secondary Klebsiella pneumoniae infection was investigated. From post-infection onwards, clinical symptoms were monitored, examined and recorded daily for 11 days. As a result, no clinical signs were observed in the mice infected with single H9N2 IAV, implying that H9N2 IAV was less pathogenic to mice. Compared to single K. pneumonia infection, K. pneumoniae infection following H9N2 IAV infection exacerbates lung histopathological lesions and apoptosis, resulting in more severe diseases. Lung index of the mice with H9N2 IAV and K. pneumoniae co-infection was significantly higher than those in the other groups. Bacterial loads in the tissues in H9N2 IAV and K. pneumoniae co-infection group were significantly higher than those in the single K. pneumoniae infection group at 7 dpi. It demonstrated that prior H9N2 IAV infection contributed to K. pneumonia proliferation and delayed bacterial clearance in mice. Secondary K. pneumoniae infection influences seroconversion of anti-H9N2 antibody titers and the cytokine profiles. The findings demonstrated that H9N2 IAV infection facilitated secondary K. pneumonia infection, causing severe the diseases in mice.

Diet-induced obesity in mice impairs host defense against Klebsiella pneumonia in vivo and glucose transport and bactericidal functions in neutrophils in vitro.

Obesity impairs host defense against Klebsiella pneumoniae, but responsible mechanisms are incompletely understood. To determine the impact of diet-induced obesity on pulmonary host defense against K. pneumoniae, we fed 6-wk-old male C57BL/6j mice a normal diet (ND) or high-fat diet (HFD) (13% vs. 60% fat, respectively) for 16 wk. Mice were intratracheally infected with Klebsiella, assayed at 24 or 48 h for bacterial colony-forming units, lung cytokines, and leukocytes from alveolar spaces, lung parenchyma, and gonadal adipose tissue were assessed using flow cytometry. Neutrophils from uninfected mice were cultured with and without 2-deoxy-d-glucose (2-DG) and assessed for phagocytosis, killing, reactive oxygen intermediates (ROI), transport of 2-DG, and glucose transporter (GLUT1-4) transcripts, and protein expression of GLUT1 and GLUT3. HFD mice had higher lung and splenic bacterial burdens. In HFD mice, baseline lung homogenate concentrations of IL-1ß, IL-6, IL-17, IFN-γ, CXCL2, and TNF-α were reduced relative to ND mice, but following infection were greater for IL-6, CCL2, CXCL2, and IL-1ß (24 h only). Despite equivalent lung homogenate leukocytes, HFD mice had fewer intraalveolar neutrophils. HFD neutrophils exhibited decreased Klebsiella phagocytosis and killing and reduced ROI to heat-killed Klebsiella in vitro. 2-DG transport was lower in HFD neutrophils, with reduced GLUT1 and GLUT3 transcripts and protein (GLUT3 only). Blocking glycolysis with 2-DG impaired bacterial killing and ROI production in neutrophils from mice fed ND but not HFD. Diet-induced obesity impairs pulmonary Klebsiella clearance and augments blood dissemination by reducing neutrophil killing and ROI due to impaired glucose transport.

Comparison between the molecular diagnostic test and chest X-ray combined with multi-slice spiral CT in the diagnosis of lobar pneumonia.

Lobar pneumonia is an inflammatory condition of the lung that mainly affects the lobes of the lungs and the alveoli, and it is usually caused by a bacterial infection. There are many ways to diagnosis this disease. But an early and accurate method for lobar pneumonia diagnosis has an important role in its treatment. Therefore, in this study, a comparison between the molecular diagnostic test and chest x-ray combined with multi-slice spiral CT was done to find out better diagnosis of lobar pneumonia. For this purpose, 122 individuals suspected of lobar pneumonia were studied by clinical examination, chest X-ray, and multi-slice spiral CT. For the molecular diagnosis test, the multiplex PCR was used for two main causes of the disease, Streptococcus pneumoniae and Klebsiella pneumoniae. Results showed that the specificity for Chest X-ray + Multi-slice Spiral CT had the highest amount (82.8%), but high sensitivity (100%) belonged to a molecular diagnostic test for both bacteria. On the other hand, the sensitivity and specificity of Streptococcus pneumoniae were better than Klebsiella pneumoniae and the possibility of error in Streptococcus pneumoniae was lower than Klebsiella pneumoniae. In general, although the Chest X-ray + Multi-slice Spiral CT method was better than the molecular diagnosis test, it could not identify the causative agent and did not show a difference between pathogens for better antibiotic treatment, and also the possibility of diagnosis is low at the beginning of the disease. Therefore, according to the results of the current study, the best way to diagnose lobar pneumonia is to use both methods, simultaneously.

