Saturday 13th August 2022

Host defense against K. pneumoniae infection | JIR

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Introduction

Klebsiella (K.) pneumoniae is a well-known human nosocomial pathogen. Most community-acquired K. pneumoniae infections involve pneumonia or urinary tract infections. However, over the past two decades, a distinct invasive syndrome known as K. pneumoniae-induced liver abscess (KLA) has been increasingly reported in Asia as a global disease, and it causes high morbidity and mortality.1 It is easy to misdiagnose the symptoms or miss them entirely, and difficult to control the development of the disease. The traditional clinical treatment of KLA mainly includes antibiotics and symptomatic management. It is of great importance to explore new effective treatments.

The gut microbiome (1000 species, with 100 trillion bacteria) is the largest and most diverse microbiome in the host.2 As an active participant in the host defense system, the intestinal flora supports mucosal immunity and regulates the immune system.3 In Streptococcus pneumoniae-induced sepsis, the intestinal microbiota protects against damage by regulating phagocytosis by alveolar macrophages.4 Meanwhile, previous studies have confirmed that the intestinal flora has an effect on K. pneumoniae colonization. For instance, a healthy gut microbiome provides an extra layer of defense and helps eliminate exogenous bacteria. However, changes in intestinal flora after antibacterial treatment increased the level of available monosaccharides in the intestinal tract and promoted the growth of pathogenic or opportunistic bacteria K. pneumoniae.5 More persuasive, we also found that antimicrobial treatment have a pervasive and long-lasting effect on the human gut microbiota in clinical practice, especially in intensive care unit patients receiving antibacterial agents.6,7 In vitro studies, gut microbiota metabolites-SCFAs directly inhibited the growth of drug-resistant enterobacteria, including carbapenemase-producing K. pneumoniae. Besides, SCFAs prevented the replication of K. pneumoniae by lowering intracellular pH.8 With the increasing understanding of the relationship between the gut microbiome and host immunity against infectious diseases, it is urgent to explore regulating the gut microbiome in order to prevent and treat infections. Recent research showed that the gut microbiota plays an important role in the regulation of mastitis, compared with those in control mice, S. aureus-induced mastitis mice were observed Increased blood-milk barrier permeability resulted to increasing abundance of pathogenic Enterobacter bacteria; however, feces microbiota transplantation (FMT) reversed these effects.9 Therefore, it is worth exploring whether the intestinal microbiota can reduce liver injury and inflammation, enhancing the ability of the liver to resist the effects of hypervirulent K. pneumoniae invasion during the pathogenesis of KLA.

We hypothesized that the intestinal microbiota can enhance the host’s defenses against K. pneumoniae. In the current study, we assessed the survival, histopathological severity, and serum biochemical and inflammatory indicators in microbiota-depleted (MD) mice infected with K. pneumoniae with or without FMT. The results suggested that the intestinal microbiota plays a protective role in the host defense against K. pneumoniae infection. This in-depth study of the relationship between the intestinal microbiota and non-mucosal hepatic immunity provides a new approach for the development of KLA treatments.

Materials and Methods

Animals and Bacterial Strains

Wild-type female C57BL/6J mice (20±2 g) were purchased from the Experimental Animal Center of Anhui Province (Hefei, China). The animals were housed in a specific-pathogen-free environment, given free access to water and food, and exposed to a 12 h light/dark cycle. The animal study was approved by the Animal Experimentation Ethics Committee of Anhui Medical University (approval NO. LLSC20190253), and experiments were carried out in strict accordance with the Animal Research: Reporting of In Vivo Experiments (ARRIVE) guidelines (the National Centre for the Replacement, Refinement and Reduction of Animals in Research, London, England) for the care and use of laboratory animals.

K. pneumoniae 181608 (serotype K1, magA+, rmpA+, aerobactin+) was isolated from patients with KLA and maintained by the Anhui Center for Surveillance of Bacterial Resistance (Hefei, China). The isolate was confirmed by time-of-flight mass spectrometry system (Clin-TOF-II, Beijing Yixin Bochuang Biotechnology Co., Ltd.). It was cultured in Luria broth at 37°C overnight before use.