Two ST11 Klebsiella pneumoniae strains exacerbate colorectal tumorigenesis in a colitis-associated mouse model.

Sequence type (ST) 11 is one of the major lineages of carbapenem-resistant Klebsiella pneumoniae (CRKP). Although the gastrointestinal (GI) carriage of CRKP predisposes individuals to subsequent infections, little is known for its impact on gut homeostasis. In this study, we investigated the association between ST11 CRKP colonization and colorectal cancer (CRC). Two ST11 CRKP, KPC160111 (KL47) and KPC160132 (KL64), were selected as the representative strains. We used azoxymethane (AOM) and dextran sodium sulfate (DSS) to initiate a colitis-associated CRC model. Both strains established prolonged colonization in the GI tract of the AOM-DSS-treated BALB/c mice and aggravated gut dysbiosis. Under this AOM-DSS-induced setting, ST11 K. pneumoniae colonization significantly promoted the growth and progression of colorectal adenomas to high-grade dysplasia. Numerous crypts were formed inside the enlarged adenomas, in which CD163+ tumor-associated macrophages accumulated. Similarly, ST11 K. pneumoniae also increased the population size of the CD163+ macrophages with the M2 phenotype in the peritoneal cavity of LPS-primed BALB/c mice. When applied to RAW264.7 cells, ST11 K. pneumoniae polarized the macrophages toward an M2 phenotype through the inhibition of IKK-NFκB and the activation of STAT6-KLF4-IL-10. Through the M2-skewing ability, ST11 K. pneumoniae promoted the accumulation of CD163+ macrophages in the adenomatous crypts to create an immunosuppressive niche, which not only accommodated the extended stay for its own sake but also deteriorated colorectal tumorigenesis.

A novel hypermucoviscous Klebsiella pneumoniae ST3994-K2 clone belonging to Clonal Group 86.

Emergent hypervirulent Klebsiella pneumoniae has been responsible for severe diseases, representing a serious threat to public health. We report the whole-genome sequencing of a novel ST3994-K2 clone, a single locus variant of ST86 K2, which is considered a worrying hypervirulent clone that emerged in several parts of the world. The strain K. pneumonia Kpi144 was isolated in 2013 from a blood culture of a 69-year-old male patient admitted to a tertiary hospital in Teresina, state of Piauí, northeastern Brazil. The strain was susceptible to 41 antibiotics tested, presented hypermucoviscous phenotype and a virulent behavior was observed in the Galleria mellonella infection model. Moreover, the virulome showed several virulence genes. To the best of our knowledge, this is the first worldwide report of a novel ST3994-K2 K. pneumoniae clone, an SLV of ST86 K2, which is considered a worrying virulent clone that has emerged in several parts of the world, including South America and Brazil.

Pentadecanoic acid against Candida albicans-Klebsiella pneumoniae biofilm: towards the development of an anti-biofilm coating to prevent polymicrobial infections.

The ability to form biofilms is a common feature of microorganisms, which can colonize a variety of surfaces, such as host tissues and medical devices, resulting in infections highly resistant to conventional drugs. This aspect is particularly critical in polymicrobial biofilms involving both fungi and bacteria, therefore, to eradicate such severe infections, new and effective anti-biofilm strategies are needed. The efficacy of pentadecanal and pentadecanoic acid as anti-biofilm agents has been recently reported against different bacterial strains. Their chemical similarity with diffusible signal factors (DSFs), plus the already known ability of fatty acids to act as anti-biofilm agents, suggested to explore their use against Candida albicans and Klebsiella pneumoniae mixed biofilm. In this work, we demonstrated the ability of both molecules to prevent the formation and destabilize the structure of the dual-species biofilm. Moreover, the pentadecanoic acid anti-biofilm coating, previously developed through the adsorption of the fatty acid on polydimethylsiloxane (PDMS), was proved to prevent the polymicrobial biofilm formation in dynamic conditions by confocal laser scanning microscopy analysis. Finally, the evaluation of the expression levels of some biofilm-related genes of C. albicans and K. pneumoniae treated with pentadecanoic acid provided some insights into the molecular mechanisms underpinning its anti-biofilm effect.

Acute vaping exacerbates microbial pneumonia due to calcium (Ca2+) dysregulation.

As electronic cigarette (E-cig) use, also known as "vaping", has rapidly increased in popularity, data regarding potential pathologic effects are recently emerging. Recent associations between vaping and lung pathology have led to an increased need to scrutinize E-cigs for adverse health impacts. Our previous work (and others) has associated vaping with Ca2+-dependent cytotoxicity in cultured human airway epithelial cells. Herein, we develop a vaped e-liquid pulmonary exposure mouse model to evaluate vaping effects in vivo. Using this model, we demonstrate lung pathology through the use of preclinical measures, that is, the lung wet: dry ratio and lung histology/H&E staining. Further, we demonstrate that acute vaping increases macrophage chemotaxis, which was ascertained using flow cytometry-based techniques, and inflammatory cytokine production, via Luminex analysis, through a Ca2+-dependent mechanism. This increase in macrophage activation appears to exacerbate pulmonary pathology resulting from microbial infection. Importantly, modulating Ca2+ signaling may present a therapeutic direction for treatment against vaping-associated pulmonary inflammation.

Human milk oligosaccharides and non-digestible carbohydrates prevent adhesion of specific pathogens via modulating glycosylation or inflammatory genes in intestinal epithelial cells.

Human milk oligosaccharides (hMOs) and non-digestible carbohydrates (NDCs) are known to inhibit the adhesion of pathogens to the gut epithelium, but the mechanisms involved are not well understood. Here, the effects of 2'-FL, 3-FL, DP3-DP10, DP10-DP60 and DP30-DP60 inulins and DM7, DM55 and DM69 pectins were studied on pathogen adhesion to Caco-2 cells. As the growth phase influences virulence, E. coli ET8, E. coli LMG5862, E. coli O119, E. coli WA321, and S. enterica subsp. enterica LMG07233 from both log and stationary phases were tested. Specificity for enteric pathogens was tested by including the lung pathogen K. pneumoniae LMG20218. Expression of the cell membrane glycosylation genes of galectin and glycocalyx and inflammatory genes was studied in the presence and absence of 2'-FL or NDCs. Inhibition of pathogen adhesion was observed for 2'-FL, inulins, and pectins. Pre-incubation with 2'-FL downregulated ICAM1, and pectins modified the glycosylation genes. In contrast, K. pneumoniae LMG20218 downregulated the inflammatory genes, but these were restored by pre-incubation with pectins, which reduced the adhesion of K. pneumoniae LMG20218. In addition, DM69 pectin significantly upregulated the inflammatory genes. 2'-FL and pectins but not inulins inhibited pathogen adhesion to the gut epithelial Caco-2 cells through changing the cell membrane glycosylation and inflammatory genes, but the effects were molecule-, pathogen-, and growth phase-dependent.

Risk Factors for Klebsiella Infections among Hospitalized Patients with Preexisting Colonization.

Klebsiella commonly colonizes the intestinal tract of hospitalized patients and is a leading cause of health care-associated infections. Colonization is associated with subsequent infection, but the factors determining this progression are unclear. A cohort study was performed, in which intensive care and hematology/oncology patients with Klebsiella colonization based on rectal swab culture were enrolled and monitored for infection for 90 days after a positive swab. Electronic medical records were analyzed for patient factors associated with subsequent infection, and variables of potential significance in a bivariable analysis were used to build a final multivariable model. Concordance between colonizing and infecting isolates was assessed by wzi capsular gene sequencing. Among 2,087 hospitalizations from 1,978 colonized patients, 90 cases of infection (4.3%) were identified. The mean time to infection was 20.6 ± 24.69 (range, 0 to 91; median, 11.5) days. Of 86 typed cases, 68 unique wzi types were identified, and 69 cases (80.2%) were colonized with an isolate of the same type prior to infection. Based on multivariable modeling, overall comorbidities, depression, and low albumin levels at the time of rectal swab collection were independently associated with subsequent Klebsiella infection (i.e., cases). Despite the high diversity of colonizing strains of Klebsiella, there is high concordance with subsequent infecting isolates, and progression to infection is relatively quick. Readily accessible data from the medical record could be used by clinicians to identify colonized patients at an increased risk of subsequent Klebsiella infection. IMPORTANCE Klebsiella is a leading cause of health care-associated infections. Patients who are intestinally colonized with Klebsiella are at a significantly increased risk of subsequent infection, but only a subset of colonized patients progress to disease. Colonization offers a potential window of opportunity to intervene and prevent these infections, if the patients at greatest risk could be identified. To identify patient factors associated with infection in colonized patients, we studied 1,978 colonized patients. We found that patients with a higher burden of underlying disease in general, depression in particular, and low albumin levels in a blood test were more likely to develop infection. However, these variables did not completely predict infection, suggesting that other host and microbial factors may also be important. The clinical variables associated with infection are readily available in the medical record and could serve as the foundation for developing an integrated risk assessment of Klebsiella infection in hospitalized patients.