KLA Mouse Model

To create a KLA mouse model (KLA group), mice were administered oral mid-log-phase K. pneumoniae 181608 (0.1 mL of 104 CFU/mL suspension) using a 21-gauge feeding needle, as described previously.10 The mice that died within 24 hours were excluded from the group. The control group (Con group) was given the same amount of phosphate-buffered saline (PBS) intragastrically. All mice were killed 48 hours after K. pneumoniae or PBS administration, and the liver, fecal, and blood were obtained for analysis. To obtain mouse fecal, first sterilize mouse anus with 95% ethanol, gently press around anus with sterile cotton ball to promote excretion of mouse fecal, and store collected fecal in refrigerator at −80°C. To obtain the blood samples and liver tissues, the mice were first anesthetized, the eyeball blood was taken and the mice were killed, the abdominal skin was disinfected after fixation with adhesive tape, and the abdominal cavity was opened. The liver was removed with sterile forceps and scissors and fixed in formalin. To assess survival and construct survival curves, the mice were monitored 4–5 times daily for reduced movement, shivering, dyspnea, or circling behavior, and these mice were killed.

Microbiota-Depleted (MD) Mouse Model

To create an MD mouse model, broad-spectrum antibiotics (ampicillin, 1 g/L; neomycin sulfate, 1 g/L; metronidazole, 1 g/L; vancomycin, 0.5 g/L; Sigma, USA) were added to the drinking water for 3 weeks to eliminate the intestinal flora, as described previously.11 During this time, weight and food intake were recorded. At 2 days after antibiotic water was discontinued, to create the MD+KLA group, K. pneumoniae was administered.

FMT

To create the MD+KLA+FMT group, fecal pellets from randomly chosen healthy mice were used to colonize the guts of MD mice 24 hours prior to K. pneumoniae infection.12 Briefly, several fecal pellets from different healthy mice were suspended together in PBS (1 fecal pellet/1 mL PBS). Next, 200 μL/day of the fecal solution was administered to the mice by oral gavage for 3 days after antibiotic water was discontinued and before K. pneumoniae infection.

16S rRNA Pyrosequencing

Microbial DNA was extracted from the feces of the mice using a QIAamp DNA Stool Mini Kit (Qiagen, Germany) according to the manufacturer’s protocol. The DNA concentration and purity was monitored on 1% agarose gels. The DNA was then diluted to 1 ng/μL using sterile water. The V3-V4 region of the bacterial 16S rRNA gene was amplified by PCR (95°C for 5 min, followed by 25 cycles at 95°C for 30 s, 60°C for 30 s, and 72°C for 25 s, and a final extension at 72°C for 5 min) using primers 338F 5′-ACTCCTACGGGAGGCAGCA-3′ and 806R 5′-GGACTACHVGGGTWTCTAAT-3′.13 The PCR products were then sequenced using an Illumina HiSeq 2500 System at Beijing Genomics Institute (BGI-Shenzhen, China).14 Adaptor sequences and low-quality reads were removed. The remaining high-quality clean data were analyzed with UPARSE software, using the UPARSE-OTU and UPARSE-OTUref algorithms. Sequences with ≥97% similarity were assigned to the same OTUs. We picked a representative sequence for each OTU and used the Ribosomal Database Project (RDP) Classifier (http://www.mothur.org/wiki/RDP_reference_files)15 to annotate each representative sequence with taxonomic information. Thereafter, this taxonomic information was used to calculate the relative bacterial abundances at various taxonomic levels.

Luminex Immunoassays

A mouse multi-factor detection kit (Univ-Biotech Technology Company, Shanghai, China) was taken out and left at room temperature for 30 min, and standard curves were then constructed using the standard substances. The kit was used to detect interleukin (IL)-1β, IL-2, IL-4, IL-6, IL-10, IL-17, tumor necrosis factor (TNF)-α, interferon (IFN)-γ, chemokine (CXCL)-1 and macrophage chemokine protein (MCP)-1….

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